Graziele Oliveira

Lorena Silva

Thiago Leão

Said Mougari

Flávio Guimarães Da Fonseca

Erna Geessien Kroon

Bernard La Scola

Jônatas Santos

Tupanvirus-infected amoebas are induced to aggregate with uninfected cells promoting viral dissemination

The discovery of giant viruses in the last years has fascinated the scientific community due to virus particles size and genome complexity. Among such fantastic discoveries, we have recently described tupanviruses, which particles present a long tail, and has a genome that contains the most complete set of translation-related genes ever reported in the known virosphere. Here we describe a new kind of virus-host interaction involving tupanvirus. We observed that tupanvirus-infected amoebas were induced to aggregate with uninfected cells, promoting viral dissemination and forming giant host cell bunches. Even after mechanical breakdown of bunches, amoebas reaggregated within a few minutes. This remarkable interaction between infected and uninfected cells seems to be promoted by the expression of a mannose receptor gene. Our investigations demonstrate that the pre-treatment of amoebas with free mannose inhibits the formation of bunches, in a concentration-dependent manner, suggesting that amoebal-bunch formation correlates with mannose receptor gene expression. Finally, our data suggest that bunch-forming cells are able to interact with uninfected cells promoting the dissemination and increase of tupanvirus progeny.

The recent discovery of tupanvirus, one of the largest and most complex viruses isolated to date, has reinforced the structural and genomic complexity of the giant viruses [START_REF] Abrahão | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] . Tupanviruses have been isolated from soda lakes, known as an extreme aquatic environments, and from ocean sediments collected at a depth of 3000 meters (m) [START_REF] Abrahão | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF][START_REF] Sorokin | Microbial diversity and biogeochemical cycling in soda lakes[END_REF] . Phylogenetic analyses have shown the clustering of the tupanvirus with members of the family Mimiviridae. However, there are many peculiarities that make the tupanviruses unique entities in the known virosphere. Since its first observation, tupanviruses showed remarkable morphological characteristics; it has optically visible particles that average about 1.2 µm in size and can reach lengths up to 2.3 µm 1 . Tupanvirus has the largest host range described so far among amoebal-infecting giant viruses and can causes a shutdown of host rRNA that is likely related to host-nucleolus degradation [START_REF] Abrahão | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF][START_REF] Scola | A giant virus in amoebae[END_REF][START_REF] Boyer | Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms[END_REF][START_REF] Philippe | Pandoraviruses: Amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes[END_REF][START_REF] Legendre | Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology[END_REF][START_REF] Legendre | In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba[END_REF][START_REF] Reteno | an Asfarvirus-Related New Lineage of Giant Viruses Infecting Amoebae[END_REF][START_REF] Andreani | Cedratvirus, a double-cork structured giant virus, is a distant relative of pithoviruses[END_REF] . The tupanviruses replication cycle is similar to those for other mimiviruses, in which viral particles attach to the host-cell surface and enter through phagocytosis. The viral inner membrane then fuses with the phagosome membrane, releasing the genome. A viral factory (VF) is formed, where particle morphogenesis occurs; the cycle ends with cell lysis and the release of progeny viruses [START_REF] Abrahão | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF][START_REF] Andrade | Filling knowledge gaps about mimivirus entry, uncoating and morphogenesis[END_REF] .

The study of the tupanvirus genome further aroused the interest of virologists, not only due of its large size (~1.5 Mb), but also because these viruses show the largest translational apparatus described. It is composed of up to 70 tRNA, 20 aminoacyl-tRNA synthetases (aaRS), 11 factors associated with translation, and factors related to tRNA/mRNA maturation and ribosome protein modification [START_REF] Abrahão | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] . In addition to the robust translation apparatus, tupanvirus also contains a gene encoding mannose-specific lectin, also called mannose-binding protein (MBP) [START_REF] Abrahão | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] . Interestingly, previous studies revealed that Acanthamoeba castellanii expresses an MBP and that free-mannose can inhibit the adhesion of A. castellanii to surfaces, suggesting that the MBP plays a role in the pathogenesis of Acanthamoeba infection [START_REF] Allen | Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast[END_REF][START_REF] Yang | Pathogenesis of acanthamoeba keratitis: Carbohydrate-mediated host-parasite interactions[END_REF][START_REF] Caot | Role of carbohydrate-mediated adherence in cytopathogenic mechanisms of Acanthamoeba[END_REF][START_REF] Garate | Cloning and characterization of a novel mannose-binding protein of Acanthamoeba[END_REF][START_REF] Garate | Biochemical characterization and functional studies of Acanthamoeba mannose-binding protein[END_REF][START_REF] Kim | Functional roles of mannose-binding protein in the adhesion, cytotoxicity and phagocytosis of Acanthamoeba castellanii[END_REF] .

In this work, we describe that tupanviruses induces the expression of cellular and viral mannose receptor genes, which are well known for promoting the adhesion of amoebas to other cells. Tupanvirus promotes the formation of large bunches of infected and non-infected cells. In this context, we have hypothesized that infected cells act like "zombies, " controlled by tupanviruses, searching and attaching to uninfected cells, and improving the chances of the newly formed viral progeny to find a new host cell in which to propagate.

Materials and Methods

Cell culture, viral production and titration. Acanthamoeba castellanii cells (ATCC 30010) were cultivated in peptone-yeast extract with glucose (PYG) medium supplemented with 25 mg/ml amphotericin B (Fungizone; Cristalia, São Paulo, Brazil), 500 U/ml penicillin (Schering-Plough, Brazil) and 50 mg/ml gentamicin (Schering-Plough, Brazil). Cell culture flasks (175 cm 2 ) containing 7 × 10 6 A. castellanii cells were infected with tupanvirus strain soda lake at an multiplicity of infection (M.O.I) of 0.1 and incubated at 32 °C. After the appearance of a cytopathic effect and evidence of cell lysis, the cells and supernatants were collected and the viruses were purified through ultracentrifugation with a 22% sucrose cushion at 36,000 × g for 30 min. After viral purification, the virus titers were determined using the endpoint method [START_REF] Reed | A simple method of estimating fifty per cent endpoints[END_REF][START_REF] Abrahão | Mimiviruses: Replication, purification, and quantification[END_REF] .

Cytopathic effect and cycle characterization.

To investigate the cytopathic effect of tupanvirus in A. castellanii cells by optical microscopy, 25 cm 2 cell culture flasks containing 1 × 10 6 A. castellanii cells were infected with tupanvirus at an M.O.I of 0.01 and 10, incubated at 32 °C and observed at different timepoints post-infection for 72 hours. The A. castellanii cells were viewed under an optical microscopy at 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 24, 32, 48 and 72 hours post-infection (h.p.i), and the more relevant times were presented. A infection control was developed using acanthamoeba polyphaga mimivirus (APMV), in which the same conditions were used at an MOI of 10. Uninfected A. castellanii cells (control) was also observed. A one-step growth curve was constructed using 25 cm 2 flasks in duplicate at an M.O.I of 10 (Fig. 1B). At different timepoints, the infected A. castellanii cells and supernatants were collected, tittered and calculated using the end point method.

For immunofluorescence, A. castellanii cells were infected with tupanvirus at an M.O.I 10 and at 0 (immediately after contact between tupanvirus and A. castellanii), 1, 8, 12, 16 and 24 h.p.i., approximately 2 × 10 5 cells were collected and centrifuged at 800 × g for 10 min. The pellet was resuspended in 50 µL of Page's amoebae saline (PAS), and the cells were attached to a slide via cytospin and fixed in methanol for 15 min. After fixation, cells were incubated with 3% bovine serum albumin (BSA)-PAS for 30 min, followed by three rinses with 0.1% PAS-Tween. Cells were then stained with polyclonal anti-tupanvirus whole particle antibody produced in mouse (1:400 diluted in 3% BSA-PAS) for 1 h at 37 °C, followed by three rinses with PAS-Tween 0.1%. After a 1 h incubation with an anti-mouse secondary antibody (1:400 diluted in 3% BSA-PAS), one drop of 0.01% Evans Blue (Sigma), which was sufficient to cover the cells, was added and incubated for 15 min at 37 °C, followed by three rinses with PAS-Tween 0.1%. Uninfected cells (control) were also fixed and prepared as described. Fluorescently labeled cells were observed using an Axio Imager Z2-Apotome 2 microscope (Zeiss). The Zen Lite software from Zeiss microscopy was used for image processing.

