Marie Plessz
email: marie.plessz@inra.fr

Emmanuelle Kesse-Guyot

Marie Zins
email: marie.zins@inserm.fr

Joane Matta
email: joane.matta@mail.mcgill.ca

Sébastien Czernichow
email: sebastien.czernichow@aphp.fr

between perceived diet healthiness and adherence to nutritional guidelines in the Constances cohort (France)

Keywords: Poverty, Diet perception, Nutritional guidelines, Socioeconomic status, Prospective cohort, Qualitative food frequency questionnaire Poverty and diet perception

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Non-communicable diseases and the social gradient in health are major public health concerns [START_REF] Wilkinson | The solid facts: social determinants of health[END_REF]. Over the last decades, in order to tackle the rising prevalence of lifestyle diseases, several countries have massively disseminated nutrition guidelines [START_REF] Mozaffarian | Dietary Guidelines in the 21st Century-a Time for Food[END_REF][START_REF]Diet, nutrition, and the prevention of chronic diseases: report of a WHO-FAO Expert Consultation[END_REF]. Their impact was often limited by the "knowledge-behaviour gap" [START_REF] Parmenter | Demographic variation in nutrition knowledge in England[END_REF][START_REF] Mcgill | Are interventions to promote healthy eating equally effective for all? Systematic review of socioeconomic inequalities in impact[END_REF]. Campaigns had inconsistent effects on diet quality and nutritional status [START_REF] Wakefield | Use of mass media campaigns to change health behaviour[END_REF][START_REF] Brambila-Macias | Policy interventions to promote healthy eating: a review of what works, what does not, and what is promising[END_REF][START_REF] Hornik | Communication and Diet: An Overview of Experience and Principles[END_REF]. Some have proven successful. For example, when oriented towards a single message [START_REF] Hornik | Communication and Diet: An Overview of Experience and Principles[END_REF]. However the main issue was to keep a sustained effect rather than a short-term change [START_REF] Wakefield | Use of mass media campaigns to change health behaviour[END_REF]. Health promotion campaigns may also take time to change attitudes [START_REF] Brambila-Macias | Policy interventions to promote healthy eating: a review of what works, what does not, and what is promising[END_REF].

Launched in 2001, the Programme National Nutrition Santé (PNNS) was a major step in French nutrition policy. Besides many actions directed to (or negotiated with) health professionals and food industries, it produced nutritional guidelines for the general population. Since 2007 any advertisement for processed food or drink must include one of the following messages: 'for a healthy life, eat at least five fruit and vegetables per day', 'for a healthy life, practice regular physical activity', 'for a healthy life, avoid eating too fat, too sweet, too salty', 'for a healthy life, avoid snacks between meals' [START_REF] Hercberg | The French National Nutrition and Health Program: 2001-2006-2010[END_REF]. These messages therefore appear in magazines, on billboards, the Internet, radio and television. By 2008 knowledge of the guidelines had significantly increased, but adherence to the guidelines had only mildly improved [START_REF] Menninger | Évaluation du programme national nutrition santé PNNS 2 2006-2010[END_REF]. Differences in knowledge across economic status were not reported.

While many factors contribute to the economic gradient in nutritional status, health education campaigns may increase this gradient because they benefit to poorer individuals less [START_REF] Mcgill | Are interventions to promote healthy eating equally effective for all? Systematic review of socioeconomic inequalities in impact[END_REF][START_REF] Viswanath | Social Determinants and Nutrition: Reflections on the Role of Communication[END_REF].

In this perspective, a range of studies have examined whether nutritional knowledge and nutrition literacy mediated the association between higher income and healthier diets [START_REF] Wardle | Nutrition knowledge and food intake[END_REF][START_REF] Mcleod | Nutrition Knowledge: A Mediator between Socioeconomic Position and Diet Quality in Australian First-Time Mothers[END_REF][START_REF] Velardo | The Nuances of Health Literacy, Nutrition Literacy, and Food Literacy[END_REF][START_REF] Carbone | Nutrition and Health Literacy: A Systematic Review to Inform Nutrition Research and Practice[END_REF].

In a British sample of 1,040 adults, nutritional knowledge was positively associated with education, age and occupational group (income was not examined) [START_REF] Parmenter | Demographic variation in nutrition knowledge in England[END_REF]. However, several studies have found that the association between knowledge and diet was weak or insignificant, or was positive only for a limited number of diet quality indicators, such as fruit and vegetable intake [START_REF] Spronk | Relationship between nutrition knowledge and dietary intake[END_REF][START_REF] Dallongeville | Association between nutrition knowledge and nutritional intake in middle-aged men from Northern France[END_REF][START_REF] Gupta | Determinants of knowledge and attitudes about sugar and the association of knowledge and attitudes with sugar intake among adults: A systematic review[END_REF].

Other studies have focused on how people perceived the quality of their diet (nutritional awareness or perceived diet quality) as key points in making dietary choices because inaccurate perception of diet healthiness may be a barrier to adopting healthier dietary habits [START_REF] Alkerwi | Association between Nutritional Awareness and Diet Quality: Evidence from the Observation of Cardiovascular Risk Factors in Luxembourg (ORISCAV-LUX) Study[END_REF][START_REF] Powell-Wiley | Perceived and objective diet quality in US adults: a cross-sectional analysis of the National Health and Nutrition Examination Survey (NHANES)[END_REF]. In France, perceiving diet as balanced correlated with more adequate intakes of fruit and vegetables, fish and dairy but not animal protein. In US National health and nutrition survey (NHANES) perceiving diet as healthy was associated with a healthier diet but participants overestimated their diet healthiness. While the poorest participants perceived their diet as less healthy, the study did not report association between perception and intakes across income categories [START_REF] Powell-Wiley | Perceived and objective diet quality in US adults: a cross-sectional analysis of the National Health and Nutrition Examination Survey (NHANES)[END_REF].

Is misperception of diet healthiness a factor that explains why poor people have less healthy eating habits in so many societies? People facing poverty may misperceive their diet healthiness because they have poorer nutritional knowledge [START_REF] Wardle | Nutrition knowledge and food intake[END_REF][START_REF] Mcleod | Nutrition Knowledge: A Mediator between Socioeconomic Position and Diet Quality in Australian First-Time Mothers[END_REF]. They may also have nutritional knowledge but have adapted their standards to their living circumstances [START_REF] Bisogni | A Biographical Study of Food Choice Capacity: Standards, Circumstances, and Food Management Skills[END_REF], or they may react to health promotion campaigns differently compared to non-poor participants.

In a qualitative study in New Zealand, such campaigns met resistance and denial, potentially reinforcing stigmatisation and the social gradient in health [START_REF] Thompson | Responses to health promotion campaigns: resistance, denial and othering[END_REF]. Some studies have tried to assess the association between perceived diet quality and adherence to nutritional guidelines, taking economic status into account. An American study compared perceived diet quality with the Healthy Eating Index: 40% of the respondents were "optimistic", perceiving their diet quality to be better than it actually was, but being optimistic was not associated with income or education [START_REF] Variyam | Consumer Misperceptions of Diet Quality[END_REF]. This study, however, only included people in charge with meal preparation in their homes and dated from 1989-90. A recent study in Luxembourg showed a significant association between perceiving diet as important for health and diet quality independently of education and income [START_REF] Alkerwi | Association between Nutritional Awareness and Diet Quality: Evidence from the Observation of Cardiovascular Risk Factors in Luxembourg (ORISCAV-LUX) Study[END_REF]. This study did not target specifically poor participants and was performed on a sample of 1,351 participants.

There is evidence that the poor are at greater risk of suboptimal diet and derive less nutritional knowledge from information campaigns, but it is still unclear whether they perceive their diet healthiness less consistently with nutritional recommendations. The aim of this study is to assess whether the association between perceived diet healthiness and adherence to nutritional guidelines is weaker among poor participants in Constances, a large, population-based epidemiologic cohort in France.

Methods

Study design

Constances is a general-purpose, population-based epidemiologic cohort. It is designed as a representative sample of French adults aged 18-69 years at inception [START_REF] Zins | The French CONSTANCES populationbased cohort: design, inclusion and follow-up[END_REF]. The random sampling protocol over-represents individuals with a higher probability of non-volunteering according to age, gender and SES, relying on the French national inter-scheme registry of health insurance beneficiaries (RNIAM). Self-employed workers have a different health insurance scheme and cannot be sampled, therefore the cohort covers 85% of the French population aged 18-69. Inclusion criteria include a health examination in one of the 19 participating Health centres scattered across the French metropolitan territory, and completion of a series of questionnaires. The data used in this paper are drawn from the 'Health and lifestyle' questionnaire, which is self-completed at home upon inclusion. The study has obtained the authorisation of the National Data Protection Authority (Commission nationale de l'informatique et des libertés-CNIL, #910486).

