Petr Bardoň

David Školoudík

Kateřina Langová

Petr Roman Herzig

Keywords: ultrasound, blood flow oscillations, transcranial probe, sonothrombolysis ultrasound, blood flow oscillations, transcranial probe, sonothrombolysis ultrasound, blood flow oscillations, transcranial probe, sonothrombolysis

Changes in blood flow velocity in the radial artery after 1-hour of ultrasound monitoring using a diagnostic 1 -5

MHz transcranial probe -a pilot study

Introduction

Multiple biological effects of ultrasound on the human body have been described. In the last 4 decades, different therapeutic effects of ultrasound have been studied experimentally. One such application involves accelerated recanalizing occluded arteries (clot lysis), especially in patients with acute strokes [1][2][3][4][5][6] . Various intensities (0.2 -2.0 W/cm 2 ) and frequencies (20 kHz -2 MHz) of the ultrasound beam were used in previous studies 1, [4][5][6][7] .

There are two possible effects of the ultrasound beam on an occluded artery: 1) mechanical destruction due to thrombus vibration with acceleration of penetration of fibrinolytics into the thrombus, and 2) stimulation of the endothelium with activation of fibrinolysis 1,7,8 . Another possible effect of the ultrasound beam was observed during our previous study in which repeated marked vasodilatation of the peripheral artery during continual Doppler monitoring was recorded 9 .

The aims of this prospective pilot study were to confirm the effect of continual ultrasound monitoring in the pulsed wave (PW) Doppler mode using a standard diagnostic 1 -5 MHz probe with a maximal diagnostic intensity (i.e., sono-thrombolysis) on blood flow in human arteries.

Materials and Methods

Fifteen volunteers were enrolled in the study during 6 months. All subjects fulfilled the inclusion and exclusion criteria, as listed in Table 1.

In all subjects, the measurement of blood flow velocities and indices in the radial artery (RA) were performed in 2-minute intervals during a 1-hour period in sessions A and B. All measurements were performed in a quiet room with a constant temperature.

Patients were lying in a comfortable position. The measurement started before noon, 4 -6 hours after the last meal, and minimally 24 hours after the last consumption of alcohol or caffeine.

Physical examinations were performed before the beginning of the session, at the end of the session, 24 hours after each session, and 30 days after the second session; all adverse events were recorded. All changes in physical examinations and all disorders requiring hospitalization were recorded as an adverse event.

Ethics committee approval

The local Ethics Committee at the University Hospital Ostrava approved the study and at the time of enrollment, each volunteer signed an informed consent form. The entire study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983 and 2008.

Statistical analysis

Data are reported as the median ± standard deviation with a coefficient of variation.

The one-sample Student´s t-test was applied when assessing statistical significance of the differences between the PSV, EDV, MFV, PI, and RI during both sessions. Statistical evaluations were performed using SPSS, version 15.0 (SPSS, Inc., Chicago, IL, USA).

Statistical significance was defined as a p value < 0.05.

Results

The sample of healthy volunteers consisted of 8 males and 7 females, with ages ranging between 30 and 78 years, and a mean age of 54.7 ± 17.0 years. Irregular changes in the measured parameters were recorded during both sessions. The changes in the blood flow parameters during the session A with intermittent measurement were similar in positive and negative deflections (Table 2). In contrast, the increase in EDV, and decrease in PI and RI were significantly higher during the session B with continual Doppler monitoring (p = 0.004, p = 0.004, and p = 0.015, respectively) with higher coefficients of variation. Several episodes of transient vasodilatation were observed during 1-hour monitoring in this session. The changes in PSV and MFV did not significantly differ between both measurements (Table 2).

The mean change in blood pressure during the first and second sessions was 6.1 ± 6.3 and 6.7 ± 7.1 mm Hg, respectively. The difference was not statistically significant. No clinically adverse events were recorded during and after the Doppler monitoring of the RA. The results of the presented study demonstrated that 60-minutes of Doppler monitoring using a 1 -5 MHz diagnostic duplex transcranial probe with maximal intensity may directly affect peripheral resistance (arterial tonus) in the territory of the monitored artery. However, the magnitude of the changes observed was not clinically significant in intact radial artery.

We hypothesize that Doppler monitoring of a peripheral artery in the human body may lead to irritation of the endothelium. This mechanical irritation with a probable local increase in temperature may stimulate nitride oxide (NO) synthesis and secretion with peripheral vasodilatation, characterized by the EDV increase and the decrease of peripheral resistance (as assessed using the PI and RI). The variation in peripheral resistance could be explained by the short half-time of NO with new re-synthesis 10 . Also, prostaglandin-2 and aspartate could play a role in this peripheral vasodilatation 11 .

One can take exception to the possibility of an increase of EDV due to variable pressure on the monitored artery caused by the ultrasound probe. However, increased pressure on the artery usually leads to an increase in the PSV and sometimes a decrease in the EDV with an increase in PI and RI. We did not record this situation in our study.

The effect of TCD monitoring on thrombus dissolution in acute stroke patients was demonstrated for the first time in 2000 12 . Subsequently, other studies demonstrated a potential effect of diagnostic ultrasound on the acceleration of spontaneous or induced recanalization of intracranial arteries with better clinical outcome after 90 days [4][5][6] . This therapeutic procedure is referred to as sono-thrombolysis. The results of the presented pilot study showed that in addition to the two possible effects of an ultrasound beam (mechanical thrombus destruction with penetration of fibrinolytics into the thrombus and activation of the endogenous fibrinolytic system) 1,7,8 , there should be another effect causing acceleration of artery recanalization and improvement of blood flow in the territory of the occluded artery (intermittent distal vasodilatation in the territory of a monitored artery). Some limitations of the current study should be mentioned. The study set was relatively small and the influence of risk factors (e.g., hypertension, diabetes mellitus, and atherosclerosis) could not be evaluated. In addition, the factors influencing blood flow, e.g., arterial blood pressure, blood oxygen saturation, and partial pressure of CO 2 and O 2 , were not continually monitored. It is also known that flow to the hand is highly variable and is governed by sympathetic neural inputs, temperature, and stress. Nevertheless, we only compared the changes of the measured blood flow parameters between two sessions in the same subject in which we presumed a similar variation of these factors and, the influence of stress was similar during both sessions, as the subjects had to remain still for an hour and were not aware of the type of sonographic insonation (intermittent versus continual) in both cases. For elimination of these limitations, a new study with continual measurement of all the mentioned factors will be initiated.

Introduction

Multiple biological effects of ultrasound on the human body have been described. In the last 4 decades, different therapeutic effects of ultrasound have been studied experimentally. One such application involves accelerated recanalizing occluded arteries (clot lysis), especially in patients with acute strokes [1][2][3][4][5][6] . Various intensities (0.2 -2.0 W/cm 2 ) and frequencies (20 kHz -2 MHz) of the ultrasound beam were used in previous studies 1, [4][5][6][7] .

There are two possible effects of the ultrasound beam on an occluded artery: 1) mechanical destruction due to thrombus vibration with acceleration of penetration of fibrinolytics into the thrombus, and 2) stimulation of the endothelium with activation of fibrinolysis 1,7,8 . Another possible effect of the ultrasound beam was observed during our previous study in which repeated marked vasodilatation of the peripheral artery during continual Doppler monitoring was recorded 9 .

The aims of this prospective pilot study were to confirm the effect of continual ultrasound monitoring in the pulsed wave (PW) Doppler mode using a standard diagnostic 1 -5 MHz probe with a maximal diagnostic intensity on blood flow in human arteries.

Materials and Methods

Fifteen volunteers were enrolled in the study during 6 months. All subjects fulfilled the inclusion and exclusion criteria, as listed in Table 1.

In all subjects, the measurement of blood flow velocities and indices in the radial artery (RA) were performed in 2-minute intervals during a 1-hour period in sessions A and B. All measurements were performed in a quiet room with a constant temperature.

