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Diego M. A. Guérin - One of the best experts on this subject based on the ideXlab platform.
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The Triatoma Virus Structural Protein VP4 Induces Membrane Permeability through Dynamic Pores
Biophysical Journal, 2015Co-Authors: Rubén Sánchez-eugenia, Julen Goikolea, David Gil-carton, Lissete Sánchez-magraner, Diego M. A. GuérinAbstract:In naked viruses, membrane breaching is a key step that must be performed for genome transfer into the target cells. Despite its importance, the mechanisms behind this process remain poorly understood. The small Protein VP4, codified in the genome of most Picornavirales order viruses, has been shown to be involved in membrane alterations. Here we have analyzed the permeabilization activity of the natively non-myristolated VP4 Protein from Triatoma virus (TrV), a virus belonging to the Dicistroviridae family within the Picornavirales order. The VP4 Protein was produced as a Maltose Binding Protein (MBP) fusion to achieve its successful expression. This recombinant VP4 Protein is able to produce membrane permeabilization in model membranes in a lipid-dependent manner. The membrane-induced permeability was also influenced by the pH, being greater at higher pH values. We demonstrate that the permeabilization activity elicited by the Protein occurs through discrete pores that are reversibly inserted on the membrane. Sizing experiments using fluorescent dextrans, cryo-electron microscopy imaging and other additional techniques showed that recombinant VP4 forms heterogeneous proteo-lipidic pores rather than common Proteinaceous channels. These results show that VP4 Protein is involved in the membrane alterations required for genome transfer or cell entry steps during dicistrovirus infection.
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Capsid Protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles.
Virology, 2011Co-Authors: Jon Agirre, Rubén Sánchez-eugenia, Kerman Aloria, Jesus M. Arizmendi, Ibon Iloro, Felix Elortza, Gerardo Anibal Marti, Emmanuelle Neumann, Félix A. Rey, Diego M. A. GuérinAbstract:Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid Protein VP4. Its cleavage sites were established by sequencing the N-terminus of the Protein precursor and MS, and its stoichiometry with respect to the other major capsid Proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyProtein. We conclude that VP4 Protein appears as a product of RNA encapsidation, and that defective processing of capsid Proteins precludes genome encapsidation.
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VP4 Protein Appears as a Product of RNA Encapsidation in Triatoma Virus (TRV)
Biophysical Journal, 2011Co-Authors: Jon Agirre, Kerman Aloria, Jesus M. Arizmendi, Ibon Iloro, Felix Elortza, Gerardo Anibal Marti, Emmanuelle Neumann, Félix A. Rey, Diego M. A. GuérinAbstract:Triatoma Virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Its non-enveloped capsid is composed of four Proteins named VP1-VP4, plus the minoritary, uncleaved Protein precursor VP0, which comprises VP4 and VP3. While the smaller Protein VP4 (5.5 kDa versus around 30 kDa for VP1-3) remained undetected in past studies by standard biochemical analyses, the icosahedral (T=1, pseudo-T=3) crystallographic structure of TrV (PDB ID: 3NAP) raised even more suspicions about its existence since no electron density could be attributed to this peptide. In the present work, mass spectrometry (MS) and Tricine-SDS gel electrophoresis were used to detect the previously elusive capsid Protein VP4. Its cleavage sites were established by sequencing the N-terminus of the Protein precursor and MS, and its stoichiometry with respect to the other major capsid Proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyProtein (P1). We conclude that VP4 Protein appears as a product of RNA encapsidation, and that defective processing of capsid Proteins precludes genome encapsidation. Our results also suggest that the TrV capsid can be built without the scaffolding aid of the nucleic acid.
Daniel N. Hebert - One of the best experts on this subject based on the ideXlab platform.
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SV40 Late Protein VP4 Forms Toroidal Pores To Disrupt Membranes for Viral Release
Biochemistry, 2013Co-Authors: Smita Raghava, Kristina Giorda, Fabian B. Romano, Alejandro P. Heuck, Daniel N. HebertAbstract:Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion Protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results found that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with bodipy-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of between 1 and 5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.