Population

This study included participants who were invited between January, 2012 and June, 2015, who had returned the 'Health and lifestyle' questionnaire (n=41,705) and had completed the material deprivation question (n=841 excluded) and at least 11 out of the 22 Food frequency questionnaire (FFQ) items (n=849 excluded). This resulted in the inclusion of 40,015 participants, including 25,507 complete cases.

Variables

Perceived diet healthiness were measured through the question 'Do you consider that your diet is balanced?'. Possible answers ranged from 1 (not at all) to 8 (perfectly). The question was used in another French nutrition survey where it correlated with more appropriate intakes of various food groups [START_REF] Escalon | Baromètre santé nutrition 2008[END_REF][START_REF] Escalon | Connection between the knowledge of the recommendations of the National Nutrition and Health Program and patterns of eating behaviour and physical activity[END_REF]. A 'balanced diet' (alimentation équilibrée) is a good French translation of a 'healthy diet' as it is frequently used in daily life and in nutrition information campaigns [START_REF] Loftfield | Construct Validity of a Single-Item, Self-Rated Question of Diet Quality[END_REF]. 'In general, how healthy is your overall diet?' was used in NHANES where it was associated with higher diet quality [START_REF] Powell-Wiley | Perceived and objective diet quality in US adults: a cross-sectional analysis of the National Health and Nutrition Examination Survey (NHANES)[END_REF]. It was recently validated as a synthetic measure of dietary quality (28).

Adherence to nutrition guidelines (PNNS score) is adapted from the PNNS-GS score [START_REF] Estaquio | Adherence to the French Programme National Nutrition Santé Guideline Score Is Associated with Better Nutrient Intake and Nutritional Status[END_REF][START_REF] Lassale | Association between dietary scores and 13-year weight change and obesity risk in a French prospective cohort[END_REF].

In another nutritional survey, PNNS-GS score has been validated for monitoring adherence to French nutritional guidelines. It was associated with healthier nutrient intakes and biomarkers [START_REF] Estaquio | Adherence to the French Programme National Nutrition Santé Guideline Score Is Associated with Better Nutrient Intake and Nutritional Status[END_REF], and lower incidence of metabolic syndrome [START_REF] Kesse-Guyot | Adherence to French Nutritional Guidelines Is Associated with Lower Risk of Metabolic Syndrome[END_REF]. Our version of the PNNS score is adapted to Constances questionnaire. Habitual food intakes were collected thanks to a selfcompleted FFQ designed by nutritionists and food sociologists. These experts intended to make it understandable by French adults of any sociocultural background and age; the FFQ was not validated. It included usual intake frequency for 22 food groups, without quantities [START_REF] Port | Association between Dietary Patterns and Depressive Symptoms Over Time: A 10-Year Follow-Up Study of the GAZEL Cohort[END_REF]. When respondents consumed the item on a daily basis, they declared how many times or servings per day. Otherwise, the answers were translated into frequency of consumption per day (Nearly never: 0 time/day; About once a week: 0.15; 2 or 3 times a week: 0.36; 4 to 6 times a week: 0.71). We also used questions on modifiable physical activity, alcohol consumption (never, less than once a week, weekly frequency), and use of fats and spreads.

Our PNNS score included 11 components: fruit and vegetables, bread cereals potatoes and legumes, milk and dairy, meat poultry seafood and eggs, seafood, added fat, sweetened foods, non-alcoholic beverages, alcohol, salt, physical activity (Online Resource Table A1).

Wholegrain cereals, added fat and water intakes, which are measured in the PNNS-GS score, could not be assessed. The maximum score is 12.5 because the score gives supplementary points for physical activity, fruit and vegetable, and salt consumption when habits are more satisfactory than current recommendations [START_REF] Estaquio | Adherence to the French Programme National Nutrition Santé Guideline Score Is Associated with Better Nutrient Intake and Nutritional Status[END_REF].

Poverty can be defined in relative terms (usually as 60% of median income) or in absolute terms as material deprivation. In this study we define it as material deprivation (33) measured through the question: 'Are there times when you experience difficulties meeting your financial needs (food, rent, electricity, loans etc.)?'. Possible answers were: Never, No but it has happened in the past, Yes less than a year ago, Yes for the past several years. We checked that material deprivation was associated with lower income by computing the monthly income per consumption unit from declared household monthly income (6 classes), household composition and the OECD equivalence scale [START_REF]Glossary: Equivalised disposable income[END_REF]. We did not derive our measure of poverty from this income variable because it had a lot of missing values (as is often the case with declared income) and did not account for the fact that the same income may allow for different standards of living according to the cost of living in the area.

Covariates Educational level was coded in 4 categories (incomplete secondary, completed secondary, bachelor level, masters and more) and age in 10-year groups. We also included variables indicating whether the respondent lived with a partner and followed a diet prescribed by a doctor [START_REF] Viswanath | Social Determinants and Nutrition: Reflections on the Role of Communication[END_REF].

Auxiliary variables were used when imputing the missing values. Body mass index was computed as weight in kilograms divided by height in meters squared, with weight and height measured during a standardised health examination [START_REF] Hanson | Work stress, anthropometry, lung function, blood pressure, and blood-based biomarkers: a cross-sectional study of 43,593 French men and women[END_REF]. The social disadvantage index of the area of residence was computed from official statistics on income, educational level, occupational groups and unemployment [START_REF] Rey | Ecological association between a deprivation index and mortality in France over the period 1997 -2001: variations with spatial scale, degree of urbanicity, age, gender and cause of death[END_REF]. It varied from -5.7 (strongest disadvantage) to 3.5. Depressive symptoms were assessed using the Center for Epidemiologic Studies Depression scale (CESD). A score above 16 indicated depression. We also used region of residence, smoking status and whether participants had foregone healthcare for financial reasons over the last 12 months.

Statistical analysis

Descriptive statistics compared the distribution of all variables across categories of material deprivation, with either chi-square or Kruskal-Wallis tests (as relevant). We also report the number of missing data for each variable and a correlation matrix for all continuous variables. We ran linear regressions with perceived diet healthiness as the dependent variable. The variables of interest were the PNNS score as a continuous variable, material deprivation categories and their interaction. We controlled for education, age, and partnership and dieting status. Diet perception and poverty risk both differ across sex, hence the analyses were separated for men and women. We reported unstandardized regression coefficients with 95% confidence intervals.

Perceived diet healthiness, material deprivation and each FFQ food item had a limited number of missing values. The PNNS score, being a combination of many variables, had 68% complete observations. Respondents in poverty have more missing values. In order to limit the possible bias, we ran multiple, multinormal imputations with 50 imputed files.

Imputations were run separately according to sex and material deprivation category, to allow for different associations in these groups of participants. Multinormal imputations assume that imputed variables follow a normal distribution. In order to get closer to this assumption, we imputed the square-root of the variables recording frequency of consumption for each food group. To improve the quality of the imputation and account as best as possible for the plausible sources of non-response bias, we used current smoking status, body mass index, foregone health care, depressive symptoms, region, and the local socioeconomic deprivation index as auxiliary variables. Models of interest where then estimated on the 50 completed files and combined according to Rubin's rules [START_REF] Rubin | Multiple imputation for nonresponse in surveys[END_REF][START_REF] Sterne | Multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls[END_REF]. Perceived diet healthiness is measured on an 8-level scale, with one third of respondents answering 7 or 8. There is a risk that the linearity assumption involved in linear regressions biases our results. For this reason, we also estimated the odds-ratios of perceiving diet as very healthy (answers 7 or 8) according to PNNS score, material deprivation, their interaction and the covariates (Online Resource table A2). We also estimated the linear regression models with continuous measurement of age (and age squared) as a sensitivity analysis (results very similar to

Results

Descriptive statistics

We excluded 1,690 cases because material deprivation was missing or because the FFQ was insufficiently completed (Figure 1). The final sample thus included 40,015 participants.

Among them, 14,508 participants had at least one missing variable, while 25,507 were complete cases.

Figure 1 about here Table 1 shows descriptive statistics according to material deprivation, among the available cases. Nearly two thirds of the participants had never experienced material deprivation; 7.1% (n=2,845) reported material deprivation only this year and 6.3% (n=2,530) for several years. Participants reporting material deprivation for several years had foregone healthcare for themselves or their partner much more often (58.4% as against 5.2% among those never in material deprivation). They also had depressive symptoms and incomplete questionnaires more often. All these associations were highly significant (p<0.005). There was a gradient in these associations: participants never deprived, deprived only in the past, currently deprived and deprived for several years had increasingly bad situations, (Kruskal-Wallis test p<0.0005, p for trend <0.001).