Patients were lying in a comfortable position. The measurement started before noon, 4 -6 hours after the last meal, and minimally 24 hours after the last consumption of alcohol or caffeine.

Physical examinations were performed before the beginning of the session, at the end of the session, 24 hours after each session, and 30 days after the second session; all adverse events were recorded. All changes in physical examinations and all disorders requiring hospitalization were recorded as an adverse event.

Ethics committee approval

The local Ethics Committee at the University Hospital Ostrava approved the study and

Statistical analysis

Data are reported as the median ± standard deviation with a coefficient of variation.

The one-sample Student´s t-test was applied when assessing statistical significance of the differences between the PSV, EDV, MFV, PI, and RI during both sessions. Statistical evaluations were performed using SPSS, version 15.0 (SPSS, Inc., Chicago, IL, USA).

Statistical significance was defined as a p value < 0.05.

Results

The sample of healthy volunteers consisted of 8 males and 7 females, with ages ranging between 30 and 78 years, and a mean age of 54.7 ± 17.0 years. Irregular changes in the measured parameters were recorded during both sessions. The changes in the blood flow parameters during the session A with intermittent measurement were similar in positive and negative deflections (Table 2). In contrast, the increase in EDV, and decrease in PI and RI were significantly higher during the session B with continual Doppler monitoring (p = 0.004, p = 0.004, and p = 0.015, respectively) with higher coefficients of variation. Several episodes of transient vasodilatation were observed during 1-hour monitoring in this session. The changes in PSV and MFV did not significantly differ between both measurements (Table 2).

The mean change in blood pressure during the first and second sessions was 6.1 ± 6.3 and 6.7 ± 7.1 mm Hg, respectively. The difference was not statistically significant. No clinically adverse events were recorded during and after the Doppler monitoring of the RA. The results of the presented study demonstrated that 60-minutes of Doppler monitoring using a 1 -5 MHz diagnostic duplex transcranial probe with maximal intensity may directly affect peripheral resistance (arterial tonus) in the territory of the monitored artery. However, the magnitude of the changes observed was not clinically significant in intact radial artery.

We hypothesize that Doppler monitoring of a peripheral artery in the human body may lead to irritation of the endothelium. This mechanical irritation with a probable local increase in temperature may stimulate nitride oxide (NO) synthesis and secretion with peripheral vasodilatation, characterized by the EDV increase and the decrease of peripheral resistance (as assessed using the PI and RI). The variation in peripheral resistance could be explained by the short half-time of NO with new re-synthesis 10 . Also, prostaglandin-2 and aspartate could play a role in this peripheral vasodilatation 11 .

One can take exception to the possibility of an increase of EDV due to variable pressure on the monitored artery caused by the ultrasound probe. However, increased pressure on the artery usually leads to an increase in the PSV and sometimes a decrease in the EDV with an increase in PI and RI. We did not record this situation in our study.

The effect of TCD monitoring on thrombus dissolution in acute stroke patients was demonstrated for the first time in 2000 12 . Subsequently, other studies demonstrated a potential effect of diagnostic ultrasound on the acceleration of spontaneous or induced recanalization of intracranial arteries with better clinical outcome after 90 days [4][5][6] . This therapeutic procedure is referred to as sono-thrombolysis. The results of the presented pilot study showed that in addition to the two possible effects of an ultrasound beam (mechanical thrombus destruction with penetration of fibrinolytics into the thrombus and activation of the endogenous fibrinolytic system) 1,7,8 , there should be another effect causing acceleration of artery recanalization and improvement of blood flow in the territory of the occluded artery (intermittent distal vasodilatation in the territory of a monitored artery). Some limitations of the current study should be mentioned. The study set was relatively small and the influence of risk factors (e.g., hypertension, diabetes mellitus, and atherosclerosis) could not be evaluated. In addition, the factors influencing blood flow, e.g., arterial blood pressure, blood oxygen saturation, and partial pressure of CO 2 and O 2 , were not continually monitored. It is also known that flow to the hand is highly variable and is governed by sympathetic neural inputs, temperature, and stress. Nevertheless, we only compared the changes of the measured blood flow parameters between two sessions in the same subject in which we presumed a similar variation of these factors and, the influence of stress was similar during both sessions, as the subjects had to remain still for an hour and were not aware of the type of sonographic insonation (intermittent versus continual) in both cases. For elimination of these limitations, a new study with continual measurement of all the mentioned factors will be initiated. Table 1. Study inclusion and exclusion criteria.

  Doppler monitoring of radial artery 4

  The peak systolic velocity (PSV), end-diastolic velocity (EDV), mean flow velocity (MFV), pulsatile index (PI; PI = [PSV -EDV] / MFV), and resistive index (RI; RI = [PSV -EDV] / EDV) were obtained from a proximal part of the left RA as a mean value from last five heart cycles during both sessions. A sample volume (SV = 10 mm) was inserted in the proximal third of the RA to a depth of 15 -35 mm, and the probe was held by hand in the same position during the entire session. During the session A, the probe was held in the same position during the whole session but the Doppler mode insonation with recording of blood flow velocities and indices was only intermittent. In contrast to the session B Thus in session A, the arterial segment was continuously insonated for a period < 10 seconds during each 2-minute interval. During the session B, the probe was also held in the same position during the whole session but continual ultrasound monitoring was performed in the Doppler mode (mechanical index [MI] = 1.4). Eight patients started with session A and 7 patients with session B. Time interval between both sessions was 14 ± 2 days. A standard diagnostic duplex transcranial 1 -5 MHz probe (P17; Aloka SSD 4000, Tokyo, Japan) was used during both sessions. The maximum diagnostic thermal and mechanical indices of the ultrasound beam (mechanical index (MI) = 1.4, tissue thermal index (Tlc) = 1.9) was set in Doppler mode. The pulsed wave (PW) Doppler mode with frequency of ultrasound beam 2.0 MHz was used. Insonation depth was between 15 and 35 mm with sample volume 10 mm. The following sonographic machine setting was used: pulse repetition period 0.2 ms, burst repetition Doppler monitoring of radial artery 5 frequency 5000 Hz, pulse duration 1.46 µs, peak rarefactional pressure 2.24 MPa, and pulse average intensity at maximum MI 208 W/cm 2 .

  Doppler monitoring of radial artery 6

Doppler monitoring of radial artery 7 Discussion

 7 This is the first study in which the changes in blood flow velocity and indices during intermittent and continual Doppler monitoring (sono-thrombolysis) were compared.

  Doppler monitoring of radial artery 8

Table 1 .

 1 Doppler monitoring of radial artery 9 Conclussion A 1-hour continuous Doppler monitoring of RA using transcranial 1 -5 MHz duplex probe may directly lead to the repetitive peripheral vasodilatation without causing any clinically relevant adverse effects. Further studies are needed for a complete explanation of the effect of various ultrasound frequencies and intensities on the human arterial system. Doppler monitoring of radial artery 11 10. Chatterjee A, Catravas JD. Endothelial nitric oxide (NO) and its pathophysiologic regulation. Vascul Pharmacol 2008;49:134-140. 11. Aird WC. Endothelium as an organ system. Crit Care Med 2004;32(Suppl 5):S271-279. 12. Alexandrov AV, Demchuk AM, Felberg RA, et al. High rate of complete recanalisation and dramatic clinical recovery during tPA infusion when continuously monitored with 2-MHz transcranial Doppler monitoring. Stroke 2000;31:610-614. Study inclusion and exclusion criteria. Table 2. Mean baseline values, coefficients of variation and mean changes in peak systolic velocity, end-diastolic velocity, mean flow velocity, pulsatility index and resistive index during intermittent Doppler measurement (session A) and continual Doppler monitoring (session B) of the radial artery.

