Absence of subviral particles and assembly activity in HeLa cells infected with defective-interfering (DI) particles of poliovirus

HeLa cells infected with defective-interfering (DI) particles of poliovirus were examined for their capacity to synthesize viral proteins, viral-related particles, and the viral factor(s) that promotes the assembly of 14 S particles into empty capsidsin vitro. As reported by C. N. Cole and D. Baltimore (1973,J. Mol. Biol.76, 325–343) cells infected with purified DI particles failed to synthesize the capsid precursor NCVP 1a or any of the capsid polypeptides VP 0, VP 1, VP 2, or VP 3. Consequently, no 14 S particles, empty capsids, or virions were formed at low to moderate multiplicities. Cytoplasmic extracts prepared from cells infected with purified DI particles do not promote the assembly of viral 14 S particles into empty capsids. We conclude that the presence of assembly activity is dependent on the formation of the capsid precursor protein (NCVP-1a) or its cleavage products.     

The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.     

Identification of a DNA encapsidation sequence for human polyomavirus pseudovirion formation.

Human polyomavirus is a naked capsid virus containing a closed circular double-stranded DNA genome. The mechanism of DNA encapsidation for the viral progeny formation is not fully understood. In this study, DNA encapsidation domain of the major capsid protein, VP1, of the human polyomavirus JCV was investigated. When the first 12 amino acids were deleted, the E. coli expressed VP1 (Delta N12VP1) failed to encapsidate the host DNA although the integrity of the capsid-like structure was maintained. In addition, capsid-like particles of Delta N12VP1 did not package exogenous DNA in vitro, which is in contrast to that of the full-length VP1 protein. These findings suggest that the N-terminal of the first 12 amino acids of VP1 were responsible for DNA encapsidation. The importance of amino acids in the DNA encapsidation domain was determined further using site-directed mutagenesis. All of the positively charged amino acids at the N-terminal region of VP1 were essential for DNA encapsidation. The results indicate that the N-terminal region of the human polyomavirus major capsid protein VP1 may be involved in viral genome encapsidation during progeny maturation.     

Baculovirus structural polypeptides

The structural polypeptides of eight insect baculoviruses were studied using vertical slab polyacrylamide gel electrophoresis. All viruses revealed a complex but unique composition of 15 to 25 bands with molecular weights ranging from 15,000 to 160,000. Since certain baculoviruses have more than one nucleocapsid per viral envelope (multiples), comparisons were made of the multiples and singles (enveloped single nucleocapsids) for each virus. Quantitative and qualitative differences were documented to exist in polypeptide composition. Where possible, the envelopes of certain baculoviruses were selectively removed in order to identify the major capsid proteins. Autographa californica MNPV (NPV: nuclear polyhedrosis virus) capsids contained two major polypeptides, VP18.5 and VP37. Rachiplusia ou MNPV capsids contained several polypeptides of which VP16, VP17, VP18, VP30, and VP36 were considered major constituents. Anticarsa gemmatalis MNPV contained one major capsid protein, VP29, and several minor polypeptides. Major capsid proteins of Heliothis zea SNPV were VP16, VP28, and VP63; as were VP16, VP17, and VP31 of Trichoplusia ni granulosis virus (GV).     

The C-terminal domain of the pVP2 precursor is essential for the interaction between VP2 and VP3, the capsid polypeptides of infectious bursal disease virus

The interaction between the infectious bursal disease virus (IBDV) capsid proteins VP2 and VP3 has been analyzed in vivo using baculovirus expression vectors. Data presented here demonstrate that the 71-amino acid C-terminal-specific domain of pVP2, the VP2 precursor, is essential for the establishment of the VP2-VP3 interaction. Additionally, we show that coexpression of the pVP2 and VP3 polypeptides from independent genes results in the assembly of virus-like particles (VLPs). This observation demonstrates that these two polypeptides contain the minimal information required for capsid assembly, and that this process does not require the presence of the precursor polyprotein.     

