Synthesis and assembly of hepatitis A virus-specific proteins in BS-C-1 cells.

To determine the mechanism for the delayed and inefficient replication of the picornavirus hepatitis A virus in cell culture, we studied the kinetics of synthesis and assembly of virus-specific proteins by metabolic labeling of infected BS-C-1 cells with L-[35S]methionine and L-[35S]cysteine. Sedimentation, electrophoresis, and autoradiography revealed the presence of virions, provirions, procapsids, and 14S (pentameric) subunits. Virions and provirions contained VP1, VP0, VP2, and VP3; procapsids contained VP1, VP0, and VP3; and pentamers contained PX, VP0, and VP3, as previously shown by immunoblotting (D.A. Anderson and B.C. Ross, J. Virol. 64:5284-5289, 1990). Under single-cycle growth conditions label was found in 14S subunits immediately after labeling from 15 to 18 h postinfection (p.i.); however, a proportion of labeled polyprotein was not cleaved and assembled into pentamers for a further 18 h. When analyzed at 72 h p.i., incorporation of label which flowed into virions was detected from 3 h p.i., with maximal uptake levels being observed from 12 to 15 h p.i. Viral antigen, infectious virus, and viral RNA were determined in parallel, with coincident peaks in these variables being observed 12 h after the period of maximum label uptake. It was also found that the lag between the synthesis of the viral polyprotein and assembly of viral particles was the same after labeling from either 12 to 15 or 27 to 30 h p.i. despite increased levels of viral RNA during this period, suggesting that factors additional to the level of RNA are involved in the restriction of viral replication. Sedimentation and immunoblot analysis revealed an additional protein of approximately 100 kDa containing both VP1- and VP2-reactive sequences and sedimenting slightly more slowly than 14S pentamers, which may represent intact P12A assembled into pentamers as has been reported for the P1 of some other picornaviruses (S. McGregor and R. R. Rueckert, J. Virol. 21:548-553, 1977). The results of this study suggest that cleavage of the hepatitis A virus polyprotein to produce pentamers is protracted (though not rate limiting) early in infection, while the assembly of pentamers into higher structures is a rapid process once sufficient viral RNA is produced for encapsidation.     

Thermosensitive Block of the Sabin Strain of Poliovirus Type I

The thermosensitive defect of the Sabin LSc2ab strain of poliovirus type I was studied. Transfer of infected KB cells from 36 to 38.5 C resulted in 30% inhibition of viral RNA replication but in 90% inhibition of formation of virions. Neither 74S procapsids nor 14S particles were detected in the cells transferred to the non-permissive temperature. However, procapsids, once accumulated at 36 C, were normally stable at 38.5 C and could transform into virions at that temperature. Viral proteins synthesized at the nonpermissive temperature were not different from those synthesized at permissive temperature, as judged from their pattern in polyacrylamide gel electrophoresis and from the fact that they normally matured into virions when the infected cells were brought back to permissive temperature, even under conditions of inhibition of protein synthesis. This leads to the conclusion that the defect in the Sabin strain studied lies in the assembly of its viral capsid proteins into capsomeres.     

Viral protein synthesis in mouse hepatitis virus strain A59-infected cells: effect of tunicamycin.

We identified eight protein species in virions of mouse hepatitis virus strain A59. Based on their sizes, prosthetic groups, and locations in virions, these proteins were designated gp180/E2, gp90/E2, pp54/N, gp26.5/E1, gp25.5/E1, p24/E1, p22/X, and p14.5/Y. The positions of the last two proteins in virions are not known. Host protein synthesis in Sac(-) cells infected with mouse hepatitis virus strain A59 was inhibited, and the following novel proteins appeared: gp150, gp90, p54, gp26.5, gp25.5, p24, p22, and p14.5. Except for gp150, these polypeptides all co-electrophoresed with mouse hepatitis virus strain A59 structural proteins. In addition, all of these proteins could be immunoprecipitated with a convalescent mouse serum or a rabbit antiserum raised against purified disrupted virus. After a 15-min pulse of infected cells with radioactive amino acids at 7h postinfection, gp90 was not detected, whereas gp26.5 and gp25.5 were only labeled to a small extent. During a subsequent chase period gp150 was processed to gp90, whereas the radioactivity in gp26.5 and gp25.5 increased concomitantly with a reduction of label in p24. Tunicamycin, an antibiotic which inhibits the synthesis of glycopeptides bearing N glycosidically linked oligosaccharides, prevented the appearance of gp150 in mouse hepatitis virus strain A59-infected cells. Instead, a 110,000-dalton protein accumulated. In contrast, the syntheses of the smaller viral glycoproteins gp26.5 and gp25.5 were resistant to this drug, indicating that these glycosylations were of the O glycosidical type. Although the production of infectious virus in tunicamycin-treated cells was inhibited by more than 99%, release of noninfectious viral particles continued. An analysis of these particles revealed that they lacked the peplomeric glycoproteins gp90/E2 and gp180/E2. Obviously, although the surface projections were not essential for budding of virus particles from the cells, they were required for infectivity.     