  Doppler monitoring of radial artery 4

  The peak systolic velocity (PSV), end-diastolic velocity (EDV), mean flow velocity (MFV), pulsatile index (PI; PI = [PSV -EDV] / MFV), and resistive index (RI; RI = [PSV -EDV] / EDV) were obtained from a proximal part of the left RA as a mean value from last five heart cycles during both sessions. A sample volume (SV = 10 mm) was inserted in the proximal third of the RA to a depth of 15 -35 mm, and the probe was held by hand in the same position during the entire session. During the session A, the probe was held in the same position during the whole session but the Doppler mode insonation with recording of blood flow velocities and indices was only intermittent. Thus in session A, the arterial segment was continuously insonated for a period < 10 seconds during each 2-minute interval. During session B, the probe was also held in the same position during the whole session but continual ultrasound monitoring was performed in the Doppler mode (mechanical index [MI] = 1.4). Eight patients started with session A and 7 patients with session B. Time interval between both sessions was 14 ± 2 days. A standard diagnostic duplex transcranial 1 -5 MHz probe (P17; Aloka SSD 4000, Tokyo, Japan) was used during both sessions. The maximum diagnostic thermal and mechanical indices of the ultrasound beam (mechanical index (MI) = 1.4, tissue thermal index (Tlc) = 1.9) was set in Doppler mode. The pulsed wave (PW) Doppler mode with frequency of ultrasound beam 2.0 MHz was used. Insonation depth was between 15 and 35 mm with sample volume 10 mm. The following sonographic machine setting was used: pulse repetition period 0.2 ms, burst repetition Doppler monitoring of radial artery 5 frequency 5000 Hz, pulse duration 1.46 µs, peak rarefactional pressure 2.24 MPa, and pulse average intensity at maximum MI 208 W/cm 2 .

  at the time of enrollment, each volunteer signed an informed consent form. The entire study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983 and 2008.

  Doppler monitoring of radial artery 6

Doppler monitoring of radial artery 7 Discussion

 7 This is the first study in which the changes in blood flow velocity and indices during intermittent and continual Doppler monitoring were compared.

  Doppler monitoring of radial artery 8

Doppler monitoring of radial artery 9 ConclussionA 1 -

 91 hour continuous Doppler monitoring of RA using transcranial 1 -5 MHz duplex probe may directly lead to the repetitive peripheral vasodilatation without causing any clinically relevant adverse effects. Further studies are needed for a complete explanation of the effect of various ultrasound frequencies and intensities on the human arterial system.

  Doppler monitoring of radial artery 11 10. Chatterjee A, Catravas JD. Endothelial nitric oxide (NO) and its pathophysiologic regulation. Vascul Pharmacol 2008;49:134-140. 11. Aird WC. Endothelium as an organ system. Crit Care Med 2004;32(Suppl 5):S271-279. 12. Alexandrov AV, Demchuk AM, Felberg RA, et al. High rate of complete recanalisation and dramatic clinical recovery during tPA infusion when continuously monitored with 2-MHz transcranial Doppler monitoring. Stroke 2000;31:610-614.

  Doppler monitoring of radial artery 12Tables

Table 2 .

 2 Mean baseline values, coefficients of variation and mean changes in peak systolic velocity, end-diastolic velocity, mean flow velocity, pulsatility index and resistive index during intermittent Doppler measurement (session A) and continual Doppler monitoring (session B) of the radial artery.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 .

 1 Study inclusion and exclusion criteria.

	Page 25 of 26
		Age 30 -80 years
	Inclusion criteria	Informed consent signed
		Known nephropathy with creatinine level > 150 µmol/L or creatinine
		clearance < 30 ml per hour
		Hepatopathy with elevation of hepatic enzymes or bilirubin > 2 times the
	Exclusion criteria	upper limit of normal
		Planned therapy using fibrinolytics or high dose heparin during 24 hours
		after the enrolment into the study
			peripheral artery disease
		History of	vascular surgery

Table 2 .

 2 Mean baseline values, coefficients of variation and mean changes in peak systolic velocity, end-diastolic velocity, mean flow velocity, pulsatile index and resistive index during intermittent Doppler measurement (session A) and continual Doppler monitoring (session B) of the Doppler monitoring (session B), EDV -end-diastolic velocity, IDM -intermittent Doppler measurement (session A), MFVmean flow velocity, PI -pulsatility index, PSV -peak systolic velocity, RI -resistive index, SD -standard deviation

	radial artery
		Mean baseline value ± SD	p value	coefficient of	Mean change ± SD	p value	Mean	95% confidence
					variation			difference	interval of the
		IDM	CDM		IDM	CDM	IDM	CDM	difference
	PSV (cm/s)	59.07 ± 10.40 58.53 ± 10.06 0.155	18.8% 18.6% -0.61 ± 8.03	-0.75 ± 8.79	0.814	-0.138	-1.372 -1.097
	EDV (cm/s)	10.47 ± 2.87 10.33 ± 2.98	0.217	29.2% 37.3% -0.41 ± 2.33	0.54 ± 3.59	0.004	0.948	0.348 -1.549
	MFV (cm/s)	26.75 ± 4.94 26.41 ± 4.98	0.120	19.0% 18.8% -0.56 ± 3.76	0.07 ± 4.65	0.086	0.629	-0.102 -1.361
	PI	1.831 ± 0.177 1.843 ± 0.180 0.287	12.7% 18.3% 0.020 ± 0.201 -0.043 ± 0.289	0.004	-0.017	-0.029 --0.006
	RI	0.819 ± 0.038 0.821 ± 0.036 0.304	5.5%	9.8% 0.004 ± 0.035 -0.013 ± 0.059	0.015	-0.018	-0.031 --0.004
	CDM -continual

John Wiley & SonsJournal of Clinical Ultrasound


Siti Azma Jusoh

Christoph Welsch

Shirley W I Siu

Rainer A Böckmann

Volkhard Helms

Contribution of charged and polar residues for the formation of the E1-E2 heterodimer from Hepatitis C Virus

Keywords:

The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1-E2 heterodimer. The simulations support the key role of the Lys370-Asp728 ion pair for mediating the E1-E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1-E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1-E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.

Introduction

Hepatitis C virus (HCV) is estimated to have infected at least 170 million people worldwide and is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma [START_REF] Appel | From structure to function: New insights into hepatitis C virus RNA replication[END_REF].

Until recently, experimental studies on HCV were limited due to lack of efficient cell culture systems for the virus amplification. However, this situation has changed with the development of novel in vitro systems [START_REF] Moradpour | Replication of hepatitis C virus[END_REF], particularly the HCV pseudoparticles [START_REF] Bartosch | Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes[END_REF] (HCVpp) and the first system for efficient production of infectious viral particles in cell culture [START_REF] Wakita | Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[END_REF] (HCVcc).

HCV is the only member of the Hepacivirus genus which belongs to evolutionary related viruses of the Flaviviridae family [START_REF] Francki | Classification and nomenclature of viruses. Fifth report of the international Committee on taxonomy of viruses[END_REF][START_REF] Lindenbach | Flaviviridae: The viruses and their replication[END_REF]. The virus genome contains a long open reading frame of more than 9600 nucleotides that is translated into a single polyprotein of approximately 3000 amino acids length [START_REF] Matsuura | The molecular biology of hepatitis C virus[END_REF]. The open reading frame between the 5'-non coding region (NCR) and 3'-NCR is composed of the structural core protein and the two envelope glycoproteins E1 and E2, the p7 ion channel and at least six non-structural proteins.