A comparative chemical and serological study of the full and empty particles of foot-and mouth disease virus.

The chemical and serological properties of the full, naturally occurring empty and artificially produced empty particles of foot-and-mouth disease virus, serotype A(subtype 10, strain 16) have been studies. The full 146S particles comprised the virus RNA, three polypeptides (VP1 to VP3) mol. wt. about 30 X 10-3, one polypeptide (VP4) mol. wt. about 13-5 X 10-3, and a small amount of a polypeptide (VPo) mol. wt. about 43 X 10-3. The naturally occurring 75S empty particles contained no RNA and much less VP1 and VP4 than were found in the fall particles. However they contained a much greater proportion of VPo than the full particles. Dialysis of purified full particles against tris-EDTA, pH 7-6, produced artificial 75S empty particles which contained only a small amount of RNA and no VP4; otherwise the polypeptide composition was similar to that of the full particles. Immunological and serological tests showed that the full particles were antigenically similar to the naturally occurring empty particles but distinct from the artificial empty particles. The latter particles, however, had serological properties similar to those of the 12S protein subunit of the virus. Both the full and naturally occurring empty particles attached efficiently to susceptible cells, whereas the artificial empty particles attached only to a limited extent. The results are related to the function of the individual polypeptides of the virus particle and compared with published work on other picornaviruses.     

Picornavirus proteins share antigenic determinants with heat shock proteins 60/65

Immunological cross-reactions between enteroviruses and islet cell autoantigens have been suggested to play a role in the etiopathogenesis of insulin dependent diabetes mellitus (IDDM). In the nonobese diabetic mouse, an autoimmune model of IDDM, one of the reactive beta cell autoantigens is the heat shock protein 60 (HSP60). These studies were prompted by sequence homology discovered between the immunogenic region in HSP60 and two regions in enterovirus capsid proteins, one in the VP1 protein and the other in the VP0, the precursor of VP2 and VP4 proteins. Possible immunological cross-reactions between enterovirus proteins and heat shock proteins were studied by EIA and immunoblotting by using purified virus preparations, viral expression proteins VP1 and VP0, and recombinant HSP60/65 proteins, and corresponding polyclonal antisera. The HSP60/65 family of proteins is highly conserved and there is a striking degree of homology between bacterial and human heat shock proteins. Rabbit antibodies to HSP65 of Mycobacterium bovis that reacted with human HSP60 were also found to recognise capsid protein VP1 of coxsackievirus A9, VP1, and/or VP2 of coxsackievirus B4. Both viruses were also recognised by antisera raised against HSP60 of Chlamydia pneumoniae. In addition to the capsid proteins derived from native virions, antisera to both bacterial HSP proteins recognised expression protein VP1 of coxsackievirus A9. The cross-reactivity was also demonstrated the other way around; antisera to purified virus particles reacted with the HSP 60/65 proteins to some extent. These results suggest that apart from the well-documented sequence homology between the 2C protein of coxsackieviruses and the beta-cell autoantigen glutamic acid decarboxylase, there are other motifs in picornavirus proteins homologous to islet cell autoantigens, which might induce cross-reacting immune responses during picornavirus infections.     

Euprosterna elaeasa virus genome sequence and evolution of the Tetraviridae family: Emergence of bipartite genomes and conservation of the VPg signal with the dsRNA Birnaviridae family

The Tetraviridae is a family of non-enveloped positive-stranded RNA insect viruses that is defined by the T = 4 symmetry of virions. We report the complete Euprosterna elaeasa virus (EeV) genome sequence of 5698 nt with no poly(A) tail and two overlapping open reading frames, encoding the replicase and capsid precursor, with ∼67% amino acid identity to Thosea asigna virus (TaV). The N-terminally positioned 17 kDa protein is released from the capsid precursor by a NPGP motif. EeV has 40 nm non-enveloped isometric particles composed of 58 and 7 kDa proteins. The 3′-end of TaV/EeV is predicted to form a conserved pseudoknot. Replicases of TaV and EeV include a newly delineated VPg signal mediating the protein priming of RNA synthesis in dsRNA Birnaviridae. Results of rooted phylogenetic analysis of replicase and capsid proteins are presented to implicate recombination between monopartite tetraviruses, involving autonomization of a sgRNA, in the emergence of bipartite tetraviruses. They are also used to revise the Tetraviridae taxonomy.     