Participants who had never faced material deprivation were also older, lived with a partner more often and had a higher educational level. About 52% were women, against 59% of the participants who declared material deprivation only this year and 55% who had faced them for several years (p<0.005 for all associations).

In the study population the PNNS score ranged from 0.5 to 12.5 with a mean of 6.33 (SD 1.54). Statistical tests indicated that it was significantly higher among women (6.53 against 6.11, p<0.0001), non-smokers (6.48 against 5.76, p<0.0001), older participants, and those with higher educational levels (Kruskal-wallis test p=0.001), reflecting healthier diets in these population categories. In Table 2, PNNS score's correlation with social disadvantage index and BMI was close to 0, with income it was 0.11 and with age 0.27.

A third of the sample rated their diet healthiness 7 or 8 on the 1-8 scale (8 meaning a perfectly balanced diet). Mean perceived diet healthiness ranged from 5.9 for those never deprived to 4.9 for those deprived several years, while the mean PNNS score ranged from 6.43 to 5.94 (p<0.0005 in both cases). Perceived diet healthiness correlated negatively with BMI and positively with age and perceived diet healthiness (0.27). This correlation did not vary much across material deprivation category (bottom line of Table 1, ranging from 0.25 among those currently deprived to 0.28 among those deprived in the past.

Models

In multivariate models for complete cases (Table 3, model 1), the association between PNNS score and perceived diet healthiness was similar for men and women who had never faced material deprivation: for each increase in PNNS score there was a +0.21 change (95% CI [0.18,0.23]) in perceived diet healthiness for men (+0.19 [0.17,0.22] for women). Among men, this increase was slightly stronger for participants who had faced material deprivation in the past, but was not significantly different for those currently facing material deprivation.

Among women who faced material deprivation, for each increase in PNNS score there was a stronger increase in perceived diet healthiness: the difference was 0.18 point and statistically significant [0.11,0.25].

Table 3 about here

When estimated on the full sample after imputation of the missing data (Table 3 model 2), the coefficients remained very close to those estimated on complete cases, with the same levels of statistical significance. Typically, the increase in PNNS score for women who have faced material deprivation for several years was 0.14 [0.09,0.20] point larger than for women who had never faced poverty.

Participants who had never faced material deprivation also had a higher intercept than the others. Figure 2 illustrates these results using predicted values from model 2 (imputed data): men who had never faced material deprivation rated their dietary habits as more balanced at every level of the PNNS score, with no difference in the slopes. Materially deprived women had lower perceived diet healthiness only if their PNNS score was low: among women with healthy diets material deprivation made no statistically significant difference.

Figure 2 about here

We estimated the odds-ratios of perceiving diet as very healthy (7 or 8 on the 1-8 scale) for men and women on the complete cases (Online Resource Table A2): none of the interaction terms were significant. Specifically, for women the changes in odds-ratios associated with each increase in PNNS score were not statistically different according to experience of material deprivation.

Discussion

Our results indicate that the association between perceived diet healthiness and adherence to national nutritional recommendations is not lower among participants facing or having faced poverty, measured as material deprivation. This result held after controlling for education and whether we imputed missing data or not.

Our results are consistent with the findings of other studies, conducted in Luxembourg [START_REF] Alkerwi | Association between Nutritional Awareness and Diet Quality: Evidence from the Observation of Cardiovascular Risk Factors in Luxembourg (ORISCAV-LUX) Study[END_REF] and in NHANES [START_REF] Gregory | How Americans rate their diet quality: an increasingly realistic perspective[END_REF]. The latter showed that materially deprived participants had less healthy diets (measured by the Healthy Eating Index) and lower perceptions of their diet healthiness which is in line with the results of this study. Our results are also consistent with another study [START_REF] Variyam | Consumer Misperceptions of Diet Quality[END_REF] which found that overestimating diet healthiness was widespread across all levels of education and income. Nutritional knowledge is associated with higher SES but correlates weakly with food intake [START_REF] Parmenter | Demographic variation in nutrition knowledge in England[END_REF][START_REF] Wardle | Nutrition knowledge and food intake[END_REF][START_REF] Mcleod | Nutrition Knowledge: A Mediator between Socioeconomic Position and Diet Quality in Australian First-Time Mothers[END_REF][START_REF] Dallongeville | Association between nutrition knowledge and nutritional intake in middle-aged men from Northern France[END_REF][START_REF] Gupta | Determinants of knowledge and attitudes about sugar and the association of knowledge and attitudes with sugar intake among adults: A systematic review[END_REF][START_REF] Darmon | Does social class predict diet quality?[END_REF] although some studies found significant associations [START_REF] Bonaccio | Nutrition knowledge is associated with higher adherence to Mediterranean diet and lower prevalence of obesity. Results from the Moli-sani study[END_REF]. This suggests that nutrition knowledge and diet perception are two different constructs. A lot of variables may mediate the link between nutritional knowledge and diet, including diet perception, home environment and access to food [START_REF] Campbell | Home food availability mediates associations between mothers' nutrition knowledge and child diet[END_REF][START_REF] Tabbakh | The home environment: A mediator of nutrition knowledge and diet quality in adolescents[END_REF] and foodmanagement skills [START_REF] Bisogni | A Biographical Study of Food Choice Capacity: Standards, Circumstances, and Food Management Skills[END_REF].

We found that the association between perceived diet healthiness and adherence to guidelines was stronger for women who had experienced material deprivation for a long period of time.

It has been shown that following nutritional guidelines is difficult for low-income people because nutritious foods are more expensive [START_REF] Castetbon | Dietary behaviour and nutritional status in underprivileged people using food aid (ABENA study, 2004-2005)[END_REF][START_REF] Maillot | Low energy density and high nutritional quality are each associated with higher diet costs in French adults[END_REF]. Poor women (with similar levels of education) may perceive better their diets' shortcomings (or qualities) because maintaining a healthy diet with a smaller budget requires more reflexivity, time and energy [START_REF] Bisogni | A Biographical Study of Food Choice Capacity: Standards, Circumstances, and Food Management Skills[END_REF][START_REF] Castetbon | Dietary behaviour and nutritional status in underprivileged people using food aid (ABENA study, 2004-2005)[END_REF][START_REF] Devine | A lot of sacrifices:" Work-family spillover and the food choice coping strategies of low-wage employed parents[END_REF]. This result, however, was not statistically significant when we modelled odds-ratios of perceiving one's diet as very healthy.

We measured poverty in terms of material deprivation, through a question regarding budget restrictions in basic needs (rent, food, energy) experienced over the last 12 months or earlier in life. We did not use income for several reasons: the collection in broad classes does not allow for precise measurement, income declarations are prone to biased or missing responses [START_REF] Moore | Income measurement error in surveys: A review[END_REF], computing income per consumption unit from household income and household composition generates even more missing responses, and the same income may allow different standards of living according to the cost of living in the area. However, we validated this measure by checking that individuals who claimed they faced material deprivation had lower declared incomes and higher chances to forego healthcare and lived in more deprived areas.

A major strength of this study is its sample. Data were collected from over 40,000 adults sampled randomly from the French population from 2012 to 2015. Low-SES adults were over-sampled in order to compensate for higher non-participation rates. The study addressed many aspects of health, and included a comprehensive and free health examination which may have lowered the overall participation rate but attracted low-income participants. We were also able to control for the most plausible confounders, to impute missing data with a comprehensive set of auxiliary variables (including depressive symptoms, smoking status and local deprivation index), and to relax the linear assumption of the first models, in order to deliver robust conclusions.

Our study has some limitations. First, as in any survey based on voluntary participation, it remains possible that the most economically deprived and hard-to-reach populations did not participate in the Constances cohort. If these populations are those for whom the association between adherence to recommendations and perception of diet healthiness is lowest, not including them may bias our results. While we could not address non-participation bias, we treated item non-response, which was associated with material deprivation. Estimating our model on complete cases or after imputation of item non-responses gave very similar results.