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The Simian Virus 40 Late Viral Protein VP4 Disrupts the Nuclear Envelope for Viral Release
Journal of virology, 2012Co-Authors: Kristina Giorda, Smita Raghava, Daniel N. HebertAbstract:ABSTRACT Simian virus 40 (SV40) appears to initiate cell lysis by expressing the late viral Protein VP4 at the end of infection to aid in virus dissemination. To investigate the contribution of VP4 to cell lysis, VP4 was expressed in mammalian cells where it was predominantly observed along the nuclear periphery. The integrity of the nuclear envelope was compromised in these cells, resulting in the mislocalization of a soluble nuclear marker. Using assays that involved the cellular expression of VP4 or the treatment of cells with purified VP4, we found that the central hydrophobic domain and a proximal C-terminal nuclear localization signal of VP4 were required for (i) cytolysis associated with prolonged expression; (ii) nuclear envelope accumulation; and (iii) disruption of the nuclear, red blood cell, or host cell membranes. Furthermore, a conserved proline within the hydrophobic domain was required for membrane perforation, suggesting that this residue was crucial for VP4 cytolytic activity. These results indicate that VP4 forms pores in the nuclear membrane leading to lysis and virus release.
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The SV40 late Protein VP4 is a viroporin that forms pores to disrupt membranes for viral release.
PLoS pathogens, 2011Co-Authors: Smita Raghava, Kristina Giorda, Fabian B. Romano, Alejandro P. Heuck, Daniel N. HebertAbstract:Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral Proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed Protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion Protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsulated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ∼3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release.
Carlos F. Arias - One of the best experts on this subject based on the ideXlab platform.
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Antirotaviral activity of bovine milk components: Extending the list of inhibitory Proteins and seeking a better understanding of their neutralization mechanism
Journal of Functional Foods, 2018Co-Authors: José Antonio Parrón, Susana López, Carlos F. Arias, Daniel Ripollés, Ana Cristina Sánchez, María D. Pérez, Miguel Calvo, Lourdes SánchezAbstract:Abstract The viral gastroenteritis mediated by rotavirus remains a major health problem, causing significant infant morbidity and mortality, and health care costs worldwide. Diverse studies have indicated that some milk-derived fractions exhibit antirotaviral activity, associated to Proteins such as immunoglobulins, lactoferrin, mucins, and lactadherin. Nevertheless, further studies are needed to enable greater understanding of the interaction mechanisms involving rotaviruses, host cells, and neutralizing agents. Our results demonstrate that bovine milk is a source of antirotaviral compounds against a wide range of strains. The neutralizing activity of milk fractions was found to be directed to the spike Protein VP4, while the purified milk Proteins inhibited through interaction with either VP4 and/or VP7. The present results highlight the huge potential of bovine milk components for their inclusion in functional foods to control rotavirus diarrhea.
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The tight junction Protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells.
Virology, 2014Co-Authors: Jesús M. Torres-flores, Daniela Silva-ayala, Marco A Espinoza, Susana López, Carlos F. AriasAbstract:Abstract Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction Proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike Protein VP4 determines the use of JAM-A as coreceptor.
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The Spike Protein VP4 Defines the Endocytic Pathway Used by Rotavirus To Enter MA104 Cells
Journal of virology, 2012Co-Authors: Marco A. Díaz-salinas, Y Hoshino, Susana López, Pedro Romero, Rafaela Espinosa, Carlos F. AriasAbstract:Rotaviruses are internalized into MA104 cells by endocytosis, with different endocytic pathways used depending on the virus strain. The bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, while the simian rhesus rotavirus (RRV) strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent. The viral surface Protein VP7 and the spike Protein VP4 interact with cellular receptors during cell binding and penetration. To determine the viral Protein that defines the mechanism of internalization, we used a panel of UK × RRV reassortant viruses having different combinations of the viral structural Proteins. Characterization of the infectivities of these reassortants in MA104 cells either transfected with a small interfering RNA (siRNA) against the heavy chain of clathrin or incubated with hypertonic medium that destabilizes the clathrin coat clearly showed that VP4 determines the pathway of virus entry. Of interest, the characterization of Nar3, a sialic acid-independent variant of RRV, showed that a single amino acid change in VP4 shifts the route of entry from being clathrin dependent to clathrin independent. Furthermore, characterizations of several additional rotavirus strains that differ in their use of cellular receptors showed that all entered cells by clathrin-mediated endocytosis, suggesting that diverse VP4-cell surface interactions can lead to rotavirus cell entry through this endocytic pathway.