Hepatitis A virus subviral particles: purification, accumulation, and relative infectivity of virions, provirions and procapsids

Summary.  Virus-specific particles were isolated from hepatitis A virus (HAV)-infected cells and the role of each particle type in the replicative cycle assessed. Mature virions, provirions (immature virions) and empty capsids (procapsids) were detected in cell lysates, and both virions and provirions were found in the culture supernatant. Particle types were separated by isopycnic caesium chloride gradient- or linear sucrose density gradient-ultracentrifugation, and their capsid proteins characterised. Virions, provirions and procapsids containing both VP1 and varying levels of the VP1 precursor protein PX were found, suggesting that trimming of PX is not essential for particle formation. Provirions (containing VP0) and virions (containing VP2) could not be clearly separated with these techniques, but sucrose gradients allowed greater separation of particle pools with distinct VP0 contents and specific infectivities which could be used for further studies of the biological role of VP0 cleavage. Virions, with a higher sedimentation coefficient and buoyant density presumably reflecting a more compact structure, had a higher relative infectivity when compared to provirions. HAV-infected cells therefore contain a heterogenous mixture of RNA-containing viral particles with characteristics between those of true provirions and virions, but all such particles are released from the cell and can participate in further rounds of infection. Received April 8, 1997 Accepted July 9, 1997     

Biochemistry of Theiler''s murine encephalomyelitis virus isolated from acutely infected mouse brain: identification of a previously unreported polypeptide

The WW strain of Theiler's murine encephalomyelitis virus (WW-TMEV) was purified from homogenates of acutely infected mouse brain. Infectious WW-TMEV was found to have an estimated sedimentation coefficient of 156 (s20,w) and a density of 1.35 g/cm3 in CsCl. Electron microscopy revealed a homogeneous population of 26-nm nonenveloped particles. Iodination of sodium dodecyl sulfate (SDS)-disrupted virions revealed four major capsid proteins with molecular weights of 58,000, 37,000, 34,000, and 27,000. A 6,000-dalton polypeptide was observed after long exposures of autoradiograms. The 37,000-, 24,000-, 27,000-, and 6,000-dalton polypeptides corresponded to picornaviral VP1, VP2, VP3, and VP4 capsid polypeptides, respectively. Comparison of autoradiograms of virions radiolabeled before and after SDS disruption indicated that the 58,000-dalton protein, VP2, and VP3 preferentially bound 125I under the labeling conditions used. Direct evidence was obtained that VP2 and VP3 were derived from the 58,000-dalton polypeptide by isolation of the 58,000-dalton polypeptide from polyacrylamide gels run under nonreducing conditions and subjecting it to reelectrophoresis under reducing conditions. The effect of trypsin on purified virions and their polypeptides was also investigated. Trypsin-sensitive sites were found in the 58,000-dalton protein, VP1, and VP2. Our results indicate that, in addition to the four typical picornaviral capsid polypeptides, there is a 58,000-dalton polypeptide present in WW-TMEV, which is sensitive to trypsin and can be reduced into two of the capsid proteins, VP2 and VP3.     