Modulation of mouse mammary tumor virus production in the MJY-alpha cell line.

Implantation of the mouse mammary tumor virus (MMTV)-producing mammary tumor cell line MJY-alpha into isogeneic mice elicited both humoral and T-cell responses against MMTV virion antigens. The carcinosarcomas which developed from the implanted cells showed a significant decrease in MMTV synthesis, compared with cells remaining in culture, which was detectable as early as 7 days after implantation and for five transplant generations. Electron microscopic examination of thin sections of the tumors revealed that intracytoplasmic A particles, budding particles, and cell-free MMTV B particles were all affected. However, immunofluorescence assays of tumor sections demonstrated the presence of MMTV viral antigens in the cells. Cell cultures initiated from first-, third-, and fourth-generation tumors were morphologically identical to the original in vitro cell line, although virus production was barely detectable. Analysis of the cultures by electron microscopy revealed a significant increase in MMTV virions after in vitro passage 3. Polypeptide profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from these cultures were identical to MMTV. Immunodiffusion demonstrated the cross-reactivity between these virions and MMTV particles obtained from mouse milk. In vitro treatment of MJY-alpha cell cultures with rabbit anti-MMTV antiserum resulted in a reduction of extracellular MMTV virions, as well as alterations in their sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide patterns.     

Chlamydiaphage Chp2, a skeleton in the phiX174 closet: scaffolding protein and procapsid identification

Chlamydiaphage Chp2 is a member of the family Microviridae, of which bacteriophage phiX174 is the type species. Although grouped in the same family, the relationship between the Microviridae coliphages and the Chp2-like viruses, which infect obligate intracellular parasitic bacteria, is quite distant, with major differences in structural protein content and scaffolding protein dependence. To investigate the morphogenesis of Chp2, large particles were isolated from infected Chlamydophila abortus by equilibrium and rate zonal sedimentation. A monoclonal antibody that recognizes only assembled viral coat proteins was used in these detection assays. Thus, the detected particles represent virions and/or postcapsid formation assembly intermediates. Two distinct particle types were detected, differing in both protein and DNA content. Filled particles lacked VP3, the putative internal scaffolding protein, whereas empty particles contained this protein. These results indicate that VP3 is a scaffolding protein and that the isolated VP3-containing particles most likely represent Chp2 procapsids.     

Mode of subacute sclerosing panencephalitis (SSPE) virus infection in tissue culture cells. I. Detection of infectious virions from cells infected with Niigata-1 strain of SSPE virus.

By the aid of freezing and thawing, cell-free infectious virions were detected from an apparently nonproductive Vero cell line infected with Niigata-1 strain of subacute sclerosing panencephalitis virus. The production of infectious virions was limited in amount and such virions were detectable only during a limited period after cell subculture. The infectious virions were filtrable through a 0.65 mu membrane filter and neutralized completely by an antiserum against measles virus. The virions were banded at the density of 1.132, while Edmonston strain of measles virus banded at 1.164 in potassium tartrate density gradients. Infectious virions were also released from infected Vero cells by treatment of the cells in a hypotonic solution to an amount comparable to that obtained by freezing and thawing. Infection of normal culture of Vero cells with the infectious virions readily established a virus-cell interaction identical to that in the original infected culture from which the virions were recovered.     

The DNA-packaging nanomotor of tailed bacteriophages

Tailed bacteriophages use nanomotors, or molecular machines that convert chemical energy into physical movement of molecules, to insert their double-stranded DNA genomes into virus particles. These viral nanomotors are powered by ATP hydrolysis and pump the DNA into a preformed protein container called a procapsid. As a result, the virions contain very highly compacted chromosomes. Here, I review recent progress in obtaining structural information for virions, procapsids and the individual motor protein components, and discuss single-molecule in vitro packaging reactions, which have yielded important new information about the mechanism by which these powerful molecular machines translocate DNA.     

Intracellular production of virus particles and viral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect of interferon.