Binding and internalization of the hepatitis C virus are essential steps in the viral replication cycle mediated by the envelope glycoproteins E1 and E2. The E1 and E2 proteins are released by host signal peptidase cleavages [START_REF] Lindenbach | Flaviviridae: The viruses and their replication[END_REF] and assemble as a non-covalent E1-E2 heterodimer which is retained in the endoplasmic reticulum (ER) [START_REF] Dubuisson | Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein[END_REF]. These two membrane proteins are type I transmembrane (TM) proteins which are composed of a large N-terminal ectodomain towards the ER lumen and a C-terminal hydrophobic anchor. The membrane-spanning segments for both E1 and E2 are located at the C-termini and predicted to be less than 30 amino acids long with two stretches of hydrophobic residues separated by a short polar segment with at least one highly conserved charged residue [START_REF] Cocquerel | Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins[END_REF]. Interestingly, two consecutive GxxxG motifs are known within the TM of E1. The presence of the GxxxG motif in glycophorin A (GpA), a membrane protein of erythrocytes, at the helix-helix packing interface is known to be involved in the GpA homodimerization [START_REF] Cuthbertson | Transmembrane helix-helix interactions: Comparative simulations of the glycophorin A dimer[END_REF]. Experimental studies demonstrated that the TM domains of E1 and E2 are not just membrane anchors, but play important multifunctional roles during the biogenesis of HCV [START_REF] Dubuisson | Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein[END_REF][START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF], e.g. virus entry [START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF],

ER retention, as an internal signal peptide and E1-E2 heterodimerization [START_REF] Cocquerel | Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins[END_REF].

In particular, Gly354, Gly358, and the conserved charged residues in the TM region Lys370, Asp728 and Arg730 were shown to be involved in E1-E2 heterodimerization. Different experiments, mutagenesis studies of alanine scanning insertion, site-directed mutagenesis and tryptophan replacement suggest a salt-bridge interaction between Lys370 and Asp728 at the helix-helix dimer interface, which strongly contributes to the E1-E2 heterodimerization [START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF][START_REF] Ciczora | Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer[END_REF]. The charged residues in the TM domain of E1 and E2 glycoproteins of bovine viral diarrhea virus (BVDV) were also claimed to be responsible for the heterodimerization [START_REF] Ronecker | Formation of bovine viral diarrhea virus E1-E2 heterodimers is essential for virus entry and depends on charged residues in the transmembrane domains[END_REF].

So far, this hypothesis has not yet been confirmed by structure determination methods. In contrast to these residues, Arg730 was shown to play a minor role for the assembly of the E1-E2 envelope glycoprotein [START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF].

Despite their relative abundance in the protein-coding regions of different genomes (2520-3025 %), only a few high-resolution structures of membrane proteins could be determined so far due to the difficulty of membrane protein crystallization in the lipid bilayer environment [START_REF] White | The progress of membrane protein structure determination[END_REF]. Yet, molecular dynamics (MD) simulations of membrane proteins embedded in lipid bilayers have become quite popular and successful in the last ten years [START_REF] Cuthbertson | Transmembrane helix-helix interactions: Comparative simulations of the glycophorin A dimer[END_REF][START_REF] Bond | Membrane protein dynamics versus environment: Simulations of OmpA in a micelle and in a bilayer[END_REF]. In particular, MD simulations were applied to study the spontaneous aggregation of phospholipids around membrane proteins [START_REF] Böckmann | Spontaneous Formation of Detergent Micelles around the Outer Membrane Protein OmpX[END_REF] or have been used to investigate the relative position of individual transmembrane helices in lipid bilayers [START_REF] Lomize | Positioning of proteins in membranes: A computational approach[END_REF] and their dynamic interactions with phospholipid bilayers [START_REF] Matthews | Dynamic Helix Interactions in Transmembrane Signaling[END_REF]. For example, the structure of the Glycoprotein A (GpA) dimer was computationally predicted [START_REF] Treutlein | The glycophorin A transmembrane domain dimer: Sequence-specific propensity for a right-handed supercoil of helixes[END_REF], including results from an extensive mutagenesis work [START_REF] Lemmon | Sequence specificity in the dimerization of transmembrane. alpha.-helixes[END_REF] to narrow the search. The prediction was later refined, using an improved global search method global [START_REF] Adams | Improved prediction for the structure of the dimeric transmembrane domain of glycophorin A obtained through global searching[END_REF]. The subsequently determined NMR structure of the GpA dimer in micelles [START_REF] Mackenzie | A Transmembrane Helix Dimer: Structure and Implications[END_REF] was in good agreement with the predicted structure. Furthermore, MD simulations were used to study the behavior of individual helices of bacteriorhodopsin [START_REF] Woolf | Molecular dynamics simulations of individual alpha-helices of bacteriorhodopsin in dimyristoylphosphatidylcholine. II. Interaction energy analysis[END_REF], the oligomerization of the helices of Vpu [START_REF] Candler | Computational analysis of mutations in the transmembrane region of Vpu from HIV-1[END_REF][START_REF] Fischer | Viral ion channels: structure and function[END_REF], the free energy for dimerization of GpA [START_REF] Hénin | Insights into the Recognition and Association of Transmembrane α-Helices. The Free Energy of α-Helix Dimerization in Glycophorin A[END_REF], and the protonation equilibrium of Arg residues within a TM helix [START_REF] Yoo | Does Arginine remain protonated in the lipid membrane? Insights from microscopic pKa calculations[END_REF].

The principal aim of the present study was to identify critical regions and crucial residues within HCV envelope proteins for the formation of the E1-E2 heterodimer. Thus, we performed atomistic MD simulations for the putative TM domain of the E1-E2 heterodimer from HCV. Our results provide, for the first time, an atomic structural and dynamic model for the TM domain of the E1-E2 heterodimer. The simulations reveal the importance of the ionpair interaction and of additional inter-helical hydrogen bonds in the middle of the helix interfacial region for the structural integrity of the heterodimer. Furthermore, we confirmed the locations of the conserved residues which are in good agreement with the experimental studies.

Methods

Sequences

The protein sequences used for MD simulations of E1 and E2 from the hepatitis C virus genome polyprotein were obtained from the UniProtKB/Swiss-Prot database (http://au.expasy.org/uniprot/) [START_REF] Wu | The Universal Protein Resource (UniProt): an expanding universe of protein information[END_REF]. The E1 sequence used in this study is G 350 AHWGVLAGIA 360 YFSMVGNWAK 370 VLVVLLLFAG 380 VDA. The E2 sequence is WAIKWEYVV 720 LLFLLLADAR 730 VCSCLWMMLL 740 ISQAEA. Both sequences are from HCV genotype 1a.

We also used test segments, named H-segments, which were used to study apparent membrane-transfer free energies of each of the 20 naturally occurring amino acids [START_REF] Hessa | Recognition of transmembrane helices by the endoplasmic reticulum translocon[END_REF]. The H-segments were prepared as ideal α-helices, which contained a charged amino acid in the middle of their TM helix. MD simulations of the H-segments were compared to the results of E1 and E2 monomer simulations (see below).

Sequence analysis

Sequences of HCV envelope proteins were retrieved from public HCV databases, UniProtKB and euHCVdb (http://www.euHCVdb.de) [START_REF] Combet | euHCVdb: the European hepatitis C virus database[END_REF]. HCV genotypes have been differentiated according to a consensus proposal for a unified system of HCV genotype nomenclature [START_REF] Simmonds | Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes[END_REF].

Sequence alignments were computed using CLUSTAL W [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF] and MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF], and subsequently improved by minor manual modifications using the SEAVIEW alignment editor [START_REF] Galtier | SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny[END_REF]. A comprehensive sequence analysis was performed in 604 HCV E1 sequences (HCV genotype 1: 476, other genotypes: 128) and in 569 HCV E2 sequences (HCV genotype 1: 444; other genotypes: 125). We deduced amino acid polymorphisms in the E1 and E2 TM domains including all sites associated with E1-E2 heterodimerization investigated in this study.