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VP7 Mediates the Interaction of Rotaviruses with Integrin αvβ3 through a Novel Integrin-Binding Site
Journal of virology, 2004Co-Authors: Selene Zárate, Carlos F. Arias, Pedro Romero, Rafaela Espinosa, Susana LópezAbstract:Rotavirus entry is a complex multistep process that depends on the trypsin cleavage of the virus spike Protein VP4 into polypeptides VP5 and VP8 and on the interaction of these polypeptides and of VP7, the second viral surface Protein, with several cell surface molecules, including integrin αvβ3. We characterized the effect of the trypsin cleavage of VP4 on the binding to MA104 cells of the sialic acid-dependent virus strain RRV and its sialic acid-independent variant, nar3. We found that, although the trypsin treatment did not affect the attachment of these viruses to the cell surface, their binding was qualitatively different. In contrast to the trypsin-treated viruses, which initially bound to the cell surface through VP4, the non-trypsin-treated variant nar3 bound to the cell through VP7. Amino acid sequence comparison of the surface Proteins of rotavirus and hantavirus, both of which interact with integrin αvβ3 in an RGD-independent manner, identified a region shared by rotavirus VP7 and hantavirus G1G2 Protein in which six of nine amino acids are identical. This region, which is highly conserved among the VP7 Proteins of different rotavirus strains, mediates the binding of rotaviruses to integrin αvβ3 and probably represents a novel binding motif for this integrin.
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Rotavirus gene silencing by small interfering RNAs.
EMBO reports, 2002Co-Authors: Miguel A. Déctor, Susana López, Pedro Romero, Carlos F. AriasAbstract:RNA interference is an evolutionarily conserved double-stranded RNA-triggered mechanism for suppressing gene expression. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of Protein, from which the spike Protein VP4 projects. Here, we show that a small interfering RNA corresponding to the VP4 gene efficiently inhibits the synthesis of this Protein in virus-infected cells. A large proportion of infected cells had no detectable VP4 and the yield of viral progeny was reduced. Most of the virus particles purified from these cells were triple-layered, but lacked VP4, and were poorly infectious. We also show that VP4 might not be required for the last step of virus morphogenesis. The VP4 gene silencing was specific, since the synthesis of VP4 from rotavirus strains that differ in the target sequence was not affected. These findings offer the possibility of carrying out reverse genetics in rotaviruses.
Mario Gorziglia - One of the best experts on this subject based on the ideXlab platform.
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The Outer Capsid Protein VP4 of Murine Rotavirus Strain Eb Represents a Tentative New P Type
Virology, 1994Co-Authors: Mitzi M. Sereno, Mario GorzigliaAbstract:The nucleotide and deduced amino acid sequence of the gene 4 of murine rotavirus strain Eb were determined. The gene is 2359 nucleotides in length and encodes for a Protein of 775 amino acids. Comparison of the VP4 amino acid sequence of the Eb strain with several human and animal rotavirus strains which represent all of the currently recognized distinct VP4 genotypes revealed amino acid identities of from 55.7-75.1% for VP4, 37.1-63.3% for VP8, and 23.9-52.1% for the B region (amino acids 84-180). In addition, antisera to recombinant VP4s of five distinct rotavirus serotypes and two subtypes failed to react significantly by neutralization assay with the Eb strain. Thus, it appears that the Eb strain should be considered a new VP4 genotype and/or serotype.
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the outer capsid Protein VP4 of equine rotavirus strain h 2 represents a unique VP4 type by amino acid sequence analysis
Virology, 1993Co-Authors: Mario Gorziglia, Michele E Hardy, Gerald N WoodeAbstract:The nucleotide and deduced amino acid sequence of G serotype 3 equine rotavirus strain H-2 was determined. A predicted 776-amino-acid H-2 VP4 shows less than or equal to 85.3% identity to other rotavirus VP4 types sequenced to date and thus represents a new P serotype. A PCR-generated probe derived from a cDNA clone of H-2 gene 4 hybridized to gene 4 of several tissue-culture-adapted equine rotavirus isolates, demonstrating that the gene 4 allele present in the H-2 strain is present in the equine rotavirus population.
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amino acid sequence analysis of bovine rotavirus b223 reveals a unique outer capsid Protein VP4 and confirms a third bovine VP4 type
Virology, 1992Co-Authors: Michele E Hardy, Mario Gorziglia, Gerald N WoodeAbstract:Abstract The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 Proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and as 257 to 271) which show high homologies between strains, demonstrate that the region 5′ to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3′ to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5′ flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of 8223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613–615 in the VP5∗ subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.
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Expression of the OSU rotavirus outer capsid Protein VP4 by an adenovirus recombinant.