The effect of interferon on the biochemical properties and the maturation process of intracellular viral particles isolated from the cytoplasmic fraction of NIH/3T3 cells chronically infected with Moloney murine leukemia virus was investigated. By labeling these virions with either [35S]methionine or [3H]glucosamine, we demonstrated that they contain the same viral proteins and glycoproteins found in extracellular virions. Interferon treatment was found to reduce the rate of intracellular virus assembly. This effect was not a consequence of an interferon inhibition of viral RNA synthesis or its translation or a consequence of an interference with the posttranslational cleavage processing of viral precursor proteins, since all of these steps were not affected by interferon. However, the reduced rate of virus assembly could be attributed to the inhibition of viral protein glycosylation observed in interferon-treated cells. Nevertheless, despite this reduced rate, virus particles accumulated in interferon-treated cells. This accumulation was probably due to the strong inhibition of their final release from such cells.     

Defective interfering passages of Sindbis virus: nature of the defective virion RNA.

Defective interfering particles of Sindbis virus contain 20S RNA identical to that found in BHK cells co-infected with standard and defective virions. We have characterized these RNAs by their oligonucleotide fingerprints. Most of the oligonucleotides were identical to those found in the mRNA (26S RNA) that codes for the virion structural proteins. Three oligonucleotides found in 20S RNA were absent from the 26S RNA pattern and may represent sequences from the 5' end of the virion RNA. Previous difficulties in describing the nature of the defective virion RNA were due to the aggregated state of the RNA. Nucleocapsids obtained from standard and defective virions were essentially the same size and had about the same density, suggesting that defective particles contain more than a single molecule of 20S RNA.     

Subgenomic mRNA of Aura alphavirus is packaged into virions.

Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined.     

Poliovirus morphogenesis. I. Identification of 80S dissociable particles and evidence for the artifactual production of procapsids.

The current model of poliovirus morphogenesis postulates a fundamental role for procapsid, 80S shells that, upon interaction with viral RNA and subsequent proteolytic cleavage, give rise to complete virus particles. Although 80S sedimenting particles can, indeed, be isolated from cytoplasmic extracts of infected cells, their physical properties differ from those reported for procapsids. Far from being stable structures, they can be dissociated by pH 8.5 and 0.1% sodium dodecyl sulfate into slower-sedimenting subunits. The reasons for this discrepancy were investigated, and two main modalities leading to the appearance of procapsids in vitro were identified. The first involves a temperature-mediated conversion of dissociable 80S particles into stable 80S procapsids, and the second involves the self-assembly of endogenous 14S subunits, also primed by an increase in the temperature of cytoplasmic extracts.     

Cytomegalovirus proteins. I. Polypeptides of virions and dense bodies.

Cytomegalovirus virions and dense bodies were purified by sucrose velocity and equilibrium centrifugation from the medium of fibroblasts infected with the strain AD169. The final virus preparations were purified more than 228-fold with respect to cellular proteins as determined by double-isotopic labeling and at least 1,600-fold on the basis of changes in the ratio of total protein to virus particles. The protein content of purified particles approximated that found for purified preparations of other herpesviruses. Twenty polypeptides ranging from 22,000 to greater than 230,000 molecular weight were detected in purified virus preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polypeptides of virions and dense bodies were allocated on the basis of analyses of preparations containing differing percentages of virions and dense bodies. Six polypeptides were represented predominantly or exclusively in virions, and four polypeptides were represented predominantly or exclusively in dense bodies, whereas the remainder appeared to be shared by both types of particles. Four polypeptides were glycosylated, and at least three of these appeared to be shared by both particles. Four polypeptides were glycosylated, and at least three of these appeared to be shared by both particle types. The protein composition of cytomegalovirus differs profoundly from that of herpes simplex virus.     

Homogeneous hepatitis A virus particles. Proteolytic release of the assembly signal 2A from procapsids by factor Xa

Among the picornaviridae, hepatitis A virus (HAV) is unique in that its assembly is driven by domain 2A of P1-2A, the precursor of the structural proteins (Probst, C., Jecht, M., and Gauss-Müller, V. (1999) J. Biol. Chem. 274, 4527-4531). Whereas infected individuals excrete in stool mature HAV capsids with VP1 as the major structural protein, its C-terminal extended form VP1-2A is the main component of immature procapsids produced in HAV-infected cells in culture. Obviously, a postassembly proteolytic step is required to remove the primary assembly signal 2A from VP1-2A of procapsids. Mutants of VP1-2A were expressed in COS7 cells to determine the cleavage site in VP1-2A and to test for the cleavage potential of viral and host proteinases (factor Xa and thrombin). Site-specific in vitro cleavage by factor Xa and thrombin occurred in procapsids that contained VP1-2A with engineered cognate cleavage sites for these proteinases. Interestingly, factor Xa but not thrombin liberated mature VP1 also from native procapsids in an assembly-dependent manner. The data show that domain 2A, which is required for pentamerization of its precursor polypeptides and thus for the primary step of HAV assembly, is removed from the surface of immature procapsid by a host proteinase. Moreover, our data open a novel avenue to produce homogeneous HAV particles from recombinant intermediates by in vitro treatment with exogenously added proteases such as factor Xa or thrombin.     