For transmission electron microscopy (TEM), the cells were pelleted for 10 min at 800 × g. The pellet was washed twice with 0.1 M phosphate buffer (pH 7.4) and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h at room temperature. The pellet was then washed twice with 0.1 M phosphate buffer and suspended in the same buffer. After, amoebas were embedded in Epon resin the using a standard method, as follows: 2 h of fixation in 2% osmium tetroxide, five washes in distilled water, overnight incubation in uranyl acetate 2% at 2-8 °C, two washes in distilled water, 10 min dehydration in increasing ethanol concentrations (35, 50, 70, 85, 95 and 100%), 20 min incubation in acetone and embedding in EPON resin. Ultrathin sections were subsequently analyzed under TEM (Spirit Biotwin FEI-120 kV).

For scanning electron microscopy (SEM) assays, 50 µL of supernatant was added to round glass coverslips coated with poly-L-lysine and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 h at room temperature. The samples were washed three times with 0.1 M cacodylate buffer and post-fixed with 1% osmium tetroxide for 1 h at room temperature. After a second fixation, the samples were washed three times with 0.1 M cacodylate buffer and immersed in 0.1% tannic acid for 20 min. The samples were then washed in cacodylate buffer and dehydrated for 10 min by serial passages in ethanol solutions (35, 50, 70, 85, 95 and 100%). Samples were subsequently subjected to critical point drying using CO 2 , placed in stubs and metalized with a 5 nm gold layer. The analyses were completed using SEM (FEG Quanta 200 FEI).

Mannose binding protein transcripts analyses and mannose assays.

To evaluate the effect of mannose during tupanvirus infection, 1 × 10 6 A. castellanii cells were infected with tupanvirus at an M.O.I of 10 in PAS medium containing different concentrations of alpha-D-mannopyranoside (25, 50, 100, 400, 600 and 1000 mM) and incubated at 32 °C. The cells were observed under an optical microscope at 8 h.p.i. In addition, the culture flasks were divided (2.5 cm 2 /field) and the counting of bunches was performed under an optical microscope. Cells infected by tupanvirus in PAS medium without mannose and PAS medium with 600 mM of mannose were titrated at 24 h.p.i. As controls, uninfected A. castellanii cells either cultivated in PAS medium without mannose or PAS medium containing 600 mM of mannose were used. To investigate the role of the mannose in the expression pattern of the A. castellanii and tupanvirus mannose binding protein genes, total RNA was purified from 5 × 10 5 A. castellanii cells that had been infected with tupanvirus (M.O.I of 10) in the presence or absence of mannose (600 mM) at 1, 2, 4, 6, or 8 h.p.i. using the RNAeasy Mini Kit (Qiagen). Uninfected A. castellanii cells were used as controls. An control of infection using APMV was used to evaluate the expression of cellular MBP in amoebae infected with APMV, total RNA was purified from 5 × 10 5 A. castellanii cells that had been infected with APMV (M.O.I of 10) at 1, 2, 4, 6, or 8 h.p.i. The samples were previously treated with DNAse (Invitrogen) and reverse transcribed using M-MLV Reverse Transcriptase (200 U/L; Thermo Fisher Scientific), according to the manufacturer's instructions. The resulting cDNAs were used as a template in a StepOne thermocycler (Applied Biosystems) quantitative polymerase chain reaction (qPCR) assay to target the mannose receptor genes of the A. castellanii using oligo-nucleotide primers sequences Forward MBP AC _ CCAGTTCAATACCACCGGCT and Reverse MBP AC _ GTGGGGCAGTCCTTGTAGTC, tupanvirus (Forward MBP TPV _ ACGATTCAAGCCAGACACAA and Reverse MBP TPV _ GAGGAGTACCTTGCCCTGTTG) and APMV (Forward MBP APMV _ ACCCGCACCAGAAAGTCAAT and Reverse MBP APMV _ GGAGTCGGATTTGACGGTGT). The accession numbers of the genomes used to design the primers were AY604039.1, KY523104.1 and NC_014649.1 respectively. The thermal cycling conditions used were: one cycle at 95 °C for 10 min, 40 cycles at 95 °C for 10 sec and 60 °C for 40 sec; a melting curve analysis at 95 °C for 15 sec and 58 °C for 15 sec, and a final cycle was completed at 95 °C for 15 sec. The results were applied to the 2 -(Δct) method and analyzed by StepOne software. The assays were carried out in biological duplicate. Graphs were created using GraphPad Prism software (v7.0 for Windows).

Evaluation of the interaction between uninfected amoebas and bunches. To evaluate whether bunches are able to interact with uninfected cells, 1 × 10 6 A. castellanii cells in 25 cm 2 culture flasks were infected with tupanvirus (M.O.I. of 10) and at 16 h.p.i. the bunches were collected and used to inoculate uninfected A. castellanii cells in suspension in a separate 25 cm 2 culture flasks. After incubation for 30 min at 32 °C the non-adherent cells were removed from the culture flasks and the cells that remained adhered were counted. As a control, 16 h.p.i. APMV-infected amoebae were used in the same procedure. The experiment was performed in triplicate. The results were plotted on a column graph, and the statistical significance was calculated using a two-tailed Student's t test, performed using Graph Pad Prism. In addition, the uninfected cells that interacted with bunches and were carried by them (the cells that did not adhere to the culture flasks again) were assayed using IF and SEM, as described above. To evaluate the importance of bunches with fresh cells for propagation of tupanvirus, 7 × 10 6 A. castellanii cells were infected (M.O.I of 10) and at 16 h.p.i. the medium was removed; the bunches were washed and used to infect new A. castellanii cells and as a control, the bunches were also added to a flask containing fresh medium. At 36 h.p.i. the supernatants were collected and titrated as described above. Graphs were constructed using GraphPad Prism software (v7.0 for Windows).

Results and Discussion

Characterization of the tupanvirus cytopathic effect: a giant virus inducing cell aggregation. In order to characterize the cytopathic effect (CPE) triggered by tupanvirus, A. castellanii cells were infected at an M.O.I. of 10 and observed at up to 72 h.p.i. The cells became rounded and lost their adherence; this effect became more evident at 4 h.p.i. (Fig. 1A). Interestingly, tupanvirus exhibits a peculiar CPE that is characterized by amoebae aggregates, called "bunches. " The formation of bunches starts at approximately 6 h.p.i. and becomes more evident at 8, 12 and 16 h.p.i., when almost all the cells are seen incorporating into large bunches (Fig. 1A). From 24 h.p.i., despite the presence of bunches, cell lysis is more evident and is concomitant with the reduction of bunches size. At 32 h.p.i., the complete disaggregation of bunches was observed, and at 48-72 h.p.i. the majority of amoebae were lysed (Fig. 1A). The formation of bunches was also observed when a low M.O.I. (0.01) was used, although there was a temporal delay in bunch appearance (Suppl. Fig. 1). The CPE triggered by tupanvirus is unlike that of the other mimivirus, as well as other known giant amoebal viruses. Acanthamoeba polyphaga mimivirus, the prototype of the genus Mimivirus, triggers rounding and lysis in A. castellanii cell cultures, being a CPE commonly described for other giant viruses (M.O.I. of 10) (Suppl. Fig. 2).

Different timepoints during infection (at an M.O.I. of 10) were selected for an immunofluorescence assay (IF) and transmission/scanning electron microscopy (TEM and SEM). IF assays using anti-tupan antibody revealed that at 0 and 1 h.p.i. tupanvirus particles appeared attached to amoebas (Fig. 2), even after the cell washing step, suggesting virion fibrils early interactions with host-cells, as demonstrated for other mimiviruses [START_REF] Rodrigues | Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire[END_REF] . VFs start to be detected by IF in amoebas' cytoplasm using anti-tupanvirus whole-particle antibodies at 12 h.p.i. (Fig. 2). Curiously, even at high a M.O.I. it was not possible to observe a synchronous infection of all cells, something that was peculiarity related to tupanvirus and that needs further investigation. Remarkably, we observed amoebas with and without VFs or viral particles in the cytoplasm by IF, especially at 12 h.p.i., suggesting that early bunches are formed by the association of cells at different stages of the viral replication cycle (Fig. 2). Even at 16 h.p.i., when the viral titer reached its plateau (Fig. 1B) and the significant majority of cells already presented viral factories and particles (Fig. 2), we were able to find a very few cells with no detectable signs of tupanvirus by IF. By SEM (Fig. 3A) and TEM (Fig. 3B), it was possible at 16 h.p.i. to observe bunches of amoebas with damaged cells filled with or releasing tupanvirus particles, which were associating with cells with an intact plasma membrane and only a few particles inside them (Fig. 3).