Intracellular visualization of precursor capsids in phage P22 mutant infected cells

The morphogenesis of the double-stranded DNA Salmonella phage P22 has been studied by electron microscopy of sections of wild type and mutant-infected cells. Previous work had shown that the precursor capsid structure that encapsidates DNA is a complex of two major protein species, the gene 5 coat protein and the gene 8 scaffolding protein. Scaffolding protein exits from the precursor capsid in coupling with DNA encapsidation and then recycles.No organized structures were seen in cells infected with amber mutants of the coat protein gene. Cells infected with an amber mutant of the scaffolding gene contained small numbers of aberrant particles, including empty petit capsids and giant nested or spiral shell structures.In cells infected with mutants blocked in DNA encapsidation (genes 1, 2, and 3) precursor capsids (proheads) accumulate amid the vegetative DNA and not along the membrane, as in phage T4. The proheads appear as double-shell structures about the size of mature phage, with the inner cell diameter about two-thirds the dimension of the outer shell.Mature phage and defective particles containing DNA form paracrystalline arrays within infected cells. Empty capsids, lacking both DNA and the inner shell of proheads, appear within the paracrystalline arrays of filled heads in cells infected with mutants blocked in head completion (genes 10 and 26). These empty capsids are presumably derived from filled but incomplete heads that have lost their DNA intracellularly.Use of temperature-sensitive mutants blocked in the encapsidation steps allowed visualization of the first filled heads upon shift to permissive temperature. These particles tended to appear at the edge of the DNA pool. Partially filled particles with dense central cores often were seen associated with the growing paracrystalline arrays, and they probably represented intermediates in encapsidation.These experiments, in conjunction with others, suggest that the scaffolding protein, which functions in prohead assembly and perhaps in DNA encapsidation, is organized into an inner shell within the precursor capsid.     

Characterization of the minor polypeptides in the foot-and-mouth disease particle.

In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.     

Purification and properties of virus particles, infectious subviral particles, and cores of bluetongue virus serotypes 1 and 4

Effective purification methods have been developed for virus particles, infectious subviral particles (ISVP), and virus cores of bluetongue virus (BTV) serotypes 1 and 4. The purified particles were analysed by indirect ELISA or PAGE using either silver staining, or fluorography of [35S]methionine-labelled preparations. No significant contamination with host cell proteins, or with the majority of BTV nonstructural proteins was detectable in any of the particle preparations. In addition to the two major outer capsid and five core proteins previously described, the purified virus particles of both serotypes were consistently found to contain small amounts of BTV protein NS2, previously regarded as exclusively nonstructural. This protein could be removed from the particle surface by treatment with a combination of chymotrypsin and sodium N-lauroyl sarcosinate, which also resulted in the cleavage of the larger of the two major outer capsid components (protein VP2). Two of the cleavage products of VP2 and the whole of the other major outer capsid component (protein VP5) formed a modified outer capsid layer in the resultant ISVP. These subviral particles were as or more infectious than the intact virus particles but had lost haemagglutinating activity. The core-associated RNA polymerase remained inactive in ISVP.     

DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus

The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300 kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.     

Ultrastructural localization of rotavirus antigens using colloidal gold

Colloidal gold was used to localize six of the ten known proteins of the simian rotavirus SA11 within infected cells by ultrastructural immunocytochemistry. Monospecific or monoclonal antibodies to selected structural and nonstructural proteins were the primary antisera. The major outer capsid glycoprotein, VP7, was associated with nonenveloped particles, with particles de-enveloped by Triton X-100 and with both nuclear and cytoplasmic inclusions. The protease-sensitive outer capsid protein, VP3, was also found on nonenveloped and de-enveloped particles. The major inner capsid protein, VP6, was accessible to antibodies on some of the nonenveloped particles (presumably single-shelled particles) and on the de-enveloped particles. A monospecific antibody to the gene 11 product, believed to be a precursor to a minor structural protein, VP9, reacted strongly with viroplasmic inclusions. Virus particles were weakly labeled by this antibody. NS35, a nonstructural SA11 protein, was found only in the viroplasms. NS29, a nonstructural glycoprotein, was localized to the cytoplasmic side of the endoplasmic reticulum membrane and to the inside of enveloped virus particles. These data support the hypothesis that NS29 facilitates budding of the virus particles and acquisition of the outer capsid layer.     

Comparison of the antibodies elicited by the individual structural polypeptides of foot-and mouth disease and polio viruses.