Second, the downside of using a general-purpose cohort rather than a nutrition survey is that adherence to nutritional guidelines is based on a 22-item FFQ. We did not measure quantities nor controlled for total energy intake. While our PNNS score is adapted from the validated PNNS-GS score, the FFQ was not validated. We found the expected associations between PNNS score and age, sex and education, as well as with material deprivation [START_REF] Castetbon | Dietary behaviour and nutritional status in underprivileged people using food aid (ABENA study, 2004-2005)[END_REF]. Nutrition recommendations addressed to the French population usually refer to frequency of consumption (five a day, twice a week), or simply advise to 'eat less salty'. Our questionnaire design is coherent with the wording of these messages, making it a relevant tool to confront with perception of diet healthiness.

Third, perceived diet healthiness was collected on an 8-point scale and treated as a continuous variable. Since assuming a linear relationship between adherence and dietary balance may induce bias, we also estimated odds-ratios of perceiving diet as very healthy: results were consistent except that the positive difference between women having faced material deprivation for several years and those never poor was no longer significant.

Conclusions

Diet contributes to the social gradient in health [START_REF] Wilkinson | The solid facts: social determinants of health[END_REF].Why do poor people keep eating such unhealthy diets, despite all the nutritional information that has been massively disseminated towards them over the last years? One possible explanation is that they misuse the information, overestimate their diet healthiness, and don't see any reason to change [START_REF] Powell-Wiley | Perceived and objective diet quality in US adults: a cross-sectional analysis of the National Health and Nutrition Examination Survey (NHANES)[END_REF]. This is an important rationale for a variety of health education campaigns. Some researchers argue that such an assumption means "blaming the victims" [START_REF] Lupton | The Imperative of Health: Public Health and the Regulated Body[END_REF][START_REF] Holm | Blaming the consumer: On the free choice of consumers and the decline in food quality in Denmark[END_REF], making them responsible for exposing themselves to well-identified health risk factors. Our results indicate that this assumption is not only ethically disputable, it is also misleading: in our study diet misperception is not stronger among poor participants. Disseminating nutritional information may improve nutritional knowledge in the whole population, but it is not sufficient to improve poor people's diets. On the contrary it may generate anxiety among the most vulnerable [START_REF] Elliott | Implementing and evaluating crime prevention and control programs and policies[END_REF][START_REF] Coveney | Morals and Meaning: The Pleasure and Anxiety of Eating[END_REF]: in our study they were equally aware of their diet healthiness, but they had less resources to improve it.

In order to make healthy eating more accessible to poor households, it is necessary to act not only on knowledge but also on the context of eating practices [START_REF] Hercberg | The French National Nutrition and Health Program: 2001-2006-2010[END_REF][START_REF] Warde | The Habits of Consumption[END_REF][START_REF]The practice of eating[END_REF] and on the food supply, so that healthier options become easier, or even unconscious for all. Research and policies developed along the perspective of the theories of practices offer promising avenues in this respect [START_REF] Warde | The Habits of Consumption[END_REF][START_REF] Blue | Theories of practice and public health: understanding (un)healthy practices[END_REF][START_REF] Hargreaves | Practice-ing behaviour change: Applying social practice theory to proenvironmental behaviour change[END_REF]. Many existing policies are in fact consistent with this 'practice' approach [START_REF] Hargreaves | Practice-ing behaviour change: Applying social practice theory to proenvironmental behaviour change[END_REF]. Improving the nutritional content of processed foods (without rising prices) [START_REF] Spiteri | Food reformulation and nutritional quality of food consumption: an analysis based on households panel data in France[END_REF], offering healthy snacks in vending machines [START_REF] Grech | Are products sold in university vending machines nutritionally poor? A food environment audit[END_REF], supplying balanced meals in schools and workplace canteens [START_REF] Vitale | A nutritional intervention programme at a worksite canteen to promote a healthful lifestyle inspired by the traditional Mediterranean diet[END_REF] or reducing portion size in restaurants and fast-foods (58) may improve diets without requiring the adoption of new behaviours. Is not clear to what extent such policies may benefit to households facing material deprivation but at least, they do not blame or stigmatize them for the shortcomings of their diets.

Declarations Ethics approval and consent to participate

The study has obtained the authorisation of the National Data Protection Authority (Commission nationale de l'informatique et des libertés-CNIL, #910486). Participants' informed consent signature validates the collection of these data and authorises their use for research purposes.

Tables and figures

Fig1

Caption: Flowchart of study population Notes: FFQ: food frequency questionnaire

Fig2

Caption: Association between perceived diet healthiness and PNNS score according to material deprivation and sex: predicted values and 95% CI.

Notes:

Linear predictions based on models in Table 3, adjusted on age (18-29 years), education (incomplete secondary), partnership status (single) and dieting status (no prescribed diet), estimated on multiply imputed data (50 imputations).

MD: material deprivation

Additional files: Online resources

Table A1. Definition of PNNS score components and their implementation in Constances Table A2. Odds-ratios of perceiving diet as very healthy according to PNNS score, material deprivation and their interaction for men and women in the Constances cohort: logistic model adjusted on age (baseline 18-29 years), education (incomplete secondary), partnership status (single), and dieting status (no prescribed diet), complete cases.

Table 3 ,

 3 not shown). All analyses were performed using the Stata software (version 15, Stata Corp., College Station,

	TX).

Table 1 about here Table 2 about

 1here2 here Material deprivation was associated with other indicators of economic deprivation: lower income per consumption unit, worsened local deprivation index, slightly higher BMI.

Table 1

 1 Sample characteristics according to declared material deprivation in the Constances cohort (column percentages unless otherwise specified) Monthly income per consumption unit using the OECD scale, in euros. c Disadvantaged areas have a lower index. Computed from median income and the percentages of blue-collar workers, unemployed and adults with less than completed secondary education in the area of residence.

			Material deprivation		Total	Missing P-value
		Never	Past only	This year Several years
	Material deprivation: row % (n)	62.2 (24,868) 24.4 (9,772) 7.1 (2,845) 6.3 (2,530) 100 (40,015)	0
	Complete case a	64.6	63.3	61.6	60	63.7	0	<0.0005
	Woman	51.8	56.6	58.9	55.4	53.7	0	<0.0005
	Age						0	<0.0005
	18-29 years	12.1	10.2	18.4	12	12.1
	30-39 years	16.9	16.9	21.1	19.9	17.4
	40-49 years	21.3	22.6	26.9	29.2	22.5
	50-59 years	22.2	27.2	20.5	24.8	23.5
	60+ years	27.5	23.1	13.1	14.1	24.6
	Educational level						591	<0.0005
	Incomplete secondary	19.3	30.7	33.1	37.8	24.1
	Completed secondary	15.5	18.6	21.3	23.5	17.1
	Bachelor level	26.7	25.9	27.2	22.5	26.3
	Masters level	38.5	24.8	18.4	16.2	32.5
	No partner	22.9	28.1	41.3	44.2	26.7	469	<0.0005
	Depressive symptoms (CESD>16)	13.9	24.4	37	43.8	19.8	0	<0.0005
	Foregone healthcare	5.2	17.9	44.7	58.4	14.1	310	<0.0005
	Prescribed diet	3.8	5.5	4.7	6.7	4.5	554	<0.0005
	Diet perceived as very healthy (7-8/8)	36.1	28.7	20.8	17.7	32.2	412	<0.0005
	Continuous variables: mean (sd)
	Age	48.2 (13.8)	48.3 (12.8)	43.4 (12.9) 45.6 (12.1)	47.8 (13.5)	0	<0.0005
	Income b	2649 (1285)	2134 (1147) 1448 (890) 1306 (758) 2358 (1278) 12,604 <0.0005
	Local social disadvantage index c	-0.9 (1.7)	-0.7 (1.6)	-0.6 (1.7)	-0.5 (1.7)	-0.8 (1.7)	19	<0.0005
	BMI (kg/m²)	24.6 (4.2)	25.5 (4.7)	25.9 (5.1)	26.4 (5.5)	25 (4.5)	2,090 <0.0005
	Perceived diet healthiness	5.9 (1.4)	5.6 (1.5)	5.2 (1.7)	4.9 (1.8)	5.7 (1.5)	412	<0.0005
	PNNS score	6.43 (1.54)	6.27 (1.55)	5.98 (1.51) 5.94 (1.51)	6.33 (1.54)	12,810 <0.0005
	Correlation PNNS x Perceived diet	0.26	0.28	0.25	0.27	0.27	12,951
	Difference between material deprivation groups has been compared by chi-square or Kruskal-
	Wallis tests as relevant.
	a Participant has valid answers on PNNS score, perceived dietary balance, material
	deprivation, age, education, partnership status, and dieting status.

b

Table 3

 3 Association between perceived diet healthiness and PNNS score according to material deprivation for men and women in the Constances cohort: unstandardized linear regressions adjusted on age, education, dieting and partnership status, on complete cases and after imputation.