Early amoebal bunches reform after mechanical separation of cells.

Another phenomenon that drew our attention was the occurrence of bunch reformation after mechanical breakdown (Fig. 4). Initially, we observed that early and late bunches could be easily disaggregated by homogenization using a pipette or by vortexing. However, 30 min after disaggregation, cells from early bunches (12 h.p.i.) were attracted to each other, forming large clusters of cells and assuming an appearance similar to early bunches before cell separation. In contrast, reformation of late bunches (24 h.p.i.) was not observed. In addition, using light microscopy we observed that some cells belonging to late bunches were larger in size, which was especially obvious after the disaggregation procedure (Fig. 4). The lack of bunch reformation at late timepoints indicates that at 24 h.p.i., cells were already killed by the viral infection, and a simple homogenization was able to permanently disaggregate the cells that formed late bunches. To confirm this hypothesis, we performed SEM on late bunches. Interestingly, we observed cells with the plasma membranes almost completely degraded and filled with intricate networks of tupanvirus particles and cell debris (Fig. 5).

Taken together, these results indicate that tupanvirus promotes the formation of large aggregates of amoebas, which are able to re-aggregate as long as cells are still alive. These findings motivated us to investigate possible factors that may interfere in bunch formation and the biological relevance of tupanvirus-promoted bunches.

Amoebal-bunch formation correlates with increased mRNA levels of mannose binding protein genes. Acanthamoeba spp. is widely distributed in the environment and can cause human diseases, mainly associated with immunocompromised patients and contact lenses wearers. Furthermore, Acanthamoeba spp. has a relevant role in ecosystems, due to its ability to act as a host for microbial pathogens [START_REF] Niederkorn | The pathogenesis of Acanthamoeba keratitis[END_REF][START_REF] Marciano-Cabral | Acanthamoeba spp. as agents of disease in humans[END_REF][START_REF] Khan | Acanthamoeba: Biology and increasing importance in human health[END_REF][START_REF] Siddiqui | Biology and pathogenesis of Acanthamoeba[END_REF] . Mannose receptor proteins are well known for promoting interactions between amoebas and other cells. In this way, previous studies revealed that mannose-binding protein (MBP) present on the surface of Acanthamoeba is one of the major virulence proteins and may be essential for pathogenesis and invasion of host cells [START_REF] Allen | Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast[END_REF][START_REF] Garate | Biochemical characterization and functional studies of Acanthamoeba mannose-binding protein[END_REF][START_REF] Garate | In vitro pathogenicity of Acanthamoeba is associated with the expression of the mannose-binding protein[END_REF] demonstrated that the potential of Acanthamoeba pathogenicity is directly dependent on the level of MBP [START_REF] Garate | In vitro pathogenicity of Acanthamoeba is associated with the expression of the mannose-binding protein[END_REF] . In addition, previous studies showed that A. castellanii treated with mannose had reduced adhesion and cytotoxicity, strongly suggesting that its pathogenicity is associated with MBP [START_REF] Allen | Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast[END_REF][START_REF] Yoo | Effects of mannose on pathogenesis of Acanthamoeba castellanii[END_REF] . Interestingly, after an in-depth analysis of the tupanvirus soda lake genome, we found that the virus has an 525 bp MBP gene. The domain involved in mannose recognition contains a three-fold internal repeat, and the consensus sequence motif is QXDXNXVXY. A previous study has suggested that the QDN subdomains may be essential for functionality of the protein. In addition, the importance of Y in subdomain of the MBP-mannose interaction was demonstrated [START_REF] Barre | Curculin, a sweet-tasting and taste-modifying protein, is a nonfunctional mannose-binding lectin[END_REF] .

This finding led us to investigate the MBP viral and cellular expression during the replication of tupanvirus and the possible effects of free mannose on the formation of bunches. An analysis of cellular MBP gene expression in uninfected A. castellanii cells showed a basal level of MBP transcripts, meaning it is likely essential for cellular processes (Fig. 6A). However, during tupanvirus infection, the levels of cellular MBP transcripts significantly increased (p = 0.0001) at earlier times during infection (1, 2 and 4 h.p.i.). The increase of cellular MBP gene expression induced by tupanvirus precedes bunch formation by a few hours (6 h.p.i.) (Figs 1A and6A). Regarding tupanvirus encoding MBP, we observed a gradual increase in (or accumulation of) transcripts through the infection time-course. High levels of viral MBP transcripts were observed from 4 until 8 h.p.i (Fig. 6B). Therefore, our data indicate that the expression of cellular and viral MBP genes is induced during tupanvirus infection, suggesting a possible relevance of these genes in the viral replication cycle (Fig. 6).

Interestingly, orthologous MBP genes were found in the genome of other members of the Mimiviridae family, such as in the prototype APMV. Phylogenetic analysis of the amino acid sequence of viral MBP showed that tupanviruses forms an out-grouping among amoebal-mimiviruses (Suppl. Fig. 3A). Considering APMV does not induces the formation of bunches in infected amoebas, an analysis was performed to determine whether APMV was able to induce the expression of cellular MBP, as well as whether this virus express its own MBP gene throughout the time-course of infection. We observed that APMV induced a decrease in cellular MBP gene expression, even when compared to uninfected cells, in contrast to tupanvirus soda lake, which significantly increases cellular MBP gene expression which may be related to the induction of bunch formation (Suppl. Fig. 4B).

The expression of APMV MBP gene transcripts was also analyzed during the time-course of APMV infection in A. castellanii, revealing that this gene was significantly expressed at 6 and 8 h.p.i. (Suppl. Fig. 4A). However, an analysis of the APMV MBP amino acid sequence revealed polymorphisms in all three of the repeats that compose the catalytic site of the protein (the mannose-binding domain), more specifically in the QDN subdomains (Suppl. Fig. 3B), which have been associated with a loss of MBP function [START_REF] Barre | Curculin, a sweet-tasting and taste-modifying protein, is a nonfunctional mannose-binding lectin[END_REF] . Polymorphisms in the Y amino acids have also been observed in the first repeat of the APMV MBP catalytic site (Suppl. Fig. 3B), which has also been associated with a loss of MBP function [START_REF] Barre | Curculin, a sweet-tasting and taste-modifying protein, is a nonfunctional mannose-binding lectin[END_REF] . In contrast, tupanvirus MBP has no polymorphisms in any of the repeats in the catalytic site, when compared to the functional protein previously studied (Suppl. Fig. 3B) [START_REF] Barre | Curculin, a sweet-tasting and taste-modifying protein, is a nonfunctional mannose-binding lectin[END_REF] . In summary, mutations in the mannose-binding domains of the APMV MBP gene could indicate that this protein is not able to bind to mannose, although more studies into structural level are needed to confirm this hypothesis. Previous work have demonstrated that free-mannose negatively affects Acanthamoeba adhesion to cell surfaces and interferes with amoeba pathogenicity [START_REF] Kim | Functional roles of mannose-binding protein in the adhesion, cytotoxicity and phagocytosis of Acanthamoeba castellanii[END_REF][START_REF] Yoo | Effects of mannose on pathogenesis of Acanthamoeba castellanii[END_REF] . Therefore, the impact of free-mannose on cellular and viral MBP gene expression was here evaluated in uninfected and tupanvirus-infected cells. Our results showed that free mannose negatively affects the expression of the cellular MBP gene, maintaining it at close to basal levels (Fig. 6A). The same negative effect was observed in relation to the expression of the tupanvirus MBP gene (Fig. 6B). Considering these results, the effect of free mannose on the cytopathic effect of tupanvirus was evaluated as well. Interestingly, when Acanthamoeba was pre-treated with free-mannose, there was a gradual inhibition of bunch formation in a dose-dependent manner (Fig. 7), suggesting that formation of amoebal-bunches correlates with the suppression of viral and cellular MBP genes (Fig. 7).

Previous studies have shown that Acanthamoeba MBP is itself a glycoprotein containing mannose [START_REF] Garate | Biochemical characterization and functional studies of Acanthamoeba mannose-binding protein[END_REF] , which may be one of the factors that explains the formation of the bunches in cells infected by tupanvirus, since this virus induces the expression of the MBP gene. Thus, the induction of mannose receptor genes in the tupanvirus replication cycle may be important for the optimization of the formation of bunches, since the interaction between amoebas may occur through interactions among their receptors.