TM protein prediction

Five prediction methods for helical membrane proteins were employed to determine the start and end points of the E1 and E2 TM regions: PHDHTM [START_REF] Rost | Topology prediction for helical transmembrane proteins at 86% accuracy[END_REF], SPLIT4 [START_REF] Juretic | Basic charge clusters and predictions of membrane protein topology[END_REF], HMMTOP2.0 [START_REF] Tusnády | The HMMTOP transmembrane topology prediction server[END_REF], TMHMM [START_REF] Krogh | Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes[END_REF], and TMMOD [START_REF] Kahsay | An improved hidden Markov model for transmembrane protein detection and topology prediction and its applications to complete genomes[END_REF]. MINS2 [START_REF] Park | Prediction of the translocon-mediated membrane insertion free energies of protein sequences[END_REF] was used to predict the membrane insertion free energy of the TM domains of E1 and E2.

MD simulations

All structures used in this study were prepared as ideal α-helices. The SCWRL program [START_REF] Canutescu | A graph-theory algorithm for rapid protein side-chain prediction[END_REF] was used to position the side chain rotamers and to generate mutants. Gromacs [START_REF] Hess | GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation[END_REF] tools were used to set up paralleled dimers aligned along the membrane normal with a salt bridge interaction at their helix-helix interfaces. In this conformation, the side chains of the charged residues were within 5 to 6 Å distance to each other (see Table 1) and Asn367 forms an interhelical hydrogen bond. MD simulations of the E1-E2 heterodimer were done twice and each simulation was assigned different starting velocities.

A snapshot of a fully hydrated equilibrated lipid bilayer containing 128 DMPC lipids [START_REF] Griepernau | 1-Alkanols and membranes: A story of attraction[END_REF] solvated with 5,673 simple point change (SPC) water molecules was used as a starting point for all MD simulations. A cavity within the bilayer was created using the protocols of reference [START_REF] Faraldo-Gómez | Setting up and optimization of membrane protein simulations[END_REF]. The solvent-accessible protein surfaces of the peptides were calculated by the program MSMS [START_REF] Sanner | Gromacs -a Message-Passing Parallel Molecular-Dynamics Implementation[END_REF] using a probe size radius of 1.4 Å. The solvent-accessible surfaces of the peptides were used as templates for estimating the volume of the necessary cavity. In each case, 4-6 lipids in the centre of the projected hole were removed to avoid overlaps of lipids with the protein. 200 ps of simulation with a modified version of the Gromacs version 3.1.4

[47] were performed to create the protein cavity in the DMPC lipid bilayer. Each peptide sequence was embedded into the DMPC bilayer using a cavity of suitable size. The mixed protein-lipid bilayer system was surrounded by approx. 45 water molecules per lipid molecule, thus ensuring full hydration of the membrane [START_REF] Siu | Biomolecular simulations of membranes: Physical properties from different force fields[END_REF]. The protein/lipid/water system was then subjected to 500 steps of energy minimization using the steepest descent algorithm.

Ions (Na + and Cl -) were added to neutralize the system and to achieve close-to-physiological conditions at ~0.1 M NaCl. This was followed by a 200 ps MD run with harmonic position restraints (force constant 1000 kJ mol nm -2 ) applied to all heavy atoms of the protein. This procedure allowed the lipids and the water molecules to relax around the protein after insertion of the protein. Subsequently, fully unrestrained production runs of 100 ns duration were performed for the protein/lipid systems.

All simulations were performed using the Gromacs 4.0.3 package [START_REF] Hess | GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation[END_REF]. All monomer and dimer simulations were performed with united atom force field based on GROMOS96 (53a6) [START_REF] Oostenbrink | A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6[END_REF] for the peptides and the Berger force field [START_REF] Berger | Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature[END_REF][START_REF] Chiu | Incorporation of surface tension into molecular dynamics simulation of an interface: a fluid phase lipid bilayer membrane[END_REF] for the phospholipids. Periodic boundary conditions were used in all directions. The system was coupled to a temperature bath at 310 K separately for the protein, the lipids, and the water/ions with a coupling constant of 0.1 ps -1 [START_REF] Berendsen | Molecular dynamics with coupling to an external bath[END_REF]. For the pressure, semi-isotropic coupling was employed separately for the lateral and for the normal directions with a coupling time τ p = 1 ps. The compressibility was set to 4.5 x 10 -5 bar -1 . Covalent bonds to H-atoms were constrained using the LINCS algorithm [START_REF] Hess | LINCS: A linear constraint solver for molecular simulations[END_REF] and an integration step size of 2 fs was used. The non-bonded pair list was generated every 10 steps with a cutoff of 1.0 nm. For short range van der Waals interactions, a cutoff distance of 1.0 nm was used. The long-range electrostatics interactions were treated using the Particle-Mesh Ewald method with a real space cutoff of 1.0 nm and a grid spacing of 0.12 nm with cubic interpolation.

Analyses of the trajectories were primarily performed with tools included in the Gromacs 4.0.3 suite [START_REF] Hess | GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation[END_REF]47]. Root mean square deviation (RMSD) analyses were based on atoms of the protein backbone. Salt bridge contacts were defined by monitoring the average distance between the side chains (see Table 1). Helix centers of mass were computed using the coordinates of C atoms only for the segments 5-25 (E1) and 35-55 (E2). All images in this work were prepared with the Pymol program (pymol.sourceforge.net).

Results

Sequence analysis of the TM domains of E1 and E2

The conserved residues Gly354, Gly358, Lys370, Asp728 and Arg730 were predicted to be located in the TM region of E1 and E2 in HCV genotype 1a from the UNIPROT database. A comprehensive sequence analysis was carried out to investigate the natural polymorphisms occurring at these particular amino acid sites.

We found the Lys370 in E1 being only once replaced by Arg in HCV genotype 1. All other genotypes investigated showed no polymorphism at this site in E1. The residues Asp728 and Arg730 are highly conserved in HCV E2 genotype 1. We found a non-conservative polymorphism only at position 728. The polar residue Asp was replaced by the aromatic and non-polar residue Tyr. Again no polymorphism at 728 or 730 was found in genotypes 2, 3 or 5, whereas Gly728 and Lys730 were found in genotype 4 once respectively, and Val728 and His730 in genotype 6 once respectively. Overall, polymorphisms at Lys370, Asp728 and Arg730 have been observed only exceptionally.

Two consecutive GxxxG motifs are present in the TM segment of E1. Gly350 and Gly354 were found to be highly conserved in all genotypes investigated. The second motif showed the conservative polymorphism Gly358Ala in genotypes 1, 5 and 6. Gly358 was conserved in genotypes 2, 3, and 4. Genotype 1 showed an Ala twice at 358. Only Ala358 was found in genotype 5. Genotype 6 showed Ala358 in the majority of sequences investigated (30 over 43).

Identification of TM residues by secondary structure prediction methods

We used five different methods for secondary structure prediction of the TM domains of E1 and E2 (see Fig. S1). This gave predicted TM helices of 21 to 31 amino acids length for the TM domain of E1. The consensus segment predicted by at least three out of five methods ranges from Val355 to Ala379. For the TM domain of the E2 glycoprotein, the consensus segment assigned by at least three methods ranges from Tyr718 to Ser742. Interestingly, all methods placed the conserved charged residues Lys370, Asp728 and Arg730 in the middle part of the TM domains.

The MINS2 [54] method that is based on amino acid frequencies and calibrated against the dataset of Hessa et al. (2007) was applied to compute membrane insertion free energies of TM segments. Compared to the threshold of 3.5 kcal mol -1 for observed TM helices in known structures of helical membrane proteins, MINS2 gives a favorable insertion free energy for the isolated TM segments of E1 (1.8 kcal mol -1 ) when using Lys370 as center, and a borderline value of 4.3 kcal mol -1 for E2 (4.3 kcal mol -1 ) when using Asp728 as center.