	Men		Women
	CC a	MI b	CC	MI

95% confidence intervals in brackets. Models adjusted on age (baseline 18-29 years), education (incomplete secondary), partnership status (single), and dieting status (no prescribed diet). a CC: complete cases only. b MI: Multiply imputed data (50 imputations).

c MD: Material deprivation.

Acknowledgements

The authors thank the Constances team, in particular Alice Guéguen for her expertise regarding missing data treatment and Marcel Goldberg for his advices.

Funding

No funding was received.

Availability of data and material

The data that support the findings of this study are available from Constances principal investigators (Marie Zins, Marcel Goldberg, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available.

Data are however available from the authors upon reasonable request and with permission of the principal investigators.

Competing interests

SC, JM, MP, EKG and MZ declared no conflict of interest.

Authors' contributions

MP conceived the hypothesis, conducted the statistical analyses and drafted the manuscript.

MZ is co-principal investigator of the Constances cohort. SC and EKG contributed to the elaboration of the nutrition guideline score. JM critically revised the drafted manuscript. All authors were involved in writing the paper and had final approval of the submitted and published versions.


Garyfallia I Makrynitsa

Dioni Ntonti

Konstantinos D Marousis

Maria Birkou

Minos-Timotheos Matsoukas

Sam Asami

Detlef Bentrop

Nicolas Papageorgiou

Bruno Canard

Bruno Coutard
email: bruno.coutard@univ-amu.fr

Georgios A Spyroulias
email: g.a.spyroulias@upatras.gr

Conformational plasticity of the VEEV macro domain is important for binding of ADP-ribose

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Venezuelan equine encephalitis virus (VEEV) has been recognized as a human pathogen since 1943 and causes flu-like symptoms and several central nervous system disorders like encephalitis and meningitis. VEEV is a new world alphavirus, ie., specific to the Americas. It follows an enzootic life cycle between mosquito Culex sp. and wild rodents, and an epizootic cycle amongst humans and equines with mosquitos Aedes sp. and Psorophora sp. as epizootic vectors [START_REF] Morrison | Venezuelan equine encephalitis virus in Iquitos, Peru: urban transmission of a sylvatic strain[END_REF]. The virus is distributed over the Americas with main outbreaks located in Central and South America. Outbreaks of VEEV can yield to fatal cases in equines and VEEV is also considered as a high-risk pathogen for humans as well. Such outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV poses a serious public health problem. It is worth mentioning that since 1995 there has not been any outbreak of the virus [START_REF] Guillén | Structural and biophysical analysis of sequence insertions in the Venezuelan Equine Encephalitis Virus macro domain[END_REF][START_REF] Morrison | Venezuelan equine encephalitis virus in Iquitos, Peru: urban transmission of a sylvatic strain[END_REF], however epidemics indicate that VEEV still represents a serious public health concern [START_REF] Aguilar | Endemic Venezuelan equine encephalitis in the Americas: hidden under the dengue umbrella[END_REF]. So far, no efficient vaccines or drugs are licensed for the treatment of alphavirus infection [START_REF] Chattopadhyay | A chimeric vesiculo/alphavirus is an effective alphavirus vaccine[END_REF].

The 12kb single-stranded positive sense RNA genome of the virus includes two large open reading frames (ORF). The first ORF encodes the four non-structural proteins (nsPs), which are necessary for virus replication, while the second is translated into the structural proteins: capsid, E1, E2 and E3 glycoproteins and 6K. In particular, nsP1 possesses guanine-7-methyltransferase and guanylyltransferase activity being responsible for the addition of a 5' cap to viral RNA [START_REF] Rupp | Alphavirus RNA synthesis and non-structural protein functions[END_REF], while nsP2 is a multifunctional protein consisting of 3 domains. The N-terminal domain acts as a helicase while it also exhibits nucleoside triphosphatase activity (NTPase). The Cterminus of nsP2 contains a protease domain and a non-functional methyltransferase-like domain.

nsP3 is comprised of three domains: the conserved N-terminal domain, which is called macro domain (also known as "X domain"), a central alphavirus unique domain (AUD) and a hypervariable, phosphorylated C-terminus. Originally, an ADPr-1''-phosphatase was associated to the macro domain, as well as nucleic acid binding but recent studies showed its implication in deribosylation of proteins. The highly conserved AUD is characterized by a zinc-binding motif. Both length and sequence composition of the C-terminal domain are hypervariable among alphaviruses [START_REF] Abu Bakar | Nonstructural Proteins of Alphavirus-Potential Targets for Drug Development[END_REF][START_REF] Rupp | Alphavirus RNA synthesis and non-structural protein functions[END_REF]. Last, nsP4 is the RNA-dependent RNA polymerase [START_REF] Shin | Structural and functional insights into alphavirus polyprotein processing and pathogenesis[END_REF][START_REF] Strauss | The alphaviruses: gene expression, replication, and evolution[END_REF].

Macro domains are highly conserved proteins throughout evolution. They consist of 130-190 amino acids. They are named after the non-histone motif of the core histone variant macroH2A, in which they were initially characterized [START_REF] Chakravarthy | Structural characterization of the histone variant macroH2A[END_REF]. High affinity binding to ADPribose (ADPr) is their most common feature and macro domains can be organized into three classes: ADP-ribosylation readers, erasers and writers. Many cellular processes are dependent on protein ADP-ribosylation which is a highly dynamic post-translational modification involved in DNA repair, apoptosis, protein degradation and chromatin remodeling [START_REF] Fehr | Viral macrodomains: Unique mediators of viral replication and pathogenesis[END_REF][START_REF] Jankevicius | A family of macrodomain proteins reverses cellular mono-ADP-ribosylation[END_REF]. However, the biological role of the viral macro domains is still poorly understood. Several recent studies have demonstrated that they exhibit de-ADP-ribosylating activity (eraser) together with their ability to bind ADP-ribose [START_REF] Fehr | Viral macrodomains: Unique mediators of viral replication and pathogenesis[END_REF]. Therefore, it was proposed that they participate in reversing the antiviral ADP-ribosylation of the host cells [START_REF] Fehr | Viral macrodomains: Unique mediators of viral replication and pathogenesis[END_REF][START_REF] Li | Viral macro domains reverse protein ADP-ribosylation[END_REF]. Mutations of viral macro domains at residues critical for ADPr binding have been shown to lead to lower affinity towards ADPr resulting in disruption of loss of virulence and/or viral replication [START_REF] Fehr | The conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection[END_REF][START_REF] Mcpherson | ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence[END_REF]. Therefore, macro domains could serve as novel therapeutic targets for the design of antiviral agents in order to control Alphavirus, Hepevirus and Coronavirus-induced diseases. The crystal structure of the VEEV macro domain has shown that the protein forms a six-stranded β-sheet surrounded by 4 αhelices [START_REF] Malet | The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket[END_REF]. The order of the β-strands within the β-sheet is β1, β6, β5, β2, β4, β3, all positioned in parallel except for β3. The N-terminus forms the β1-strand while the C-terminus forms the last α-helix (α4), which is located close to the N-terminus. This structural motif is quite conserved among the macro domains of different organisms [START_REF] Cho | Macro domain from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an efficient ADP-ribose binding module: crystal structure and biochemical studies[END_REF][START_REF] Malet | The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket[END_REF]) [START_REF] Peterson | Orphan macrodomain (human C6ORF130) is an o-acyl-ADP-ribose deacylase: solution structure and catalytic properties[END_REF]. The same study revealed the structural basis of the binding affinity of ADPr to the macro domain. The binding site of ADPr is located at the top of the β2, β4 and β5 strands surrounded by the loops connecting β2 to α1 and β5 to α3 defining a conserved binding pocket [START_REF] Malet | The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket[END_REF].

In this study we provide the solution structure and the dynamic properties of the VEEV macro domain both in the apo-and the ADPr-bound state. Using NMR spectroscopy, we demonstrate a local conformational transition on the µs-ms time scale between two states, involving residues in the crevice occupied by ADPr. The data presented here suggest that the flexibility of defined regions nearby the macro domain active site is crucial for recognition and binding of ADPr.

Material and Methods

Protein expression and purification

The experimental protocol for the expression, purification and NMR sample preparation of the VEEV nsP3 macro domain (A1330-E1489) has been reported previously [START_REF] Makrynitsa | NMR study of non-structural proteins-part II: 1 H, 13 C, 15 N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)[END_REF].