Bunch-forming cells are able to interact with uninfected cells. As previously characterized in this

study, the formation of the bunches starts at approximately 6 h.p.i. and persists until at least 24 h.p.i. At 16 h.p.i., almost all the cells are infected and clustered forming large bunches (Fig. 1A). An experiment was designed in order to evaluate whether bunch-forming cells were able to interact with uninfected cells (Fig. 8A). For this, A. castellanii cells were infected with tupanviruses (at an M.O.I. of 10), and at 16 h.p.i. the bunches were collected and used to inoculate a flask containing uninfected A. castellanii cells in suspension. After incubation, the supernatant (containing bunches and eventual non-infected amoebas interacting with bunches) were removed. The cells that remained adhered to the flask (the ones that did not interact with bunches) were counted to evaluate if the bunches were able to hijack the uninfected cells (Fig. 8). In addition, the supernatant was submitted to SEM and IF, using tupanvirus-specific antibodies. APMV-infected amoebae, which do not induce bunch formation, were used as a control in order to compare whether APMV-infected cells were also able to interact with and hijack uninfected cells (Fig. 8B).

Our results revealed that tupanvirus-induced bunches are able to bind to and hijack many uninfected cells. The number of cells remaining in the flasks after interaction with tupanvirus-induced bunches was significantly lower than those in flasks inoculated with APMV-infected cells (Fig. 8B). The interaction among tupanvirus-induced bunches and uninfected cells was also observed by SEM; it was possible to observe infected cells, which were rounded, interacting with fresh trophozoites presenting their typical pseudopods (Fig. 9). IF assays also showed bunches containing a mix of infected and uninfected cells (data not shown). Lastly, considering the ability of tupanvirus-induced bunches to bind and hijack uninfected cells, we evaluated whether such interactions could promote an increase in viral titer. For this, bunches were collected at 16 h.p.i. and inoculated in flasks containing fresh, uninfected amoebas, as previously described above. After incubation, the supernatant containing bunches attached to uninfected cells was collected and transferred to a new flask. Infectious particles in these population of cells were tittered at 0 and 36 h.p.i, revealing an of almost 2 log increase in viral titer (Suppl. Fig. 5).

Tupanviruses has the broadest host spectrum known among amoebal giant viruses 1 . While most of the giant viruses, such as cedratvirus, marseilleviruses, mollivirus, pandoraviruses, mimivirus, faustovirus and kaumoebavirus, are known to be able to replicate only in a single amoeba genus, tupanvirus is able to replicate in Acanthamoeba spp., Vermoameba vermiformis, Dysctiostelium discoideum and Willartia magna [START_REF] Abrahão | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF][START_REF] Philippe | Pandoraviruses: Amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes[END_REF][START_REF] Legendre | Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology[END_REF][START_REF] Legendre | In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba[END_REF][START_REF] Reteno | an Asfarvirus-Related New Lineage of Giant Viruses Infecting Amoebae[END_REF][START_REF] Andreani | Cedratvirus, a double-cork structured giant virus, is a distant relative of pithoviruses[END_REF][START_REF] Aherfi | The expanding family Marseilleviridae[END_REF][START_REF] Bajrai | a new virus that clusters with Faustoviruses and Asfarviridae[END_REF] . The generalist profile displayed by tupanviruses may be related to the extreme aquatic environments where they were isolated (high salinity, pH and depths); within inhospitable environments there is a lower species richness and abundance, meaning there are more hosts available and a better chance of a future infection [START_REF] Sergeev | The Proterozoic history and present state of cyanobacteria[END_REF] . In this way, bunch formation induced by tupanvirus may be important to the recruitment of cells, improving the chances of viral progeny to find a new host cell that may be scarce in those environments. In addition, bunch formation can reduce the dilution effect in aquatic environments, thereby allowing the release of more particles near the host, which could also facilitate the encounter between viral particles and their hosts.

Concluding Remarks

Here we described a new virus-host interaction mechanism in which tupanvirus-infected amoebas are induced to aggregate with uninfected cells, forming bunches that can increases tupanvirus progeny. In addition, our results demonstrated that mannose reduced the transcript levels of cellular and viral mannose receptor genes, as well as inhibited bunch formation in a concentration-dependent manner, suggesting that amoebal-bunch formation correlates with MBP gene expression. Taken together, our results provide insights into the ecology of these intriguing viruses.

Figure 1 .

 1 Figure 1. Characterization of tupanvirus' cytopathic effect: formation of bunches. (A) A. castellanii monolayer was infected by tupanvirus using an M.O.I. of 10 and visualized by light microscopy. The induced cytopathic effects (CPEs) were observed by cell rounding at approximately 4 h.p.i. At approximately 6 h.p.i. the agglomeration of cells culminating in the formation of a characteristic effect, referred to as "bunches, " was observed. The presence of bunches becames more evident at 8, 12 and 16 h.p.i., when almost all the cells are clustered, forming large bunches. Cell lysis becomes more evident at 24 h.p.i. At about 32 h.p.i. a disaggregation of bunches was observed. The later timepoints are characterized by cell lysis (48 h.p.i.) and a large amount of viral particles dispersed throughout the medium (72 h.p.i.). Scale bar, 200 µm. (B) Tupanvirus one-step growth curve at an M.O.I. of 10. Error bars indicate standard deviation.

Figure 2 .

 2 Figure 2. Characterization of the tupanvirus replication cycle in A. castellanii by IF. A. castellanii monolayer was infected by tupanvirus at an M.O.I. of 10 and visualized by IF. At early timepoints, tupanvirus particles are observed attaching to the amoebae surfaces, even after th cell washing step was done. The VF forms between 8 and 12 h.p.i. At 16 and 24 h.p.i. the A. castellanii cytoplasm is filled with viral particles and cell lysis is observed at 24 h.p.i. (at which time the cytoskeleton of lysed amoebas is visualized, although these cells remain adhered).The viral particles are in green (anti-tupan particle antibody) and amoeba cytoskeleton in red (stained by Evans Blue). Scale bar, 100 µm.

Figure 3 .

 3 Figure 3. Detailed images of a 16 h.p.i. bunch. The bunches induced by tupanvirus in A. castellanii cells at different stages of infection were analyzed by: (A) Scanning electron microscopy and (B) transmission electron microscopy. White arrows indicate some tupanvirus particles being released from damaged amoebas. Scale bars, 2 µm and 10 µm.

Figure 4 .

 4 Figure 4. Early bunches re-aggregate after disassociation. A. castellanii cells infected with tupanvirus. Early (12 h.p.i.) and late (24 h.p.i.) bunches are shown, which were mechanically homogenized for cell disaggregation. Thirty minutes later, the cells were analyzed. (A) Re-aggregation of the early bunches 30 min after disassociation. (B) Re-aggregation of late bunches was not observed. Scale bar, 200 µm.

Figure 5 .

 5 Figure 5. Late bunches are composed of highly damaged dead A. castellanii cells. A. castellanii cells shown intensely damaged at 24 h.p.i., as observed by SEM. Scale bars, 5 µm and 10 µm.

Figure 6 .

 6 Figure 6. Tupanvirus upregulates the expression of viral and cellular mannose binding protein (MBP) genes. A. castellanii cells were infected with tupanvirus and collected at 1, 2, 4, 6 and 8 h.p.i. and MBP mRNA levels were measured by qPCR: cellular (A) and viral (B) genes with and without the presence of mannose. The data were calculated using the 2 -(Δct) method and are shown as the standard deviation of two independent biological assays. Error bars indicate SDs. The statistical significance was calculated using a two-tailed 2-way ANOVA test, Tukey's range test, using GraphPad Prism. ****p < 0.0001.

Figure 7 .

 7 Figure 7. Free-mannose affects tupanvirus-induced bunch formation in a dose-dependent manner. A. castellanii cells were treated with free-mannose at concentrations ranging from 25 to 1000 mM in PAS solution, and then infected with tupanvirus (M.O.I. of 10). Eight hours post-infection, cells were observed by optical microscopy. As controls, A. castellanii cells cultivated in PAS medium without mannose and uninfected cells were cultured with 600 mM mannose. Bar graphs represent the number of bunches found per ml in different concentrations of mannose. Error bars indicate SDs. The treatment of amoebas with 600 mM mannose did not affect the replication of tupanviruses in amoebas and did not induced cytotoxic effects in uninfected A. castellanii. Scale bar, 200 µm.

Figure 8 .