Antibody produced against preparations of VP1, one of the four structural polypeptides of foot-and-mouth disease virus, neutralized the virus and reacted with both full and empty particles in radioimmunoassays (RIA). Antiserum against VP2 reacted with artificial empty particles of the virus but not with full particles. In contrast, none of the individual polypeptides of poliovirus produced antisera which neutralized the virus nor reacted with it in RIA. However, antisera produced with VP1 and VP2 reacted with artificial empty particles in RIA.     

Efficient generation of cowpea mosaicvirus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants

To elucidate the mechanism of formation of cowpea mosaic virus (CPMV) particles, RNA-2-encoded precursor proteins were expressed in Spodoptera frugiperda cells. Processing of the 105K and 95K polyproteins in trans to give the mature Large (L) and Small (S) coat proteins required both the 32K proteinase cofactor and the 24K proteinase itself, while processing of VP60, consisting of the fused L-S protein, required only the 24K proteinase. Release of the L and S proteins resulted in the formation of virus-like particles (VLPs), showing that VP60 can act as a precursor of virus capsids. Processing of VP60 expressed in plants also led to efficient production of VLPs. Analysis of the VLPs produced by the action of the 24K proteinase on precursors showed that they were empty (RNA-free). This has important implications for the use of CPMV VLPs in biotechnology and nanotechnology as it will permit the use of noninfectious particles.     

The morphogenesis of Theiler’s murine encephalomyelitis virus is strain specific

Summary.  During a single cycle infection with the neurovirulent GDVII- and demyelinating DA-strain of Theiler’s murine encephalomyelitis virus (TMEV) in L-929 cells, different subviral particles were found for both strains. Early in the assembly process, the DA-strain generated 14 S pentamers composed of the viral proteins VP0, VP1 and VP3, while in GDVII-infected cells, particles with the same protein composition but with a sedimentation coefficient of 20 S were found. These newly discovered 20 S particles are probably virion assembly precursors considering their capsid protein composition and their early time of appearance in infected cells. Near the end of the assembly process, VP0, VP1 and VP3 containing 80 S empty capsids became apparent in GDVII-infected cells, while these particles could not be found in DA-infected cells. The significance of these empty capsids will be discussed. After virion assembly, 14 S particles were observed for both strains. These 14 S particles resulted from the degradation of the 160 S virions as indicated by their protein composition (VP1, VP2, VP3) and time of appearance. Our results demonstrate that the assembly of the GDVII-strain differs from that of the DA-strain. In addition, the strain-specific assembly of TMEV implies that not all picornaviruses assemble as proposed by the poliovirus morphogenesis model and thus rendering its general validity questionable. Received October 14, 2002; accepted January 3, 2003 Published online March 21, 2003     

Immune response to hepatitis A virus capsid proteins after infection.

This study was undertaken to determine the immune response of humans to viral capsid polypeptides of hepatitis A virus (HAV) after natural infection, which is very important for vaccine development. Antiviral capsids in 73 serum samples from patients with acute and chronic HAV infections were analyzed by immunoblotting against individual HAV capsid polypeptides (VP1, VP2, VP3, and VP4) by using a cell culture-based HAV antigen. For reference, total anti-HAV immunoglobulin G (IgG) and anti-HAV IgM were also determined by radioimmunoassay. As a result, a dominant immune response against VP1 (98% IgG, 94% IgM) was found in the acute phase. However, many other sera also reacted with VP0 (88% IgG; 35% IgM) and VP3 (81% IgG and 29% IgM). In contrast to the acute phase, anti-VP1, anti-VP0, and anti-VP3, IgG antibodies against all three viral proteins (29, 29, and 73% respectively), especially those against VP3, were found years after onset of HAV disease and over long periods in the sera of hepatitis patients. These results suggest that antibodies for capsid polypeptides are present over an extended period in the sera of HAV-infected patients. They are likely of importance in maintaining long-term immunity.     

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS–PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.