Journal of virology, 1992Co-Authors: Mario Gorziglia, Albert Z KapikianAbstract:Abstract Full-length cDNA of the VP4 gene of porcine rotavirus strain OSU was cloned into adenovirus type 5 (Ad5) downstream of the E3 promoter. The plaque-purified recombinant (Ad5-OSU VP4) expressed apparently authentic VP4 rotavirus outer capsid Protein. The Protein had the same molecular size (85 kDa) and electrophoretic mobility as did native OSU VP4 and was immunoprecipitated by a polyclonal antiserum raised to OSU VP4. Cotton rats that possessed prechallenge rotavirus antibodies that may have been acquired either passively or actively developed neutralizing antibodies against the OSU strain following intranasal administration of the live Ad5-OSU VP4 recombinant. The neutralizing activity was enhanced by a parenteral booster injection with baculovirus-expressed OSU VP4 antigen. In addition, a high titer of neutralizing antibodies was induced by parenteral administration of the latter antigen and subsequent intranasal administration of the Ad5-OSU VP4 recombinant. These observations indicate that the VP4 outer capsid Protein of a rotavirus strain can be expressed by a recombinant adenovirus vector. This approach warrants further exploration for immunization against rotavirus disease.
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VP4 monotype specificities among porcine rotavirus strains of the same VP4 serotype.
Journal of Virology, 1991Co-Authors: Ferdinando Liprandi, I Rodriguez, C Piña, G Larralde, Mario GorzigliaAbstract:The porcine rotavirus OSU strain was used to produce monoclonal antibodies (MAbs) directed against the outer capsid Protein VP4. From two separate fusions, eight MAbs that inhibited hemagglutination activity of the OSU strain were selected. All MAbs immunoprecipitated both the OSU VP4 Protein derived from a lysate of infected MA104 cells and the OSU VP4 Protein expressed in Sf9 cells by a recombinant baculovirus. By immunoprecipitation of in vitro-translated OSU gene 4 transcripts of different length, the eight MAbs were found to be specific for the VP8 subunit of VP4. All MAbs neutralized the OSU strain but failed to neutralize human, bovine, and simian rotavirus strains. Antiserum to the expressed OSU VP4 Protein was used to study the distribution of VP4 antigenicity among porcine rotaviruses. At least two distinct specificities were identified among 14 rotavirus strains that had been previously assigned to four distinct VP7 serotypes. Five groups of monotype specificities of the VP4 Protein were identified by the eight anti-VP4 MAbs among 11 porcine strains that share the same VP4 serotype.
Jon Agirre - One of the best experts on this subject based on the ideXlab platform.
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Capsid Protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles.
Virology, 2011Co-Authors: Jon Agirre, Rubén Sánchez-eugenia, Kerman Aloria, Jesus M. Arizmendi, Ibon Iloro, Felix Elortza, Gerardo Anibal Marti, Emmanuelle Neumann, Félix A. Rey, Diego M. A. GuérinAbstract:Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid Protein VP4. Its cleavage sites were established by sequencing the N-terminus of the Protein precursor and MS, and its stoichiometry with respect to the other major capsid Proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyProtein. We conclude that VP4 Protein appears as a product of RNA encapsidation, and that defective processing of capsid Proteins precludes genome encapsidation.
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VP4 Protein Appears as a Product of RNA Encapsidation in Triatoma Virus (TRV)
Biophysical Journal, 2011Co-Authors: Jon Agirre, Kerman Aloria, Jesus M. Arizmendi, Ibon Iloro, Felix Elortza, Gerardo Anibal Marti, Emmanuelle Neumann, Félix A. Rey, Diego M. A. GuérinAbstract:Triatoma Virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Its non-enveloped capsid is composed of four Proteins named VP1-VP4, plus the minoritary, uncleaved Protein precursor VP0, which comprises VP4 and VP3. While the smaller Protein VP4 (5.5 kDa versus around 30 kDa for VP1-3) remained undetected in past studies by standard biochemical analyses, the icosahedral (T=1, pseudo-T=3) crystallographic structure of TrV (PDB ID: 3NAP) raised even more suspicions about its existence since no electron density could be attributed to this peptide. In the present work, mass spectrometry (MS) and Tricine-SDS gel electrophoresis were used to detect the previously elusive capsid Protein VP4. Its cleavage sites were established by sequencing the N-terminus of the Protein precursor and MS, and its stoichiometry with respect to the other major capsid Proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyProtein (P1). We conclude that VP4 Protein appears as a product of RNA encapsidation, and that defective processing of capsid Proteins precludes genome encapsidation. Our results also suggest that the TrV capsid can be built without the scaffolding aid of the nucleic acid.