 8 Figure 8. Tupanvirus-induced bunches may bind to and hijack uninfected cells. (A) A. castellanii cells were infected with tupanvirus at an M.O.I. of 10 and at 16 h.p.i. bunches were collected and used to inoculate flasks containing fresh, uninfected A. castellanii cells. After 30 min of incubation, the supernatant in flasks containing bunches and uninfected cells was removed. The remaining cells attached to the flask were then quantified. As a control, APMV-infected amoebae (which does not induce bunches formation) were used in parallel with tupanvirus-infected ones. (B) Remaining cells attached to flasks after incubation with tupanvirus-induced bunches or cells infected with APMV were then counted. Tupanvirus-induced bunches attached to and hijacked uninfected cells, causing a significant decrease of remaining adherent cells in the flasks, compared to APMVinfected cells. The statistical significance was calculated using a two-tailed Student's t-test. *p < 0.05, performed using GraphPad Prism. Error bars indicate SDs.

Figure 9 .

 9 Figure 9. SEM showing the interaction between uninfected A. castellanii cells and tupanvirus-induced bunches. SEM images of uninfected cells that interacted with bunches and were carried by infected cells (experiment described in Fig. 8). Scale bars, 10 µm and 20 µm. Arrows indicate infected A. castellanii cells (rounded) and asterisks indicate uninfected A. castellanii cells (most with pseudopods).

SCIentIFIC REPORTS | (2019) 9:183 | DOI:10.1038/s41598-018-36552-4

SCIentIFIC REPORTS |

Acknowledgements

We are grateful to our colleagues from Laboratório de Vírus of Universidade Federal de Minas Gerais. In addition, we thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FAPEMIG (Fundação de Amparo à Pesquisa do estado de Minas Gerais), PRPq-UFMG (Pro-Reitoria de Pesquisa of UFMG) and the Microscopy Center of UFMG. F.G.F; E.G.K and J.S.A are CNPq researchers. E.G.K.; B.L.S. and J.S.A. are members of a CAPES-COFECUB project.

Author Contributions

Additional Information

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36552-4.

Competing Interests:

The authors declare no competing interests.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Charlotte Vaurs

Jean-Frédéric Brun

Monelle Bertrand

Rémy Burcelin

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Post-prandial hypoglycemia results from a non-glucose-dependent inappropriate insulin secretion in Roux-en-Y gastric bypassed patients

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  hal.umontpellier.fr/hal-01819191 Submitted on 10 Dec 2019     Post-prandial hypoglycemia results from a non-glucose-dependent inappropriate insulin secretion in Roux-en-Y gastric bypassed patients Charlotte Vaurs, Jean-Frédéric Brun, Monelle Bertrand, Rémy Burcelin, Maël Chalret Du Rieu, Yves Anduze, Hélène Hanaire, Patrick Ritz To cite this version: Charlotte Vaurs, Jean-Frédéric Brun, Monelle Bertrand, Rémy Burcelin, Maël Chalret Du Rieu, et al.. Post-prandial hypoglycemia results from a non-glucose-dependent inappropriate insulin secretion in Roux-en-Y gastric bypassed patients. Metabolism, WB Saunders, 2016, 65 (3), pp.18 -26. 10.1016/j.metabol.2015.10.020. hal-01819191


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Segmental bioelectrical impedance analysis (SBIA) and blood rheology: Reducing the gap between in vivo and in vitro?

Emmanuelle Varlet-Marie, Céline Joré, Jean-Frédéric Brun

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Emmanuelle Varlet-Marie, Céline Joré, Jean-Frédéric Brun. Segmental bioelectrical impedance analysis (SBIA) and blood rheology: Reducing the gap between in vivo and in vitro?. Clinical Hemorheology and Microcirculation, 2017, 64 (4), pp.603 -611. 10.3233/CH-168041. hal-01804152


Louis Zukofsky'

Homophonic Practice

Abigail Lang

Luis D'

Antin Van Rooten

Luis

"To 'Tune in' to the Human Tradition"

Lorsque le poète américain Louis Zukofsky (1904-1978) commence à recourir à la traduction homophonique, elle n'existe pas en tant que telle : Zukofsky parle indifféremment d'homonymes, d'imitation sonore ou de translitération. Contrairement à Howard Chace (Anguish Languish, 1956) ou Luis van Rooten (Mots D'Heures: Gousses, Rames, 1967) qui furent des amateurs passionnés, Zukofsky consacra sa vie à l'écriture ; la traduction homophonique n'était pas pour lui une obsession singulière mais une technique parmi d'autres. Retracer l'apparition et l'évolution de la pratique homophonique chez Zukofsky montre qu'elle est en cohérence avec l'ensemble de sa poétique, à commencer par son matérialisme ; qu'elle participe d'une recherche prosodique et reproduit à l'échelle individuelle un fonctionnement structural des langues qui empruntent parfois le système prosodique ou les caractères d'écriture à leur voisine ; et qu'elle cherche à offrir l'accès le plus objectif possible à la tradition, en créant un contact matériel, mais médiatisé, avec les oeuvres du passé.

1 "To breathe the 'literal' meaning with him" Catullus (Gai valeri veronensis Liber), Celia and Louis's translation of Catullus, came out in 1969, published simultaneously in the United States by Grossman and in Britain in a deluxe edition by Cape Goliard. 1 A translation of the complete works of Catullus as provided by the Loeb Classics edition, both editions are bilingual, with the original Latin printed on the facing page. The Zukofskys devoted eight years to the project, leaving no doubt that the work was meant as a genuine translation and an earnest endeavor.

or a shrug from the classicists, well used to their own students' calques or hereby reminded of their own pranks as schoolboys learning Latin. 3 It was the manifest earnestness of the Zukofskys' endeavor that triggered the classicists and the critics' almost unanimous hostility. They disparaged it as "knotted, clumsy, turgid and ultimately silly", as so much "unreadable", "gibberish" 4 . Worse than inept, it conspired against civilization: "The Hun is at play-worse still, at work-among the ruins", wrote one reviewer. 5 Lawrence Venuti's analysis of the reviews perfectly brings out the implicit ideology governing acceptable translation, which the Zukofskys eagerly violated by promoting sound and a varied lexicon at the expense of communication and homogeneous diction, by foregrounding the body of the original at the expense of its paraphraseable soul, and by claiming homophonic translation as a legitimate approach to canonical authors.

Not that the Zukofskys called it homophonic, which might have defused its disruptive potential by identifying it too readily and sealing it off. Their very short translators' preface says the following: "This translation of Catullus follows the sound, rhythm, and syntax of his Latin-tries, as is said, to breathe the 'literal' meaning with him" 6 . What the inverted commas around "literal" indicate is that the word is to be taken literally, signifying not a verbatim translation, one that remains close to the text, but a translation 'to the letter', both as grapheme and phoneme, script and sound. Much of the success and enduring interest of the Zukofskys' Catullus stems from the fact that they are constantly trying to do three or four things at the same time and not just following the sound as a more one-dimensional procedure might propose to do. While the first dozen or so poems follow mostly the semantic meaning, from poem 8 ("Miser Catullus"), Zukofsky begins to select words according to four criteria: "sound, rhythm, and syntax" as mentioned in his preface, and meaning.

Poets typically overemphasize sound but rarely to the extent of Zukofsky. Introducing his current collaborative translation in a preparatory note for his November 1960 Library of Congress reading, Zukofsky explains that his wife supplied a pony of the grammar "to the n th inflection", and put a seal on his sensitivity when he was faced with a multiplicity of choices in his obstinacy of trying to render Catullus' sound as English. In the process, Zukofsky explains, he must "sharp" and "flat" those sounds to make sense at all. Never academically trained as a Latinist, Zukofsky would have perceived the sound and script of the original Latin over and above its meaning. This enabled him to treat Catullus not only as a text to be translated but also as a score to be performed, with the same leeway that the contemporary aleatoric composers (Cage, Feldman . . . ) were allowing their performers, a comparison Zukofsky makes in a July 30 th 1963 note. While listening to Catullus as music, Zukofsky never gives up on Gouses, Rames: The d'Antin Manuscript. As Jeff Twitchell-Waas remarks, "Van Rooten's work would have intrigued Zukofsky because, although it purports to be an obscure book of poems in archaic French, it is actually Van Rooten's homophonic translation of English nursery rhymes into French".

3. French students of Greek, for instance, have long chuckled at the homophonic translation of this fragment attributed to : "Ουκ έλαβον πόλιν, αλλά γαρ ελπίς εφή κακά" ("Ouk elabon Polin alla gar elpis Ephe Kaka").