MD Simulations of E1 and E2 monomers

MD simulations were carried out to investigate the behavior of the monomeric TM segments containing a charged residue in the middle of the helices. We observed that during the 100 ns MD simulations the charged residues Lys370 from E1 and Asp728 from E2 were attracted towards the lipid bilayer interface. Only Lys370 was able to comfortably anchor to the interfacial region without affecting the helix stability (Fig. 1). Due to its shorter side chain, Asp728, which is positioned in the centre of the TM domain of E2, was not able to anchor to the interface region. Moreover, its strained conformation led to disruption of the α-helical conformation of the N-terminal half of the E2 monomer. In contrast the E1 helix segment was stable along the simulation time. Analogous simulations of H-segment monomers containing Lys and Asp amino acids in the middle of the TM segments, respectively, gave similar results (see Fig. S2) thus confirming our observations and providing further evidence that they are caused by the charged Lys370 and Asp728 residues. YFSMVGNWAK 370 VLVVLLLFAG 380 VD). The E2 TM domain was observed to contain 27 residues between two polar residues at both N-and C-termini (EYVV 720 LLFLLLADAR 730 VCSCLWMMLL 740 ISQ). These are Glu717 and Tyr718 at the N-terminus and Ser742 and Gln743 within the C-terminal region. The consensus from the secondary structure prediction methods agrees closely with the consensus of the MD simulations sequences (Fig. 2).

MD simulations of E1-E2 heterodimers

To investigate the dynamics of the TM domain of the modeled E1-E2 dimer structure (see methods section), we performed two MD simulations of the E1-E2 wild type heterodimer with different starting velocities. These were named WT1 and WT2 in the subsequent tables and figures. Both simulations resulted in similar stable final E1-E2 conformations (Fig. S4). Fig. S3 shows root-mean-square deviations (RMSDs) of each monomer in the simulations of the E1-E2 heterodimer with respect to the perpendicular starting conformation. The RMSD values of the entire structures stabilize after around 20 ns between 0.5 and 0.7 nm which is mainly due to a tilting motion of one peptide with respect to its initial perpendicular orientation in order to find an optimal position in the membrane environment. The tilting motion observed matches with the fact that secondary structure prediction assigned TM segments of 25 to 30 residue length (see above). RMSD analyses also indicate that both simulations of the E1-E2 heterodimer showed smaller fluctuations than the simulations of the E1 and E2 monomers.

The heterodimerization was clearly mediated by the salt bridge interaction of the charged Lys370 and Asp728 at the helix-helix interface. Table 1 shows average distances between the functional groups (atoms Lys370-NZ:HZ and Asp728-CG:OD) to measure the stability of the Lys-Asp ion-pair. The distance was found to be stable at 0.30 -0.32 nm in the WT1 and WT2 simulations. Due to the helical periodicity, Arg730, being two positions away from the central Asp728, pointed into the opposite position and faced the hydrophobic lipid bilayer to anchor to the lipids polar interface.

Apart from formation of the central ion pair, we also observed formation of additional interhelical H-bonds (see Table S1). This appears to be a novel finding related to the formation of the E1-E2 dimer. For the wild-type, about 1 ± 0.4 H-bonds are formed between Asn367 and Asp728.

Mutational analysis

MD simulations of E1-E2 single and double mutants were carried out to analyze the naturally occurring polymorphisms and to confirm the contributions of the conserved amino acids of the E1 and E2 TM segments. All three single mutants with a salt-bridge (R730K, K370R, and D728E) were set-up independently as for wild-type and maintained stable heterodimers during the simulations (Fig. 3) as for wild-type that are stabilized by an ion-pair interaction when started from a salt-bridged conformation. This behavior can be expected due to the conservative nature of the mutation. The charged residues of all single mutants with an ionpair interaction were in close atomic contact as for the wild-type (0.27 -0.34 nm distance) (Table 1). Also, the RMSD values are of similar magnitude than those of the wild-type simulations (Fig. S3). Interestingly, despite having a longer side chain than Asp, the replaced Glu residue of the D728E mutant showed the shortest average distance (0.27 nm). The longer side chain of Glu apparently allows for an optimal contact with the Lys730 side chain. On the other hand, the K370R mutant had the largest average salt-bridge distances (0.34 nm), which may be caused by the long and bulky side chain of the mutated Arg.

The MD simulations of the three double mutants (N367L & K370L, D728L & R730L, G354A & G358A) resulted in different conformations with intact TM helices (Fig. 3 and Fig. 4). The largest structural fluctuations compared to the starting structure were observed for the D728L & R730L mutant (Fig. S3c). Mutating the conserved residues N367 and K370 in E1, and D728 and R730 in E2 led to a partial separation of the two helices, see C distances in Table 2 and Fig. 4. However, the double mutant G354A & G358A was as stable as the wildtype during the simulation (Fig. 4c), probably due to the presence of the salt-bridge interaction.

The tight heterodimerization of the ion-pair stabilized helix dimers is also reflected by a close distance between the centers of mass of the two helices (Table 2). All single mutants with a salt-bridge (K370R, R730K and D728E), and the G354A & G358A double mutant showed close distances (1.08 -1.23 nm) as the two wild type simulations (1.08 -1.10 nm). On the other hand, the double mutants (N367L & K370L and D728L & R730L) showed much larger separations (1.31 -1.76 nm) reflecting the absence of an ion pair interaction or of other stabilizing inter-helical interactions (Table 2 and Fig. 4). Most simulations showed tilting angles of the two helices around 40º to 60º. The only exception is the E1 monomer in the double mutant D728L & R730L that is almost straight (13º) as this mutant dissociated.

As discussed before, additional inter-helical H-bonding was observed to stabilize the helix dimer and prevent helix kinking or partial unfolding. For the mutants with a salt bridge (R730K and G354A & G358A) a similar average number of inter-helical H-bonds was found as for wild-type (0.91 -1.02) (Fig. 5). The highest average number of inter-helical H-bonds resulted from the D728E mutant (1.86) followed by the K370R mutant (1.30) indicating more favorable contacts.

To clarify the function of the salt bridge interaction at the helix-helix interface, we mutated Lys370 to Ala which removes the ability to form an ion-pair between the helix monomers.

Interestingly, even in the absence of an ion-pair interaction, the K370A mutant was heterodimerized during the simulation. The distance between the helix monomers is the closest one found (0.97 nm) and the average number of H-bond interactions between both monomers was similar to the other heterodimerized conformations (1.21 nm) (Table S1 and Fig. 5). We found that Asp728 made very stable contacts with Arg730 so that Arg730 turned around and was now located at the helix-helix interface. Arg730 then formed an H-bond with Asn367 with 72% occupancy. This behavior caused local unfolding in the centre of the E2 of K370A mutant (Fig. S4). On the other hand, if Asp728 is mutated into Ala, no rotation of Arg730 in E2 was observed (Fig. S5).

Comparison of MD structures versus the NMR structure

As the only NMR structure available for the HCV envelope glycoproteins is a segment of E1 consisting of 21 residues [START_REF] Beeck | The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization[END_REF] with the PDB-code 1EMZ.pdb, RMSD analyses were done on the same segment during the MD simulations with respect to the NMR structure (Fig. 6). We compared the RMSD of the backbone atoms of the E1 TM segment (Gly350 -Lys370). The central part (354-370) formed a well defined α-helix in the simulation. The average conformation from residues 359-367 in the simulation of the E1-E2 dimer has an RMSD of 0.06 nm compared to the NMR structure (Fig. 6), whereas the RMSD of the structural ensemble derived from NOE restraints was 0.03 nm [START_REF] Beeck | The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization[END_REF]. Although we found a somehow larger RMSD of the 21 residue segment (Gly350 to Lys370) of 0.15 nm, these deviations are still smaller to the variation within the NMR ensemble of 24 structures (0.29 nm) [START_REF] Beeck | The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization[END_REF]. Thus, the E1 helical conformation derived from MD simulations is quite similar to the conformation determined by NMR in trifluoroethanol (TFE) [START_REF] Beeck | The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization[END_REF]. As expected, the largest RMS fluctuations were observed for the residues at the helix ends near the membrane bilayer interface. Although previous NMR studies did not show the segment Gly354 to Gly358 to be α-helical, we found that this segment is in stable α-helical conformations in the MD simulations on the investigated timescale.