The NMR samples, which were used for the structure determination of the macro domain and the macro domain-ADPr complex, contained 0.4mM 15 N-labeled protein or 0.8mM 13 C/ 15 N-labeled protein and 0.18mM 15 N-labeled protein or 0.21mM 13C/15N-labeled protein along with 0.26mM ADPr, respectively.

NMR spectroscopy and structure determination

NMR measurements were performed at 298K with an Avance III High-Definition four-channel 700 MHz spectrometer equipped with a cryogenically cooled 5mm 1 H/ 13 C/ 15 N/D Z-gradient probe (TCI) (Bruker). Proton chemical shifts were referenced to internal 4,4-dimethyl-4-silapentane-1sulfonic acid (DSS). Sequence specific resonance assignments of the VEEV macro domain were obtained as reported previously (BMRB ID 25132) [START_REF] Makrynitsa | NMR study of non-structural proteins-part II: 1 H, 13 C, 15 N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)[END_REF]. For the structure determination of the complex the same set of NMR experiments [START_REF] Makrynitsa | NMR study of non-structural proteins-part II: 1 H, 13 C, 15 N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)[END_REF] was used and the 1 H, 15 N and 13 C resonance assignments were deposited in the BioMagResBank under the accession number 26753. However, in order to define the 3D structure of the VEEV macro domain-ADPr complex, a sufficient number of intermolecular distance restraints had to be obtained through 2D 13 C-filtered/edited NOE experiments [START_REF] Peterson | New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes[END_REF]. NOE-derived distance constraints for the VEEV macro domain were obtained by the analysis of a 3D 15 N-NOESY and two 3D 13C-NOESY spectra, which were recorded with the proton carrier frequency in the aliphatic and the aromatic region, respectively. DYANA's routine CALIBA was used to generate distance restraints from the volume of each NOESY crosspeak. Dihedral angles derived from TALOS+ [START_REF] Makrynitsa | NMR study of non-structural proteins-part II: 1 H, 13 C, 15 N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)[END_REF][START_REF] Shen | TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts[END_REF] were incorporated in the structure calculations as well. The calculations were conducted with DYANA [START_REF] Güntert | Torsion angle dynamics for NMR structure calculation with the new program Dyana1[END_REF] and the 20 conformers with the lowest target function values were subjected to energy minimization with AMBER [START_REF] Case | The Amber biomolecular simulation programs[END_REF]. The solution structure of the apoprotein was determined from 1648 unique, meaningful NOE constraints derived from a total number of 1486 NOEs from the 15 N-NOESY and 1043 NOEs from the 13 C-NOESY spectra (SI, Table S1). The coordinates of the 21 energy-refined conformers (including the average model) were deposited in the RCSB Protein Data Bank (PDB ID: 5ISN). For the solution structure of the biomolecular complex, the same procedure and software were used and it was defined by 1988 unique, meaningful NOE constraints: 1361 from the 15 N-NOESY, 1991 from the 13 C-NOESY spectra and 18 intermolecular NOE correlations from the 13 C-filtered/edited NOESY. The coordinates of the 21 energy-refined models (20 DYANA models and the mean model) were deposited in the RCSB Protein Data Bank (PDB ID: 5MQX) as well.

NMR titration experiments

To monitor the behavior of the individual amino acids of the 15 N-labeled VEEV macro domain in the presence of ADPr, we calculated the changes of their chemical shifts in 1 H-15 N HSQC spectra during the NMR titration experiment. The unlabeled ligand (ADPr stock concentration = 5mM) was added in 16 steps in order to reach the following protein/ligand ratios: 1/0.125, 1/0.25, 1/0.4, 1/0.5, 1/0.75, 1./0.8, 1/0.9, 1/1, 1/1.125, 1/1.25, 1/1.4, 1/1.5, 1/1.75, 1/2, 1/3 and 1/4. Chemical shift perturbation values were calculated using the equation ∆δ ppm = ( ) + ( ) [START_REF] Garrett | Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system[END_REF]. After the first addition (1/0.125) the ADPr concentration was 0.0225 mM;

(
after the last addition (1:4) it was 0.64mM.

Protein Dynamics

The backbone dynamics of the VEEV macro domain and the VEEV macro domain-ADPr complex on the ps-ns time scale were studied through the analysis of 15 N R1 and R2 relaxation rates and heteronuclear { 1 HN}-15 N NOEs. 1 H-15 N HSQC experiments were recorded with relaxation delays of 7,18,40,85,150,230,350,500,680, and 900 ms for obtaining the R1 relaxation rates and with delays of 18,32,50,68,86,100,120,150,190, and 240 ms for the R2 relaxation rates. All the relaxation data were analyzed with the Tensor2 [START_REF] Dosset | Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data[END_REF] program in order to define the dynamic properties of the apoprotein and the complex by obtaining the values of S 2 order parameters for each amino acid.

The µs-ms dynamics of the amide backbone of VEEV macro domain was probed by SQ CPMG relaxation dispersion experiments. Dispersion profiles were obtained at 298 K and external magnetic field strengths of 14.1 T (600 MHz 1 H) and 21.1 T (900 MHz 1 H), respectively. 1 H continuous wave decoupling was employed throughout both constant-time echo periods with a rf amplitude of 12.5 kHz [START_REF] Hansen | An improved 15N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins[END_REF]. The CPMG field CPMG was varied from 25 to 1000 Hz, while rel was set to 40 ms and the pre-scan delay to 2 s. 15 N 180° refocusing pulses were applied at ~5.6 kHz. The equilibration delay to achieve equilibrium populations of ground and excited states prior to the CPMG train was set to 5 ms. Amide resonance intensities ( CPMG )

were converted to transverse relaxation rates by ,eff ( CPMG ) = -1 rel ln ( CPMG ) , where I 0 is the intensity when omitting the CPMG train. The experimental error was set to two times the standard deviation of the spectral noise.

Dispersion curves were fitted employing the Carver-Richards equation for a system undergoing chemical exchange in two states, while fitting four independent parameters ( ex, α, |Δ |, 2,0) [START_REF] Carver | A general two-site solution for the chemical exchange produced dependence of T2 upon the Carr-Purcell pulse separation[END_REF]. For each residue curves at both available fields were fitted simultaneously. To quantitatively discriminate exchanging from non-exchanging residues, all curves were additionally fit by a linear function. The model, which represents the data the best, was selected based on the comparison of AICc values (d'Auvergne and Gooley, 2003). To improve the fitting performance, several residues localized on the same secondary structure element were grouped together, while employing the χ^2 ratio for group over individual fitting [START_REF] Mulder | Studying excited states of proteins by NMR spectroscopy[END_REF]. Residues with ratios larger than two were discarded, assuming they undergo conformational fluctuations distinct from the global process.

Normal Mode Analysis

The free starting structure of the VEEV macro domain was obtained from the Protein Data Bank entry 5ISN and the ADPr-bound target from the calculated mean structure of the current study. In order to assess the effects of ligand binding, normal mode analysis (NMA) between the two states was performed using the iMOD program and specifically the imorph_gcc tool, which calculates potential transition pathways between two different conformations of a protein [START_REF] Lopéz-Blanco | iMod: multipurpose normal mode analysis in internal coordinates[END_REF]. Elastic connections between all protein heavy atoms and their neighbors were considered, whilst sigmoid function was used, with a distance cut off of 10Å. The step size used to control the iteration number was set at 10, the maximum stiffness constant at 1, the inflexion point at 3.8Å and the cut-off for removing ineffective very weak springs from calculations at 10Å. The RMSD to trigger NMA was set at 0.01Å.

Results and Discussion

3.1 Solution structure of the VEEV macro domain (A1330-E1489)

The NMR solution structure of the VEEV macro polypeptide shows that the domain falls into the α/β class of proteins and adopts the typical macro domain fold, consisting of a central six-stranded β sheet flanked by 4 α-helices namely α1 (V33-S44), α2 (E78-D98), α3 (L121-D133) and α4

(K147-R158). The βsheet topology is β1β6β5β2β4β3 with the β3 strand being antiparallel to its neighbors (Fig. 1A). The NMR structure of the VEEV macro domain in apo form superimposes well with the corresponding X-ray structure [START_REF] Malet | The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket[END_REF] with a rmsd of 2.1Å for the backbone atoms (Fig. 1B). Although the two structures are very similar regarding the secondary structure elements and topology, loops Q28-A36, G116-R120 and the loop connecting the β6 strand with the α4 helix exhibit different conformations due to the lack of NOEs and unassigned residues in these regions [START_REF] Makrynitsa | NMR study of non-structural proteins-part II: 1 H, 13 C, 15 N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)[END_REF]. Taking into consideration that the NMR structure is of high resolution (target function: 1.49Å, rmsd: 1.07Å), the high value of the rmsd is the result of the behavior of the aforementioned loops. Amino acids without detectable spin patterns in the NMR spectra are located around the highly positively charged surface area, which defines the ADPr binding site [START_REF] Malet | The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket[END_REF]. Similar behavior has been observed in the NMR study of the human orphan macro domain C6orf130 in which the amino acids of corresponding loops around the ADPr binding pocket were also missing in the NMR spectra [START_REF] Peterson | Orphan macrodomain (human C6ORF130) is an o-acyl-ADP-ribose deacylase: solution structure and catalytic properties[END_REF].