4. semantic meaning. In fact, the power of Zukofsky's poetics is very much predicated on his refusal to separate sound and sense, a consequence of his consistent materialism: there is no such thing as a free-floating meaning waiting to be incarnated in one linguistic body or another. Sound was hardly the opposite of sense for Zukofsky who constantly revived the other meanings of the word, notably as an adjective meaning solid and sensible and as a verb meaning to test and to fathom. Praising a sixteenth century translator of the Aeneid, Zukofsky had written in 1948:

The sound of the words is sometimes 95% of poetic presentation. One can often appreciate the connotations of the sound of words merely by listening, even if the language is foreign. And on the previous page Zukofsky had stated his faith in the power of pronunciation out loud to make sense: "The spelling should help sound the words. Sounded, they make sense" (ibid., p. 57).

What does Zukofsky imply when he writes he follows rhythm, his second criteria? Clearly, he strives to retain the number of syllables of the original line, a constraint which preserves the visual layout of the poem and produces condensation.

In a 1978 letter to Burton Hatlen recapitulating what their working procedure had been, Celia Zukofsky explains she "wrote out the Latin line and over it, indicated the quantity of every vowel and every syllable, that is long or short; then indicated the accented syllable" 8 . Whether Zukofsky was successful in rendering quantity and accents and approximating the characteristic tune of the original Latin meter is hard to tell, and commentators disagree. 9 . It is not all that clear that classicists agree on what Latin hendecasyllabics sounded like; certainly, no distinct pattern emerges from the recorded readings of Catullus available online. The issue of translating the rhythm and prosody of a foreign language will be pursued further down.

In Conniving Thallus, mulley, you, cony cully, cop below-Wee, anserous medulla, well immolated auricula-Wee, pain and languid older sense, see, too extraneous-o so-And then qua Thallus, turbid air, rape packing off, a hell of Come diva mull over Aries-oscine oscitancy (Anew, p. 256)

11. For a commentary on Klossowki's translation see Berman, Antoine, La traduction à la lettre ou l'auberge du lointain, Paris, Seuil, 1999.

12. The Poems of Catullus and Tibullus, and the Vigil of Venus, A Literal Prose Translation with Notes by Walter K. Kelly, to which are added The Metrical Versions of [George] Lamb and [James] Grainger, and a selection of versions by other writers, London, George Bell, 1878. Kelly supplies extensive annotations to the poems (Jeffrey Twitchell-Waas: www.z-site.net/notes-to-poetry/ catullus-1969-with-celia-zukofsky/).

The translation of the famous Carmina 16 belongs to the second category which balances sense and sound: Piping, beaus, I'll go whoosh and I'll rumble you pathic Aurelius and catamount Furius, who mix my versicles with your poor tastiesthe sound is a mollycoddle's, I'm not up to par for chasteness. But the pious poet is chaste, his versicles not nailed to his need, quick to themselves with no lack of decorum, if the sound models not quite pure for pudency what incitement it carries passes into now I won't say hairless boys', but such hoary necks as endure not quite up to feel lumbar. Milling thousands of kisses are base or make me out some mare of a male-you impute that? Piping, beaus, I'll go whoosh and I'll rumble you. (Anew, p. 253) One can very well and very enjoyably read this poem on its own for its sonic profusion and the innuendoes aroused by rare words and their juxtaposition. As Venuti has shown, the homophonic constraint uncovers "a dazzling range of Englishes" 13 , juxtaposing levels of diction, mixing the archaic and the scientific, joining the vulgar and the precious. Where translators typically seek to recreate a voice by establishing a homogeneous diction, Zukofsky mines the dictionary to present heterogeneous Englishes. A tease to the linguistic imagination, Zukofsky's poem also invites the reader to scour the dictionary and come out with a new poem. I always took "The sound's a mollycoddle" (line 4) as a commentary on homophonic translation, based on the assumption that "mollycoddle" must mean a muddle, a tangle, a jumble. But the Oxford English Dictionary reveals it means "an effeminate man or boy; a milksop". Does this imply that sound is effeminate, mere matter as opposed to the father's informing seme, as a structuring metaphor of the western tradition would have it? Following in the footsteps of Lori Chamberlain 14 who revealed the gendered nature of the Western metaphorics of translation-riddled with issues of fidelity, paternity, authority, rightful progeny, and rape-, one can consider Zukofsky's indulgence in sound and scattering of semantics as a form of queering. Performed at the linguistic level, it chimes with Catullus' erotic contents, especially in this poem whose first line flaunts oral and anal sex. Carmina 16 is famous for its indecent contents and was never fully translated before the second half of the twentieth century. While Zukofsky stated that he didn't want his "Cats" to "titillate the passion" 15 , homophonic translation allowed him to provide the innuendoes and let his reader's imagination do the rest. Carmina 16 is also famous for asserting the distinction between the author and the persona. Catullus argues that tender or lewd verse does not imply an effeminate or lecherous author, and that while the author must be chaste, his poems need not be.

The poem was referenced by later Latin poets as an authority on this matter. The poet abuses two male friends who, finding his verse molliculi (tender, delicate), imply that Catullus is an effeminate poet. Catullus's warning hasn't prevented generations of scholars from reconstructing his life from his poems, since "we know next to nothing about the poet and his actual biography" 16 . Catullus's warning would have struck a chord in Zukofsky who similarly discouraged his readers from wanting to reconstruct the author's intentions from the works: "I hope everybody would read me the same way-that is, not wonder whether I was afraid of a draft as I am at the moment, but just read the words" 17 . This emphasis on words at the expense of a voice, on the dictionary at the expense of a coherent diction, is the basis of Zukofsky's translation method and poetics.

Per-sona

There is only one entry for the word translation in the index to Prepositions, Zukofsky's collection of essays, and it refers to one of his earliest, devoted to Ezra Pound. It was Pound, of course, who had insisted on the importance of translation and, by his own achievements and authority, convinced several generations of American poets to translate. In his essay, Zukofsky praises Pound the translator for "the distinction of rendering into English unexplored poetic forms", and for "translating himself through personae" 18 . While Zukofsky praises Pound for translating, "not merely dramatizing", himself into a persona, by the end of the section the dramatization seems to disqualify Pound's use of persona, because it is historically ungrounded, implicitly a mere psychological projection: "What after all, asks Zukofsky, does Ezra Pound know about En Bertrans [twelfth-century troubadour Bertran de Born] to attempt a persona?" 19 . As is so often the case, Zukofsky arrives at his own solution by subverting Pound, here by taking him at his word, literally; in this case, by attending to the etymology of persona-at least, the generally accepted nineteenth-century etymology of persona-: "to sound through", shifting the focus from the actor's mask to the voice that filters through: "En Bertrans, as Pound's measure reflects him, and as he reflects Pound, can bear but the relation of a veracious actor to his historic original, and act him, mask penetrated, per sonum-through sound" (ibid.). The rest of the paragraph deserves to be quoted in full as Zukofsky advances an early (1929) and prescient formulation of his own approach to historical material, articulating the poetics he will carry out some forty years later in "A"-22 & 23, the final movements of his 800-page lifework:

Only speech transforms whatever skeleton remains of the past and conveys judgment of it to the intelligence. Try as a poet may for objectivity, for the past to relive itself, not for his living the historical data, he can do only one of two things: get up a most brief catalog of antiquities (people become dates, epitaphs), or use this catalog and breathe upon it so that it lives as his music. This latter action need not falsify the catalog. (ibid.)

But of course, the second branch of the alternative also foreshadows the Catullus translation and its ambition "to breathe the literal meaning with him"-not impersonating Catullus but mouthing his sounds and encouraging the English-speaking reader to do the same, thus each time resuscitating the skeleton of Catullus with live breath. By taking the demiurge's creative breath literally, Zukofsky turns idealism on its head. As against humanist translators and critics who wish for the spirit of the original-and that would include Pound whose ambition, for all his archaisms and literalisms, is to evoke the power (virtù) of the original-Zukofsky focuses on the letter (as sound and script), and takes spirit literally, not as the distinctive style of the dead author but as the breath of the living reader. Homophonic translation offers Zukofsky a materialist approach to the past that he will fully carry out in the final movements of "A".

Fluorescence

If Zukofsky formulated an argument in favor of homophonic translation as early as the late 1920s, he didn't enact it fully before 1958, ten poems or so into Catullus.

