scholarly journals The Autographa californica Multiple Nucleopolyhedrovirusac54Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

2016 ◽  
Vol 90 (8) ◽  
pp. 4115-4126 ◽  
Author(s):  
Zhanwen Guan ◽  
Ling Zhong ◽  
Chunyan Li ◽  
Wenbi Wu ◽  
Meijin Yuan ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Autographa Californica ◽  
Capsid Proteins ◽  
Capsid Assembly ◽  
Filamentous Actin ◽  
Content Type ◽  
Minor Capsid Protein ◽  
Virogenic Stroma ◽  
Assembly Site

ABSTRACTBaculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate theac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma,ac54ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasind-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected byac54deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly.IMPORTANCEBaculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of capsids into the capsid assembly site requires virus-induced nuclear F-actin; the inhibition of nuclear F-actin formation results in the retention of capsid structures at the periphery of the nucleus. In this paper, we note that the minor capsid protein VP1054 is essential for the localization of capsid structures, the major capsid protein VP39, and the minor capsid protein 38K into the capsid assembly site. Moreover, VP1054 is crucial for correct targeting of the nuclear F-actin factors BV/ODV-C42 and PP78/83 for capsid maturation. However, the formation and distribution of nuclear F-actin are not affected by the lack of VP1054. We further reveal that VP1054 interacts with BV/ODV-C42 and a capsid transport-related protein, VP80. Taken together, our findings suggest that VP1054 plays a unique role in the pathway(s) for transport of capsid proteins.

scholarly journals The Minor Capsid Protein VP11 of Thermophilic Bacteriophage P23-77 Facilitates Virus Assembly by Using Lipid-Protein Interactions

2015 ◽  
Vol 89 (15) ◽  
pp. 7593-7603 ◽  
Author(s):  
Alice Pawlowski ◽  
Anni M. Moilanen ◽  
Ilona A. Rissanen ◽  
Juha A. E. Määttä ◽  
Vesa P. Hytönen ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Building Blocks ◽  
Capsid Proteins ◽  
Major Protein ◽  
Protein Species ◽  
Virus Family ◽  
Content Type ◽  
Minor Capsid Protein ◽  
Capsid Shell

ABSTRACTThermus thermophilusbacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrostatic interaction with negatively charged molecules, including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function of VP11 is elucidated by demonstrating the interactions of VP11 withThermus-derived LUVs and with the major capsid proteins by means of the dynamic-light-scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers the binding of the major capsid protein VP17, thus facilitating a controlled incorporation of the two different major protein species into the assembling capsid.IMPORTANCEThe study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein charge is important in order to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.

scholarly journals Modulation of a Pore in the Capsid of JC Polyomavirus Reduces Infectivity and Prevents Exposure of the Minor Capsid Proteins

2015 ◽  
Vol 89 (7) ◽  
pp. 3910-3921 ◽  
Author(s):  
Christian D. S. Nelson ◽  
Luisa J. Ströh ◽  
Gretchen V. Gee ◽  
Bethany A. O'Hara ◽  
Thilo Stehle ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Feature ◽  
Host Cell ◽  
Capsid Protein ◽  
Major Capsid Protein ◽  
Capsid Proteins ◽  
Jc Polyomavirus ◽  
Minor Capsid Protein ◽  
X Ray ◽  
Infectious Entry

ABSTRACTJC polyomavirus (JCPyV) infection of immunocompromised individuals results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). The viral capsid of JCPyV is composed primarily of the major capsid protein virus protein 1 (VP1), and pentameric arrangement of VP1 monomers results in the formation of a pore at the 5-fold axis of symmetry. While the presence of this pore is conserved among polyomaviruses, its functional role in infection or assembly is unknown. Here, we investigate the role of the 5-fold pore in assembly and infection of JCPyV by generating a panel of mutant viruses containing amino acid substitutions of the residues lining this pore. Multicycle growth assays demonstrated that the fitness of all mutants was reduced compared to that of the wild-type virus. Bacterial expression of VP1 pentamers containing substitutions to residues lining the 5-fold pore did not affect pentamer assembly or prevent association with the VP2 minor capsid protein. The X-ray crystal structures of selected pore mutants contained subtle changes to the 5-fold pore, and no other changes to VP1 were observed. Pore mutant pseudoviruses were not deficient in assembly, packaging of the minor capsid proteins, or binding to cells or in transport to the host cell endoplasmic reticulum. Instead, these mutant viruses were unable to expose VP2 upon arrival to the endoplasmic reticulum, a step that is critical for infection. This study demonstrated that the 5-fold pore is an important structural feature of JCPyV and that minor modifications to this structure have significant impacts on infectious entry.IMPORTANCEJCPyV is an important human pathogen that causes a severe neurological disease in immunocompromised individuals. While the high-resolution X-ray structure of the major capsid protein of JCPyV has been solved, the importance of a major structural feature of the capsid, the 5-fold pore, remains poorly understood. This pore is conserved across polyomaviruses and suggests either that these viruses have limited structural plasticity in this region or that this pore is important in infection or assembly. Using a structure-guided mutational approach, we showed that modulation of this pore severely inhibits JCPyV infection. These mutants do not appear deficient in assembly or early steps in infectious entry and are instead reduced in their ability to expose a minor capsid protein in the host cell endoplasmic reticulum. Our work demonstrates that the 5-fold pore is an important structural feature for JCPyV.