Structural basis of ADP-r binding to VEEV macro domain

Macro domains are ADPr-binding molecules and contain a highly conserved binding-pocket [START_REF] Karras | The macro domain is an ADP-ribose binding module[END_REF][START_REF] Li | Viral macro domains reverse protein ADP-ribosylation[END_REF]. To further investigate the binding of ADPr, we titrated 15 Nlabeled VEEV macro domain incrementally with ADPr and monitored backbone amide chemical shift changes in the 1 H-15 N HSQC spectra. During the titration experiments, 21 residues exhibited chemical shift perturbations (CSPs) above the threshold (calculated according to the standard deviation of all CSP values as determined by the equation ∆δ ppm = ( ) + ( ) [START_REF] Garrett | Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system[END_REF] with the majority of these residues located around the binding groove (Fig. 2A,B).

Interestingly, 1 H-15 N HSQC spectra of the complex did not display any further changes regarding the chemical shifts of the perturbed amino acids after a 1:1 protein-ligand ratio had been reached.

This strongly indicates that the VEEV macro domain binds specifically one ADPr molecule in its well-defined binding cleft. Crosspeaks of residues affected by ADPr gradually vanished during the titration experiments and simultaneously appeared in a new region of the HSQC spectrum (Fig. 2C). This behavior is characteristic of a slow exchange process and is indicative for high affinity ligand binding (K d of 3.9 ± 0.65 µM as measured by isothermal titration calorimetry [START_REF] Malet | The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket[END_REF]).

Analysis of 13 C-filtered/edited NOESY spectra along with the three-dimensional structure of the VEEV-ADPr complex gave insights into the interactions between the residues in the binding pocket and the ADPr moieties (SI, Figure S1). In particular, the adenine moiety is accommodated in a hydrophobic cavity (P1 pocket) and shows NOE contacts with I11, V33, Y142, and R144.

Concerning the proximal ribose (the adenosine ribose), we identified NOE contacts with V33, L108, T111, and R144 (P2 pocket), while the distal ribose of ADPr is hydrogen bonded with the backbone NH of G32 and shows NOEs with A22 and I113, fitting into a pocket (P3 pocket) comprising the N-terminal part of the α1 helix and the loop preceding the α3 helix (residues L108-R120). The phosphate binding site is stabilized through hydrogen bonds with the main chain NH groups of V33 and I113. Analysis of the VEEV/ADPr complex by molecular dynamics also revealed that the above residues participate strongly in the ADPr binding with the distal ribose playing a key role in the complex's formation [START_REF] Rungrotmongkol | Molecular insight into the specific binding of ADP-ribose to the nsP3 macro domains of chikungunya and Venezuelan equine encephalitis viruses: molecular dynamics simulations and free energy calculations[END_REF]. Due to exchange with the bulk solvent the assignment of the ADPr OH groups was not feasible. The residues that map the binding sites for each ADPr moiety are not random. A sequence alignment of macro domains from different organisms reveals the high degree of conservation for the amino acids residing in the binding pocket [START_REF] Fehr | Viral macrodomains: Unique mediators of viral replication and pathogenesis[END_REF][START_REF] Mcpherson | ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence[END_REF]) (SI, Figure S2).

Mapping amino acids with CSP values above threshold to the surface of the complex revealed that the majority of these residues form the ADPr cavity. However, despite their high CSPs residues K26, L37, K40, H64, A68, V69, F70, N72, and G116 do not participate directly in the binding of ADPr (Fig. 2D). Comparison of the structures in the free and complex state revealed conformational differences in loop S25-G31 and the C-terminal helix α4 although ADPr binding to the VEEV macro domain does not dramatically alter the overall structure of the protein (Fig. 2E). Likewise, the solution structure of the VEEV macro domain-ADPr is very similar to the corresponding crystal structure except for the aforementioned regions that exhibit conformational differences [START_REF] Malet | The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket[END_REF] (Fig. 2F). These differences are coupled with the binding of the ADPr molecule to the macro domain cavity. The loops comprising S25-G31 and L109-R120 undergo conformational changes resulting in a slight widening of the crevice where the distal ribose is located. Helices α1 and α4 helices seem to exhibit a displacement towards the binding pocket as well, but in this case the conformational changes cause a closure of the pocket around the ligand (Fig. 3A,B).

Dynamic and exchange properties of the VEEV macro domain (apo state and complex with

ADPr)

So far, a remarkable wealth of studies has focused on the structure and function of macro domains [START_REF] Götte | The enigmatic alphavirus non-structural protein 3 (nsP3) revealing its secrets at last[END_REF]. However, information about their dynamics and the conformational changes triggered by substrate binding is limited.

Model-free analysis of 15 N-NMR relaxation data as implemented in the Tensor2 program [START_REF] Dosset | Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data[END_REF] (Fig. 3C, SI Figure S3) shows that the VEEV macro domain in solution exhibits a rather rigid structure on the ps-ns time scale. However, a lot of residues in the sequential stretches G30-Y38 and G112-R120 could not be assigned [START_REF] Makrynitsa | NMR study of non-structural proteins-part II: 1 H, 13 C, 15 N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)[END_REF] suggesting that these residues undergo an exchange between different conformational states on an intermediate µs-ms time scale, where the signals are broadened.

To quantify the protein dynamics on this timescale, we performed 15 N CPMG relaxation dispersion experiments (SI, Figure S4). There are essentially two regions at the N-and C-terminus that undergo µs-ms motion. We simultaneously fit both clusters with residues 1-44 and 103-168, respectively, and found exchange rates kex of ~3500 and ~2580 s -1 and populations of the excited state pB of around 0.35-0.5%.A23 as well as loops β5-α3 (L108-R120) and α3-β6 (T134-D138)

exhibit the largest exchange contribution to R2, being responsible for the flexibility of the nearby residues (Fig. 4A,B). Remarkably, after ADPr binding, the flexibility of loop β5-α3 was reduced as all residues of this loop were assigned in the 1 H-15 N HSQC spectrum and showed relatively high order parameters S 2 .

Besides populations and exchange rates, relaxation dispersion curves are sensitive to the absolute chemical shift difference between ground and excited state, with the chemical shift being a valuable structural probe reporting on secondary and tertiary structure. We determined absolute 15 N chemical shift differences from HSQC spectra in the apo and bound state and plotted them against CPMG derived values (Fig. 4C). Residues in the ADPr binding crevice (L108-G116)

showed a linear correlation suggesting that the ADPr-bound pose can be nearly adopted by the excited state of the apo protein, however, most likely with a more open conformation compared to the ground apo and ADPr-bound state to allow entering of the ligand. We note that deviations are in part also attributed to chemical shift contributions imposed by the ligand, which are not present in the CPMG experiment.

To study a potential transition pathway from the apo to the bound form of the domain during binding of ADPr, we performed Normal Mode Analysis (NMA) calculations. The most pronounced topological differences observed between the two NMR resolved states of the domain are in the two loops L108-R120 and Q28-A36 and in helices α1, α2, and α4 (Fig. 5A), which relates well to the experimental CPMG data (Fig. 4). Using NMA to address these conformational changes, the structure converged smoothly to the target ADPr-bound conformation with final heavy atom RMSD of 0.55Å (initial RMSD: 1.8Å), indicating a pathway of backbone and side chain transitions to reshape the binding cavity of the domain and accommodate the ligand.

Specifically, the Q28-A36 loop undergoes a significant translational movement of ~3Å with respect to the L108-R120 loop, increasing the distance between the Cα atoms of G31 and I113 from ~7 to ~10Å in order to admit the distal ribose of ADPr (Fig. 5B). Consequently, F114 also moves to interact with the same ribose. Mutational insertions in the Q28-A36 loop can conditionally alter virus replication and, at the protein scale, destabilize the macro domain and partly affect its ADPr binding property [START_REF] Beitzel | High-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing[END_REF][START_REF] Guillén | Structural and biophysical analysis of sequence insertions in the Venezuelan Equine Encephalitis Virus macro domain[END_REF], highlighting the role this loop in the transition mechanism required for the accommodation of ADPr.