But he had already experimented with it in the 1930s, without identifying it as such, while composing the first canzone of "A"-9. Like Catullus, "A"-9 is a work that hovers indefinably between translation and creation. Following in the footsteps of Pound, who had himself devoted more than a decade to Guido Cavalcanti's thirteenth century doctrinal canzone and translated it twice, Zukofsky spent seven years considering and two years translating Cavalcanti's canzone on love (1938-40), successfully retaining its rigorous formal features in a way Pound had never been able to, but replacing the treatise on love by a treatise on labor, couched in phrases drawn from Marx's Capital.

In a decade when their political views were increasingly at odds, this constituted both a homage and a challenge from Zukofsky to his mentor. Even more than the later Catullus, "A"-9 considers the translation of form as a perfectly acceptable alternative to the translation of paraphraseable content. Zukofsky retains the rhyme-scheme, approximates the Italian hendecasyllable with eleven syllable lines and retains as much of the original sound as he can. Juxtaposing the Italian original and the English translation of the fifth stanza shows that approximations of sound begins with the rhyming words, suggesting that transliteration can be viewed as a continuation of rhyme by other means-a species of translingual rhyme.

Dissemble-pledging complexions so guarded

Da ssimil tragge complessione e sghuardj 20

In the first, privately printed edition of the canzone (1940), in which Zukofsky compiled the various sources for his poem, he makes no reference to his homophonic 20. Zukofsky, "A", New York, New Directions, 2011 [1978], p. 108; Guido Cavalcanti, "Donna mi priegha", in Ezra Pound, Translations, New York, New Directions, 1963, p. 138.

practice. The "fluorescence" he evokes in the foreword is as close as he gets: "These aids are presented in the foregoing order, the poem last, so that if the intention to have it fluoresce as it were in the light of seven centuries of interrelated thought has at all been realized the poem will explain itself" 21 . But twenty-five years later (in 1965, at the time he was translating Catullus), when he describes the poem in view of a German edition of "A"-9, Zukofsky revises his 1939 formulation and introduces the term "transliteration".

"A"-9 consists of two canzoni, the first a definition of value, the second a definition of love. Both are profiled after Guido Cavalcanti's Donna mi priegha. The rhymes of the second canzone of "A"-9 are the same as those of the first in the linear sequence which in its discourse attempts to transliterate the sound (or "Noise") of the original Italian as English. The intention is to have "A"-9 fluoresce as it were in the light of seven centuries of interrelated thought (the sound a part of it). 22 The term "transliterate" suggests that the thirteenth century Italian seeps through the twentieth century American English, while "fluorescence" implies that the heterogeneous material condensed formally into the poem shines, delighting the senses. In Marxian terms, Zukofsky's invisible labor resuscitates the dead labor already objectified by Cavalcanti into his poem, bringing it back to life. 23 So that Zukofsky's "breathing the literal meaning" doesn't only echo creation myths but also involves his Marxist view of labor and value.

Prosody and Accents, or "new-old quantities" 24

Readers of Zukofsky know that he was constantly sounding script and transcribing sound, "to try the sound of words" 25 . Zukofsky signed his first discarded book of poems "Dunn Wyth (pronounced 'done with')" (1928). He named a 1963 volume of poems I's (pronounced 'eyes'). In his partly autobiographical fiction Little, the character of the poet (Dala) is shown making homophonic translations from medieval Welsh poetry. Dala, on his own time before another evening class completed his long day, transliterated, on his wallet's memo pad, from Gorhoffedd of Hywel ab Owain Gwynedd : 21. Zukofsky, First Half of "A"-9, New York, Louis Zukofsky, 1940, p. 1. A form of luminescence, fluorescence is the emission of (typically visible) light by a substance that has absorbed (typically invisible) light or other electromagnetic radiation.

22. Zukofsky archive, HRC, University of Texas, Austin, Works, "A", box 2. 23. Or, to quote Zukofsky quoting Marx, "by incorporating living labour power with their dead substance, the capitalist transforms value (past labour, objectified labour, dead labour) into capital, into self-expanding value, into a monster quick with life, which begins to 'work' as if love were breeding in its body". (Zukofsky, First Half of "A"-9, op. cit. p. 14, quoting Capital, Book I, section 3, The Production of Absolute Surplus-Value). In several preparatory notes Zukofsky calls Catullus his collaborator and the translation his chore, his beloved drudgery.

24. Jeffrey Twitchell-Waas reveals that the line "Cue in new-old quantities" ("A"-23, p. 563), at the very end of the last movement Zukofsky composed, is an oblique reference to his work on Catullus with his wife and their attempt to homophonically mimic Catullus' quantitative verse.

25. Zukofsky, "A", op. cit., p. 177.

is the dent roc towered [. . . ] After an Institute function Dala was walking home with an equally questionable variant of Gwynedd: is that eye hant rack toward- [START_REF] Zukofsky | Collected Fiction[END_REF] The novella, composed between 1950 and 1969, opens with A Note on the Pronunciation of Names:

As in Latin: names of two syllables are accented on the first (with a few exceptions, Dríscháy, Uńzúng Phárétte); of more than two syllables, on the penult if that is long (Verchádet, Babállo, Sweetsíder); otherwise on the antepenult (Teárilee, Beármeout). 27 Zukofsky clearly enjoys transcribing exactly the various foreign accents of his son's successive violin professors throughout:

(Aside to Verchadet [Celia Zukofsky] and Baballo [Louis Zukofsky]: "No, no? you no American?" They and Little [Paul Zukofsky, their son] unison, "But of course we are, what else?" Proba: "Maybe, no?" then back to Little-) "Play premier, third exercise Svecik. Bis, more once. Probier. Moment (pronounced as in French, probably to mean momentum.) Più ah più, ma! side of hair-happily! 28

In his essay on Charlie Chaplin entitled "Modern Times" (1936), Zukofsky reveals why transcribing foreign accents is so pleasurable. Commenting on the famous scene at the end of Modern Times when having lost the lyrics of the song Chaplin launches into a mish-mash of European languages, [START_REF]On Chaplin's 'nonsense' songs, see also Charles Bernstein[END_REF] Zukofsky writes: "Singing, Charlie concocts his words internationally. The result is the effect of words conveying a familiar feeling of being spoken without the least affectation" 30 . We tend to think of the 'mother tongue' as our linguistic home but for Zukofsky, it is this gibberish of European languages that feels 'familiar', a feeling perhaps shared by first generation immigrants, exiles, and those who live near borders or in cosmopolitan cities. Zukofsky was born in the Lower East Side of Manhattan from Russian Jewish parents whose first language was Yiddish. An oft-repeated anecdote pictures him reciting Longfellow's Hiawatha in Yiddish to a gang of Italian bullies. Because his parents could not decipher the birth certificate filled out in English by the midwife, Zukofsky never knew what his birth date was until he applied for a passport. Then he found out that the midwife had written his name down as Sallikowsky, practical proof that transliteration is how different languages tune into each other.

6 "to 'tune in' to the human tradition" It is this musical horizon of poetry [. . . ] that permits anybody who does not know Greek to listen and get something out of the poetry of Homer: to 'tune in' to the human tradition, to its voice which has developed among the sounds of natural things, and thus escape the confines of time and place, as one hardly ever escapes them in studying Homer's grammar. In this sense poetry is international. [START_REF] Zukofsky | Prepositions +[END_REF] For Zukofsky, as we have seen, this international character is not to be achieved by a universally translatable transparency but concocted by an idiosyncratic sonic obduracy.

Three hypotheses

Having now delineated the emergence and stakes of transliteration in Zukofsky's work, I would like to draw three hypothetical implications for homophonic translation more generally.

First, that transliteration blurs the customary boundary between creation and translation. As we have seen, Zukofsky increasingly incorporated unassigned quotations and translations in his compositions while making his own collaborative input manifest in his translations. In his obstinacy to render the sound of the original, Zukofsky brought out the productive and creative power of translation, its ability to uncover a new diction or lexicon, to help achieve a new prosody. The implicit refusal to discriminate between composition and translation rests on a radical materialism which also refuses to oppose sound and sense, and justifies, even requires, transliteration.

Secondly, that Zukofsky's transliterations seem part of a wider quest for an alternate prosody after the demise of metered verse and the (real or imagined) dulling of free verse invites us to view homophonic translation as a modern outlet for the phonological impulse. Translingual, and expanding its field from consecutive endwords to entire poems printed on facing pages, homophonic translation can be viewed as a limit case of rhyme. But linguistic sound is sense, "matter that thinks" [START_REF] Zukofsky | A[END_REF] . In "A Statement for Poetry" (1950), having defined the three components of poems as "A. Image B. Sound, C. Interplay of Concepts", Zukofsky associates puns and "the homophonic devices of syllabaries" not with the second but with the third category, "in which words involve other words in common or contrasting logical implications, and to this end it [Interplay of Concepts] employs sound, and sometimes image, as an accessory" 36 .