scholarly journals Modification of Human Papillomavirus Minor Capsid Protein L2 by Sumoylation

2010 ◽  
Vol 84 (21) ◽  
pp. 11585-11589 ◽  
Author(s):  
Martina Bergant Marušič ◽  
Nina Mencin ◽  
Mia Ličen ◽  
Lawrence Banks ◽  
Helena Šterlinko Grm
Keyword(s):  
Human Papillomavirus ◽  
Infected Cell ◽  
Host Cell ◽  
Capsid Protein ◽  
Major Capsid Protein ◽  
Capsid Assembly ◽  
Complex Interplay ◽  
Hpv 16 ◽  
Minor Capsid Protein ◽  
Viral Particles

ABSTRACT The human papillomavirus (HPV) minor capsid protein L2 plays important roles in the generation of infectious viral particles and in the initial steps of infection. Here we show that HPV-16 L2 protein is sumoylated at lysine 35 and that sumoylation affects its stability. Interestingly, the sumoylated form of L2 cannot bind to the major capsid protein L1, suggesting a mechanism by which capsid assembly may be modulated in an infected cell. Additionally, L2 appears to modulate the overall sumoylation status of the host cell. These observations indicate a complex interplay between the HPV L2 protein and the host sumoylation machinery.

scholarly journals Parvovirus LuIII transducing vectors packaged by LuIII versus FPV capsid proteins: the VP1 N-terminal region is not a major determinant of human cell permissiveness

2004 ◽  
Vol 85 (5) ◽  
pp. 1251-1257 ◽  
Author(s):  
Ian H. Maxwell ◽  
Françoise Maxwell
Keyword(s):  
Capsid Protein ◽  
Human Cell ◽  
Major Capsid Protein ◽  
Human Cells ◽  
Terminal Region ◽  
Major Determinant ◽  
Capsid Proteins ◽  
Entry Level ◽  
Capsid Protein Vp1 ◽  
Post Entry

Human cell lines are permissive for LuIII, a member of the rodent group of autonomous parvoviruses. However, LuIII vectors pseudotyped with feline panleukopaenia virus (FPV) capsid proteins can transduce feline cells but not human cells. Feline transferrin receptor (FelTfR) functions as a receptor for FPV. Transfection of Rh18A, a human rhabdomyosarcoma cell line, with FelTfR enabled transduction by vector with FPV capsid. This was not true of other human lines, suggesting restriction at some additional, post-entry, level(s) in human cells other than Rh18A. It seemed a reasonable hypothesis that a second blockage might be in nuclear delivery mediated by the N-terminal region of the minor capsid protein, VP1. We therefore generated virions containing an LuIII–luciferase genome, packaged using chimaeric VP1 molecules (N-terminal region of LuIII VP1, fused with body of FPV, and vice versa) together with the major capsid protein, VP2, of FPV or LuIII. The virions were tested for ability to transduce feline and human cells. Our hypothesis predicted that the N-terminal region of LuIII VP1 should allow transduction of human cells expressing FelTfR, while the FPV N-terminal region should not allow transduction of human cells (except for Rh18A). The experimental results did not bear out either of these predictions. Therefore, the VP1 N-terminal region appears not to be a major determinant of permissiveness for LuIII, versus FPV, capsid in human cells.

scholarly journals The Abundant Nuclear Enzyme PARP Participates in the Life Cycle of Simian Virus 40 and Is Stimulated by Minor Capsid Protein VP3

2003 ◽  
Vol 77 (7) ◽  
pp. 4273-4282 ◽  
Author(s):  
Ariela Gordon-Shaag ◽  
Yael Yosef ◽  
Mahmoud Abd El-Latif ◽  
Ariella Oppenheim
Keyword(s):  
Amino Acids ◽  
Life Cycle ◽  
Capsid Protein ◽  
Membrane Integrity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Capsid Proteins ◽  
Dependent Manner ◽  
Minor Capsid Protein ◽  
Stimulation Of