The segment between Gly354 and Gly358 was observed to be in the TM region during the MD simulations (Fig. 2), but the GxxxG motif was not located at the helix-helix interface (see discussion below).

Discussion

In the viral Flaviviridae family [START_REF] Cocquerel | Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins[END_REF], at least one positively charged residue is highly conserved in both putative TM domains of the envelope glycoproteins. Polymorphism analysis of the conserved residues G354, G558, Lys370, Asp728 and Arg730 in all HCV genotypes indicates that mutations rarely occur at these particular sites. The data analyzed in this study confirms previous findings [START_REF] Cocquerel | Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins[END_REF][START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF] that these conserved residues are crucial for the viral specific functions of the E1 and E2 envelope proteins.

Having a charged residue in the middle of a TM domain would be energetically unfavorable for an isolated -helix. Instead, these residues would probably appear neutral by shifting their pKa values at an energetic expense or they try to position their charged side-chain into the polar head-group region [START_REF] Yoo | Does Arginine remain protonated in the lipid membrane? Insights from microscopic pKa calculations[END_REF]. In spite of these considerations, an experimental study by Hessa et al. [START_REF] Hessa | Recognition of transmembrane helices by the endoplasmic reticulum translocon[END_REF] proved that single transmembrane segments with a polar or charged residue in the middle of the domain were able to be inserted as membrane proteins via Sec61 translocon.

Simulation of monomers

The MD simulations of helix monomers revealed that the charged Lys370 and Asp728 had different effects on the TM segments of E1 and E2 monomers, respectively, if they were placed as isolated helices in a membrane lipid bilayer. The TM segment of the E1 helix was stable during the simulation, whereas the N-terminal half of the TM segment of E2 was disrupted, possibly due to the shorter side chain of Asp730. Subsequent MD simulations of Hsegments containing a charged Lys or Asp showed a similar behavior. Asp residues were previously shown to induce stronger distortions in α-helices compared to basic residues [START_REF] Johansson | Amino-Acid Solvation Structure in Transmembrane Helices from Molecular Dynamics Simulations[END_REF].

Moreover, Hessa et al. [START_REF] Hessa | Recognition of transmembrane helices by the endoplasmic reticulum translocon[END_REF] found that the biological apparent insertion free energy scale showed the highest value (3.49 kcal mol -1 ) when Asp was placed in the middle of the TM domain of the H-segment compared to other amino acids.

Simulation of E1-E2 wild type

Dubuisson and co-workers suggested that the E1 and E2 TM helices are inserted cooperatively into the lipid bilayer based on mutagenesis results [START_REF] Cocquerel | Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins[END_REF][START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF][START_REF] Ciczora | Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer[END_REF]. Here, we put this hypothesis on stable energetic and structural grounds based on extensive MD simulations of wild-type and mutant heterodimers. Indeed, favorable salt-bridge and H-bonding interactions between the TM segments of E1 and E2 contribute to stabilization of the dimer conformation in lipid bilayers. As mentioned above, the E2 monomer containing the charged Asp728 unfolded partially during MD simulations. However, when simulated as part of the E1-E2 heterodimer, the E2 maintained its stable α-helical structure. This is a strong indication that the dimer conformation of the E1 and E2 envelope glycoproteins is a favorable arrangement even in a hydrophobic environment. If the ion-pair of Lys370 and Asp728 at the helix-helix interface is already established in the translocon or near to its exit, as suggested before [START_REF] Cocquerel | Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins[END_REF], this should facilitate the entry of the E1-E2 heterodimer into the lipid bilayer environment [START_REF] Cocquerel | Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins[END_REF]. Moreover, the stability of this ion-pair interaction may serve as a kinetic barrier against the E1-E2 heterodimer dissociation. This role is in agreement with the suggested function of one or more hydrophilic residues which were observed in other TM domains to be responsible for the ER retention [START_REF] Bonifacino | Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum[END_REF]. Since Lys370 and Asp728 were located at the helix-helix interfacial region in our model, Arg730 was positioned oppositely where it faced the lipid tails. To optimize its position, the positively charged side chain of Arg730 oriented its guanidinium group towards the polar region of the lipid bilayer. Molecular simulations previously showed that Arg adjust energetically in the membrane environment and its long side chain is likely to remain positively charged in lipid bilayers [START_REF] Yoo | Does Arginine remain protonated in the lipid membrane? Insights from microscopic pKa calculations[END_REF]. Also, the charged Arg residues in the voltage sensor domain of potassium channels behaved such that the Arg residues were stabilized by the polar head groups of lipids and water molecules [START_REF] Freites | Interface connections of a transmembrane voltage sensor[END_REF].

The atomistic observation from the MD trajectories also reveals a so far unreported interhelical H-bond contributed by Asn367 which also contributes to stabilize the structure of the E1-E2 heterodimer. Inter-helical H-bonds are known to be of particular importance for the formation of secondary or tertiary structure in the hydrophobic membrane center with low dielectric environment [START_REF] Joh | Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins[END_REF]. A recent report from von Heijne and co-workers also demonstrated that engineered TM domains with inter-helical interactions mediated by polar residues are more efficiently inserted into the lipid bilayer [START_REF] Meindl-Beinker | Asn-and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly[END_REF].

We now discuss the relevance of the sampled dimer conformations. At the start of each simulation, the two helices were arranged parallel to the membrane normal with the ionpairing residues facing each other. The simulations then showed that these initial orientations are stable on the time scale of the simulations what supports the experimental finding that the E1-E2 helices are inserted by the translocon with the ion-pair already formed. A situation of an E1-E2-dimer with one or both of the helices turned by 90 or 180 degrees, for example, likely never occurs in nature. However, as we clearly did not sample the range of possible orientations, we cannot address whether the generated models correspond to the thermodynamically most favorable orientation of the isolated E1-E2 helices. That would require sampling a large range of orientations over long simulation times what is currently infeasible by plain MD simulations in explicit bilayers. The simulated 100 ns time scale is clearly not sufficient for entire helices to turn around their axis in a lipid bilayer. As an alternative, using an implicit-solvent representation of the membrane [START_REF] Im | An implicit membrane generalized Born theory for the study of structure, stability, and interactions of membrane proteins[END_REF][START_REF] Tanizaki | A generalized Born formalism for heterogeneous dielectric environments: application to the implicit modeling of biological membranes[END_REF][START_REF] Bu | De Novo Prediction of the Structures of M. tuberculosis Membrane Proteins[END_REF][START_REF] Bu | Membrane assembly of simple helix homo-oligomers studied via molecular dynamics simulations[END_REF][START_REF] Lazaridis | Effective energy function for proteins in lipid membranes[END_REF][START_REF] Mottamal | Energetics of the native and non-native states of the glycophorin transmembrane helix dimer[END_REF][START_REF] Ulmschneider | A generalized born implicit-membrane representation compared to experimental insertion free energies[END_REF] would allow for a more complete sampling and for faster orientational relaxation. Besides, replica-exchange simulations allow to speed up the penetration into membranes and re-orientations [START_REF] Nymeyer | Folding is not required for bilayer insertion: replica exchange simulations of an a-helical peptide with an explicit lipid bilayer[END_REF]. However, it is not clear from experiment what is the thermodynamically most favorable state of the two isolated helices because the experiments [START_REF] Cocquerel | Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins[END_REF][START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF][START_REF] Cocquerel | Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins[END_REF] were always performed on the full E1-E2 proteins with the external domains present. So it is in fact possible that dimerization is only stable with the external domains present.