Thirdly, Zukofsky's reference to the final scene of Modern Times in which Chaplin's voice can be heard for the first time suggests that the increasing use of sound recording technologies, with their foregrounding of speech as noise, has probably favored the spread of homophonic translation. Its boost in the 1950s and 60s certainly corresponds to the marketing of tapes and tape-recorders. Zukofsky's 1962 preface to the Catullus translations published in Kulchur appears strangely consonant with William Burroughs's statement in his almost contemporary introduction to "The Cut-Up Method of Brion Gysin", soon applied to splicing tape:

This version of Catullus aims at rendition of his sound. By reading his lips, that is while pronouncing the Latin words, the translation-as his lips shape-tries to breathe with him. L.Z. Shakespeare Rimbaud live in their words. Cut the word lines and you will hear their voices. Cut-ups often come through as code messages with special meaning for the cutter. Table tapping? Perhaps. Certainly an improvement on the usual deplorable performance of contacted poets through a medium. [START_REF] Zukofsky | The Cut-Up Method of Brion Gysin[END_REF] Both convert the spiritualist lure of an immediate contact with dead writers into a mediate and material transformation of previous texts. If, as Zukofsky once defined it, "poetry is information" 38 -form preserving the clarity and allowing the complexity of the message-, then transliteration, predicated as it is on noise, makes palpable the loss of information which besets any transmission while offering a manner of experimenting with it.

  The Scotch of Gawin Douglas [in his 1513 translation of the Aeneid ] can hardly be called foreign, if one reads English. What is foreign to poetry is the word which means little or nothing-either as sound, image, or relation of ideas. 7

  Catullus. Wray is both a distinguished Latinist and an informed reader of Zukofsky. For an upto-date list of commentaries on the Zukofskys' Catullus see Jeffrey Twitchell-Waas's Z-site: A companion to the Works of Louis Zukofsky (www.z-site.net/notes-to-poetry/catullus-1969-with-celia-zukofsky/). 10. Hatlen, op. cit., p. 347.of the words juxtaposed supersedes the foreignness of the syntax. That syntax isn't Zukofsky's prime criterion can be felt by comparison if one turns to Pierre Klossowski's 1964 French translation of the Aeneid which some critics disparaged as calque for remaining too close to the Latin syntax. 11Finally, although he does not refer to it in his preface, Zukofsky remains committed to semantic meaning. The Zukofskys consulted several historical translations 12 as the two notebook pages for Catullus 85 reproduced on the Cape Goliard cover indicate.In most cases one would be incapable of reconstituting a paraphrase of the original poem based solely on Zukofsky's translation, but all commentators have shown that the juxtaposition of Zukofsky's translation with the original Latin or a more standard translation yields countless semantic echoes and complexification, Zukofsky's translation functioning as a commentary or variation or riff on the original.

	a 1960 note, Zukofsky claims that following Catullus's sounds syllable for syllable
	and line for line has involved him in his syntax, his third criteria. In her 1978 letter
	to Hatlen, Celia Zukofsky explains that "[b]elow the Latin line I wrote the literal
	meaning or meanings of every word indicating gender, number, case and the order
	or sentence structure. I used Lewis & Short Latin Dictionary (Oxford UP) and
	Allen & Greenough Latin Grammar (Ginn & Co.)" 10 . But Zukofsky's claim can only
	be partly true since he appears more committed to retaining sounds than parts of
	speech. And even when he does follow the syntax closely, the semantic strangeness
	7. Zukofsky, A Test of Poetry, Hanover, Wesleyan University Press, 2000 [Objectivist Press, 1948],
	p. 58.
	8. Hatlen, op. cit., p. 347.
	9. See notes 26 and 55 in David Wray's " 'cool rare air': Zukofsky's Breathing with Catullus and
	Plautus", Chicago Review, vol. 50, No. 2/3/4 (2004/05), p. 52-99, one of the most illuminating readings of
	Zukofsky's

2 "The sound's a mollycoddle"

Depending on the criteria that Zukofsky foregrounds, the translations sound quite different. Until poem 8, Zukofsky foregrounds semantics and, to some extent, syntax: Whom do I give my neat little volume slicked dry and made fashionable with pumice? Cornelius, to you [. . . ] (Anew, p. 245) Then comes the oft-quoted poem 8, rather typical in its mixing of sound and semantics and it its creation of a noble if tortuous species of English. It begins: Miser Catulle, desinas ineptire et quod vides et quod vides perisse perditum ducas.

Miss her, Catullus? don't be so inept to rail at what you see perish when perished is the case. (Anew, p. 248) Sonics lead but semantics resist in "what you see" for "quod vides". Finally, beginning with poem 25 and increasingly toward the end of the volume the reader must ford through a delightfully if disconcertingly strange English riddled with Latin sounding words:

  International Journal of the Classical Tradition, vol. 13, No. 3 (Winter 2007), p. 409-430, here p. 417 (www.jstor.org/ stable/30222155).

16. Ziolkowski, Theodore, "Anglo-American Catullus since the Mid-Twentieth Century", 17. Zukofsky, Prepositions +, The Collected Critical Essays, ed. Mark Scroggins, Hanover, Wesleyan University Press, 2001 [1967], p. 24. 18. Zukofsky, Prepositions +, op. cit., p. 71. 19. Ibid., p. 73.

  That the recourse to homophonic translation is enmeshed with the quest for new prosodic forms is further demonstrated by Zukofsky's mature poetics which emerged in the early sixties, while he was deep in Catullus. Starting with A-"14" (completed in 1964), Zukofsky's poetry demonstrates two striking features. First, Zukofsky introduced a new prosody based on word-counted line: "A"-14, -18, -19, -21, -22 and -23 are all composed of word-counted lines. Second, he incorporated homophonic translation at the service of his compositional strategy: "A"-15 opens with a transliteration from the Book of Job ("He neigh ha lie low h'who y'he gall mood", p. 359); "A"-19 contains transliterations and free translations from Mallarmé ("Place / it futile", p. 409); "A"-21 is a translation in five-word lines of "Rudens", a play by Latin poet Plautus. While not strictly homophonic it keeps an eye and ear in the Latin, as David Wray has shown ("Your mendacious tool of tongue / magnified auscultation worked my mendacity", p. 458). And "A"-22 & 23 are a pair of universal histories, the former scientific the second literary, each composed of 1000 lines, 5 words to a line. Hebrew and Old English, with shorter passages from Sanskrit, Welsh, Provençal, and Spanish. In addition, Jeffrey Twitchell-Waas has identified individual lines, phrases and words homophonically generated from close to ten further languages. 80 Flowers 31 , Zukofsky's final book of poems (1978) perfects this systematic association of word-count and homophonics. Apparently independent these two features work in cooperation. The word-counted line is Zukofsky's ultimate answer to the quantitative quest launched earlier in the century: a numerical yet flexible constraint. Inaudible and invisible, it is a metrics for the intellect, which is why it needs to be supplemented by homophonics (along with other compositional techniques) for the sake of sound, syntax and diction.

	Charred roots: why you goad
	Loved weed loam more than
	Harm'll frame (why) whom now
	Wind's woodpeople move, rue, ache,
	Choir shocked call rest, pause
	Renew-whirligig punning tempest, cut ("A"-23, op. cit., p. 545)
	Zukofsky condenses his readings, collaging and transliterating freely. "A"-22 & 23,
	particularly the latter, include substantial passages of homophonic renderings from
	Greek, Latin, "A"-22 & 23 accomplish the second alternative that Zukofsky had drawn in 1929 for
	the poet trying "for the past to relive itself": instead of getting up a catalogue of
	antiquities, he "uses this catalog and breathe[s] upon it so that it lives as his music" 32 .
	"History's best emptied of names" claims "A"-22, which Zukofsky conceived as "a
	history without (the) dates" 33 . I find the justification for this final crowning project
	best expressed in "A Statement for Poetry" (1950), where Zukofsky vindicates a poetry
	that reaches out across time, place and languages.

31. Zukofsky, 80 Flowers, reprinted in Anew: Complete Shorter Poetry, op. cit. 32. Zukofsky, Prepositions +, op. cit., p. 73. 33. Zukofsky, A"-22, op. cit., p. 511; notes for "A"-22, 23, 24, HRC, University of Texas, Austin.