ABSTRACT The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.

scholarly journals A Newly Isolated Reovirus Has the Simplest Genomic and Structural Organization of Any Reovirus

2014 ◽  
Vol 89 (1) ◽  
pp. 676-687 ◽  
Author(s):  
Albert J. Auguste ◽  
Jason T. Kaelber ◽  
Eric B. Fokam ◽  
Hilda Guzman ◽  
Christine V. F. Carrington ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Structural Characteristics ◽  
Infected Cell Line ◽  
Asymmetric Unit ◽  
Loss Of Function ◽  
Content Type ◽  
Mosquito Cells ◽  
A Genome ◽  
Pathogenic Viruses

ABSTRACTA total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genusDinovernavirus) were detected by culturing mosquito pools onAedes albopictus(C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events.IMPORTANCEWe describe the detection, genetic, phenotypic, and structural characteristics of a novelDinovernavirusspecies isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.

scholarly journals Self-Assembly of Epstein-Barr Virus Capsids

2009 ◽  
Vol 83 (8) ◽  
pp. 3877-3890 ◽  
Author(s):  
Brandon W. Henson ◽  
Edward M. Perkins ◽  
Jonathan E. Cothran ◽  
Prashant Desai
Keyword(s):  
Amino Acids ◽  
Capsid Protein ◽  
Self Assembly ◽  
Epstein Barr Virus ◽  
Major Capsid Protein ◽  
Mutational Analysis ◽  
Capsid Assembly ◽  
Barr Virus ◽  
Capsid Shell ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV), a member of the Gammaherpesvirus family, primarily infects B lymphocytes and is responsible for a number of lymphoproliferative diseases. The molecular genetics of the assembly pathway and high-resolution structural analysis of the capsid have not been determined for this lymphocryptovirus. As a first step in studying EBV capsid assembly, the baculovirus expression vector (BEV) system was used to express the capsid shell proteins BcLF1 (major capsid protein), BORF1 (triplex protein), BDLF1 (triplex protein), and BFRF3 (small capsid protein); the internal scaffold protein, BdRF1; and the maturational protease (BVRF2). Coinfection of insect cells with the six viruses expressing these proteins resulted in the production of closed capsid structures as judged by electron microscopy and sedimentation methods. Therefore, as shown for other herpesviruses, only six proteins are required for EBV capsid assembly. Furthermore, the small capsid protein of EBV (BFRF3), like that of Kaposi's sarcoma-associated herpesvirus, was found to be required for assembly of a stable structure. Localization of the small capsid protein to nuclear assembly sites required both the major capsid (BcLF1) and scaffold proteins (BdRF1) but not the triplex proteins. Mutational analysis of BFRF3 showed that the N-terminal half (amino acids 1 to 88) of this polypeptide is required and sufficient for capsid assembly. A region spanning amino acids 65 to 88 is required for the concentration of BFRF3 at a subnuclear site and the N-terminal 65 amino acids contain the sequences required for interaction with major capsid protein. These studies have identified the multifunctional role of the gammaherpesvirus small capsid proteins.

scholarly journals The 4.4 Å structure of the giant Melbournevirus virion belonging to the Marseilleviridae family

2021 ◽  
Author(s):  
Raymond N Burton-Smith ◽  
Hemanth K N Reddy ◽  
Martin Svenda ◽  
Chantal Abergel ◽  
Kenta Okamoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Capsid Protein ◽  
Major Capsid Protein ◽  
Capsid Proteins ◽  
Atomic Model ◽  
Penton Base ◽  
Cryo Electron Microscopy ◽  
Internal Membrane ◽  
Structural Details ◽  
Giant Viruses

Members of Marseilleviridae, one family of icosahedral giant viruses classified in 2012 have been identified worldwide in all types of environments. The virion shows a characteristic internal membrane extrusion at the five-fold vertices of the capsid, but its structural details need to be elucidated. We now report the 4.4 Å cryo-electron microscopy structure of the Melbournevirus capsid. An atomic model of the major capsid protein (MCP) shows a unique cup structure on the trimer that accommodates additional proteins. A polyalanine model of the penton base protein shows internally extended N- and C-terminals, which indirectly connect to the internal membrane extrusion. The Marseilleviruses share the same orientational organisation of the MCPs as PBCV-1 and CroV, but do not appear to possess a protein akin to the ″tape measure″ of these viruses. Minor capsid proteins named PC-β, zipper, and scaffold are proposed to control the dimensions of the capsid during assembly.

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