Simulations of heterodimer mutants

To further confirm the location of the important residues of the TM domains of E1 and E2, we carried out simulations of several conservative mutants. The single mutants R730K, K370R and D728E, which contain a salt bridge between the helices, were all heterodimerized during the simulations with stable α-helical conformations (Fig. 3). These results strongly support that the ion pair interaction between the particular charged amino acids is responsible for the inter-helix interaction. The distances between the charged side chains of these single mutants are similar to the E1-E2 wild-type (0.27-0.34 nm). These results are in perfect agreement with experimental findings which reported that R730K and D728E mutants form heterodimers similarly to the E1-E2 wild-type [START_REF] Ciczora | Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer[END_REF]. Our analysis of natural polymorphisms indicates that the R730K mutant occurred once in genotype 4 of HCV which is very rare. On the other hand, the R730K mutants only resulted in a slightly reduced incorporation and infectivity of E1-E2 proteins into HCVpp compared to the E1-E2 wild-types. Here, Lys led to a similar dimer conformation than with Arg730 since both are positively charged amino acids. We note, however, that in this structural model, with a salt-bridge stabilized heterodimer, Arg730 is not located at the helix-helix interface and its mutation should not affect dimerization. In contrast, the infectivity of the D728E mutant was strongly reduced, however without affecting the formation of heterodimers [START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF][START_REF] Ciczora | Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer[END_REF]. This indicates that even conservative mutations that can be expected to maintain the salt-bridge interaction may lead to different biological function such as viral entry. One may therefore speculate that placing the longer side chain of Glu between the two helices may affect the helical packing although this is not apparent in the simulations.

In a second set of mutant simulations, we mutated Lys370 to Ala to investigate the effect of removing the salt-bridge on the E1-E2 heterodimerization. Interestingly, the K370A mutant still managed to remain heterodimerized during the simulation. Arg730 turned around to interact with Asn367 so that the average number of H-bonds between the E1-E2 helices increased compared to the wild-type. Inter-helix H-bonding of polar amino acids was recently studied experimentally by systematically constructing H-segment dimers [START_REF] Meindl-Beinker | Asn-and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly[END_REF]. This work concluded that polar inter-helix interactions increase the translocon insertion efficiency of both helices. However, this rotation of Arg730 caused severe rearrangements of the backbone conformation in the central part of the E2 TM helix. In the experimental setting, mutation of Lys370 led to reduced heterodimerization to about 50% [START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF]. On the other hand, mutation of Asp728 severely reduced the E1-E2 heterodimer biogenesis to about 10 to 20% when replaced with hydrophobic amino acids such as Leu, Ala or Trp [START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF][START_REF] Ciczora | Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer[END_REF][START_REF] Beeck | The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization[END_REF].

In a third, final set of double mutants, the central residues at 367 and 370 or 728 and 730 were replaced by leucine residues. Both double mutants resulted in significantly enlarged distances between the TM helix monomers compared to the wild-type and to the mutants containing a salt-bridge. The E2 double mutant D728L/R730L residue gave a larger average distance (1.76 Å) between the helix dimer than the E1 double mutant N367L/K370L (1.31 Å). Interestingly, these results are again in line with the experimental study, which reported a differential effect of both double mutations [START_REF] Cocquerel | Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins[END_REF]. For soluble proteins, there exist several computational methods that can qualitatively predict the effect of protein mutations on their stability [START_REF] Benedix | Predicting free energy changes using structural ensembles[END_REF][START_REF] Potapov | Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details[END_REF]. It is certainly feasible to transfer these methods to the area of TM proteins. Up to now, however there is a lack of quantitative experimental data on the thermodynamics stability of TM helix bundles and respective mutants against which such computational methods can be calibrated.

GxxxG motif

For the GpA homodimer, the GxxxG motif at the helix dimer interface has been shown to play an important role for the homodimerization [START_REF] Langosch | Dimerisation of the Glycophorin A Transmembrane Segment in Membranes Probed with the ToxR Transcription Activator[END_REF][START_REF] Senes | Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with ßbranched residues at neighboring positions[END_REF]. Also for the E1-E2 heterodimer, mutating either Gly354 or Gly358 impaired the E1-E2 assembly [START_REF] Ciczora | Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry[END_REF]. In the structural model of the E1-E2 heterodimer developed in this study, however, the Gly350, Gly354 and Gly358 residues are not located at the helix interfacial region. Therefore, we did not observe any possible interaction between the GxxxG motif of E1 and the residues from the TM domain of E2. However, this does not exclude the probability of GxxxG segments to heterodimerize at the ectodomain region of the E2 glycoprotein. The E1 helix conformation agrees nicely with an experimental structure of E1 solvated in TFE. Whereas the NMR analysis revealed an unwinding of the N-terminal end of the E1 helix between Gly354 and Gly358, this region stayed intact in an α-helical conformation during the heterodimer simulations.

Conclusions

This study puts the assignment of the TM domains of E1 and E2 on a firm basis. The structural model explains the roles of the highly conserved positively and negatively charged residues in the family of Flaviviridae glycoproteins. The stability of the ion pair supports the hypothesis [START_REF] Cocquerel | Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins[END_REF] that membrane insertion at the translocon complex occurs cooperatively for the E1 and E2 helices. Otherwise, having unpaired charged residues in the middle of a membrane bilayer would be thermodynamically unfavorable. The emerging structural model of the helix dimer shows the importance of the Lys370-Asp728 ion pair at the center of the lipid bilayer for the formation of the E1-E2 heterodimer.

Fig. 2

 2 Fig. 2 shows the consensus TM assignment based on the analysis of all MD simulations. Both the MD simulations of the individual helices and of the heterodimers indicate that the TM domain of E1 consists of 29 residues ranging from Gly354 to Gly380 (G 354 VLAGIA 360

Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6

 13456 Fig. 1 Final snapshots of MD simulations from the E1 and E2 monomers (a) E1 TM segment with charged Lys370 and (b) E2 TM segment with charged Asp728. Lipid tails and ions are not shown for clarity. The charged Lys370 and Asp728 are shown as stick representation

  Figure 1 Click here to download colour figure: Fig1.eps

Table 2

 2 Structural parameters for the data between 80 and 100 ns of the MD simulations of E1-E2 wild-type heterodimer and E1-E2 mutants  Average number of H-bonds per time frame. An H-bond (D-H•••A) is defined to have an H•••A distance less than 3.5 Å and a D-H•••A angle greater than 120º  Interacting residues at the helix-helix packing are shown for each dimer

		CA distances	TM	TM
		between E1 and E2	Tilt Angles E1	Tilt Angles E2
	wild-type E1-E2	helices	( ° )	( ° )
		(nm)
	wild-type 1 (K•••D)	1.10  0.03	41.3  5.1	40.8  4.7
	wild-type 2 (K•••D)	1.08  0.02	47.3  4.0	50.2  4.1
	Single mutants with a salt-bridge
	R730K (K•••D)	1.08  0.03	50.0  3.7	67.2  1.4
	K370R (R•••D)	1.17  0.03	59.5  2.2	42.8 4.1
	D728E (K•••E)	1.23  0.03	41.0  3.1	41.9  1.7
	Single mutants without salt-
	bridge
	K370A	0.97  0.02	60.0  3.0	Kinked
	D728A	1.07  0.03	40.4  4.1	Kinked
	Double mutants
	D728L & R730L	1.76  0.04	13.3  4.2	43.3  4.1
	N367L & K370L	1.31  0.05	47.7  3.9	52.0  5.2
	G354A & G358A (K•••D)	1.11  0.04	45.7  4.0	45.1  3.7

Acknowledgments

We are grateful for the financial support of the Universiti Teknologi MARA, Malaysia and for the research facilities from The Center for Bioinformatics Saarland University. We also thank the Deutsche Forschungsgemeinschaft Graduate School 1276/1 "Structure Formation and Transport in Complex Systems" for collaboration.

Tables

 Table 1 Salt-bridges between E1-E2 wild-types and mutants. Given are average values for the data between 80 and 100 ns of the MD simulations  A salt-bridge distance is calculated by averaging the distances between the hydrogen and oxygen atoms from an amine/carboxyl group of E1 and the carboxyl group of E2  Only mutants with a predicted salt-bridge at the helix-helix interface are calculated