Conclusions

Our study demonstrates that ADPr binding on the VEEV macro domain results in a conformational exchange process of loops S25-G31 and L109-R120 in order to adopt an open conformation.

Similar rearrangements have been found in human C6orf130, an orphan macro domain protein [START_REF] Peterson | Orphan macrodomain (human C6ORF130) is an o-acyl-ADP-ribose deacylase: solution structure and catalytic properties[END_REF], and in the macro domain from SARS coronavirus [START_REF] Götte | The enigmatic alphavirus non-structural protein 3 (nsP3) revealing its secrets at last[END_REF]. The data reveals that the VEEV macro domain exhibits high adaptability to bind ADPr. The ligand does not simply bind to a well-defined, highly conserved binding pocket of the protein, but several regions of the protein contribute to the formation of the biomolecular complex. A number of residues undergo both backbone and side-chain conformational changes to adopt a suitable geometry that allows ADPr to enter the cavity and bind strongly to the protein.

Regarding the mobility analysis, the movement of helix α4 results in a reorientation of the W148 and L108 side chains which upon binding interact with the proximal ribose of ADPr. V33, as part of the moving helix α1, plays a crucial role in binding, positioning itself to a σ-π interacting arrangement with the aromatic group of adenine. The charged residues R144 and D10 also undergo certain rearrangements, the first to widen the binding pocket and the second to form a crucial hydrogen bond with the adenine amine. R144 in particular showed minor but significant conformational exchange as determined by the CPMG experiment (Fig. 4, SI Figure S4). The conformational changes not only contribute to the ADPr binding but also to the efficient deribosylation activity by properly positioning the reactive molecule, likely a water molecule, close to the substrate. Indeed, it was evidenced that a mutation in the corresponding Q28-A36 loop of Hepatitis E virus macro domain or O. iheyensis macro domains greatly impaired the catalysis without affecting significantly the substrate recognition [START_REF] Li | Viral macro domains reverse protein ADP-ribosylation[END_REF][START_REF] Zapata-Pérez | Structural and functional analysis of Oceanobacillus iheyensis macrodomain reveals a network of waters involved in substrate binding and catalysis[END_REF].

These conformational changes along the transition path thus provide a framework to understand the dynamic nature of the macro domains and to dissect the events during ADPr binding and deribosylation.

Taken together, our structural and mobility data strongly suggest that in solution the VEEV macro domain shows dynamics on the µs-ms timescale in the absence of ADPr, while the VEEV macro-ADPr complex seems to adopt a compact structure. The analysis of the 3D structures allows the identification of key conformational changes around the binding cleft that are critical for ADPr We identified a conformational exchange process between the solution structure of the apoprotein presented herein and another, low-populated conformational state (~0.5%). A comparison of the structures of the VEEV macro domain in the apo and ADPr bound state reveals that flexibility of the loop β5-α3 is crucial for the key conformational changes around the active site favoring ADPr binding. The high exchange rates of loops β5-α3 and α3-β6 contribute to the flexibility of these segments, which may serve as micro-switches that allow ADPr to enter the ligand-binding cleft.

The correlation of chemical shifts of the excited apo to the bound ground state suggests that ADPr binding occurs through a conformational selection mechanism. It is well established that polypeptide segments with high adaptability play a key role in partner or ligand identification, selectivity and binding [START_REF] Peterson | Orphan macrodomain (human C6ORF130) is an o-acyl-ADP-ribose deacylase: solution structure and catalytic properties[END_REF][START_REF] Zapata-Pérez | Structural and functional analysis of Oceanobacillus iheyensis macrodomain reveals a network of waters involved in substrate binding and catalysis[END_REF]. This may also be the function of these regions in many macro domains, including the one from VEEV. It is worth mentioning, that so far most of the studies concerning the structure of macro domains have focused on the characterization on the ADPr binding pocket and the open-close conformation during the binding of the ligand [START_REF] Egloff | Structural and functional basis for ADPribose and poly (ADP-ribose) binding by viral macro domains[END_REF]. In the present study, for the very first time, it is demonstrated the exchange process between two states of certain regions crucial for ADPr binding, in the apo form of a macro domain via experimental data.

  binding and depend on the plasticity of the loops β5-α3 and α3-β6. The sensitivity of loop β5-α3 to ADPr binding in combination with the mobility of loop α3-β6 indicates that they serve as gates of the binding cleft and assist the ADPr entrance. ADPr binding drives the protein molecule to switch from an open conformation with flexible segments to a rigid closed one.

Fig. 1 .

 1 Fig. 1. Solution structure of VEEV macro domain. A) Representation of VEEV macro domain NMR solution structure with secondary structure elements labeled. B) Superposition of ribbon representations of VEEV macro domain solved by NMR spectroscopy (purple) and X-ray diffraction (light blue). C) Surface rendering of the calculated electrostatic potential of VEEV macro domain generated with the Adaptive Poisson-Boltzmann Solver (APBS) plug-in in PyMOL (Schrödinger, 2010) where blue and red indicate positively and negatively charged regions respectively (scale -3 kT/e to +3 kT/e).

Fig. 2 .

 2 Fig. 2. Interaction of VEEV macro domain with ADPr. A) Overlay of 1 H-15 N HSQC spectra of the VEEV macro domain in the apo (purple) and ADPr bound state (ratio 1:4) (orange). The response of the NMR resonances of A22(bottom) and N72 (top) to increasing ADPr concentrations. Peaks corresponding to the apo state decrease in intensity as the ones of the bound state increase (purple: free, red: 1:0.250, light blue: 1:0.5, grey: 1:0.750, green: 1:1, yellow: 1:2, orange: 1:4. B) Chemical shift perturbation (CSP) in response to ADPr binding (ratio 1:4) with threshold value 0.26. Residues with CSP up to 0.2 from the threshold value are colored yellow, residues with CSP from 0.2 to 0.5 above the threshold are colored green and residues with CSP higher than 0.5 from the threshold are colored red. C) Surface representation of the VEEV macro domain in complex with ADPr. The residues with CSPs above the threshold are mapped onto the surface using the same color code as in B. D) Superposition of ribbon representations of VEEV macro domain in the apo (purple) and ADPr bound states (grey) as determined by NMR spectroscopy. E) Superposition of ribbon representations of the VEEV macro domain-ADPr complex as determined by X-ray diffraction (orange) and NMR spectroscopy (grey).

Fig. 3 .

 3 Fig. 3. Dynamic properties of VEEV macro domain. A) Surface representations of the VEEV macro domain in the apo state (purple) and the ADPr-bound state (grey). Regions G30-V33, I113-R120 and helix α4 are colored cyan. B) Close-up view of the ADPr binding pocket -left: VEEV macro domain apo state, right: VEEV macro domain/ADPr complex. Panels A and B display the conformational changes upon ADPr binding and some of the affected residues. C) S 2 values plotted as a function of residue number of VEEV macro domain in the apo state (top row) and complex (bottom row).

Fig. 4 .

 4 Fig. 4. µs-ms dynamics of VEEV macro domain without ADPr as probed by SQ 15 N CPMG relaxation dispersion experiments. A) The residues undergoing significant µs-ms conformational exchange were mapped to the structure. The tube radius corresponds to the exchange contribution to the R2 rate (Rex) as plotted in (B). Inset C) Linear correlation of absolute experimental 15 N chemical shift differences between 1) apo and ADPr bound macro domain (xaxis) and 2) ground and excited state of apoprotein from relaxation dispersion (y-axis).

Fig. 5 .

 5 Fig. 5. Normal mode analysis of VEEV macor domain. A) Differences between the NMR solution structures of VEEV macro domain in the apo state (purple) and the ADPr-bound state (grey) are indicated by red arrows (minimum length of the displayed vectors is 2 Å). B) Normal mode analysis from the apo (purple) to the bound state (grey) shows a transition pathway in the binding cavity. Transition states are shown in transparent white to dark grey colors as they progress to the bound state. Red arrows indicate significant reorientations of amino acids participating in binding of ADPr.

Acknowledgements

CPMG experiments were performed at the Bavarian NMR center (BNMRZ, http://www.bnmrz.org). EU FP7 REGPOT CT-2011-285950 -"SEE-DRUG" project is acknowledged for financial support (DB, GAS) as well as for the purchase of UPAT's 700 MHz NMR equipment. This work was also supported by the European program H2020 under the EVAg Research Infrastructure (grant agreement No. 653316) Appendix A. Supplementary data