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus.

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus were studied. Electron micrographs of the infected cells showed that most of mutant virions bud from the plasma or vacuolar membrane as a multiploid particle containing a various number of nucleocapsids enclosed with a defined common envelope. The mutant virions contained three polypeptides which migrated to the position identical with those of wild type on discontinuous acrylamide gels. Cells infected with the mutant virus synthesized the same intracellular viral RNA species as was made after infection of wild type. Cytoplasmic nucleocapsids of the mutant sedimented at 140S and contained 42S virion RNA as those of wild type; they were indistinguishable from those of wild type in an electron microscope examination. On the other hand, mutant nucleocapsids isolated from extracellular virions sedimented as heterogeneous particles larger thant 140S and were shown to be pleomorphic and aggregate in electron micrographs. The budding process of this mutant seemed to be modified, so that it might form the multiploid with the alteration of its nucleocapsids.     

Proteins Specified by bovine herpesvirus 1 (infectious bovine rhinotracheitis virus)

An electrophoretic analysis of radioactively labeled, purified, "empty" and DNA-containing infectious bovine rhinotracheitis virions revealed the presence of 25 to 33 structural (virion) polypeptides. A total of 11 of these polypeptides could be labeled with [3H]glucosamine and were identified as glycoproteins. In addition to the 25 structural polypeptides, infectious bovine rhinotracheitis virus infected cells also contained at least 15 nonstructural (nonvirion) polypeptides that were not present in purified virions. Expression of the viral polypeptides in infected cells was controlled temporally. Thus, most viral polypeptides could be categorized as "alpha" (immediate early), "beta" (early), or "gamma" (late) on the basis of their order of appearance in infected cells and whether their syntheses were dependent upon prior viral protein or DNA synthesis. None of the glycoproteins belongs to the alpha class, although at least one (GVP11) was synthesized in the absence of viral DNA synthesis. Serum from a cow in which infectious bovine rhinotracheitis virus lesions were reactivated by dexamethasone precipitated both structural and nonstructural polypeptides.     

Expression of the influenza A virus M2 protein is restricted to apical surfaces of polarized epithelial cells.

The M2 protein of influenza A virus is a small, nonglycosylated transmembrane protein that is expressed on surfaces of virus-infected cells. A monoclonal antibody specific for the M2 protein was used to investigate its expression in polarized epithelial cells infected with influenza virus or a recombinant vaccinia virus that expresses M2. The expression of M2 on the surfaces of influenza virus-infected cells was found to be restricted to the apical surface, closely paralleling that of the influenza virus hemagglutinin (HA). Membrane domain-specific immunoprecipitation indicated that the M2 protein was inserted directly into the apical membrane with transport kinetics similar to those of HA. In polarized cells infected with a recombinant vaccinia virus that expresses M2, we found that 86 to 93% of surface M2 was restricted to the apical domain compared with 88 to 90% of HA in a similar assay. These results indicate that the M2 protein undergoes directional transport in the absence of other influenza virus proteins and that M2 contains the structural features required for apical transport in polarized epithelial cells. The ultrastructural localization of the M2 protein in influenza virus-infected MDCK cells was investigated by immunoelectron microscopy using M2 antibody and a gold conjugate. In cells in which extensive virus budding was occurring, the apical cell membrane was labeled with gold particles evenly distributed between microvilli and the surrounding membrane. In addition, a significant fraction of the M2 label was apparently associated with virions. A monoclonal antibody specific for HA demonstrated a similar labeling pattern. These results indicate that M2 is localized in close proximity to budding and assembled virions.     

In vivo assembly of an archaeal virus studied with whole-cell electron cryotomography

We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.     

Virions released from cells transfected with a molecular clone of human T-cell leukemia virus type I give rise to primary and secondary infections of T cells.

The ability of molecular clones of human T-cell leukemia virus type I (HTLV-I) to direct the synthesis of infectious virions has not previously been demonstrated. An HTLV-I provirus originating from an adult T-cell leukemia patient was cloned into a plasmid vector and is designated pCS-HTLV. This molecular clone was shown to direct the synthesis of viral mRNA and proteins in transiently transfected cells; in addition, virus structural proteins were released into the culture medium. Viral proteins were assembled into virions that sedimented at a buoyant density characteristic of retrovirus particles and whose morphology was verified by electron microscopy. Virions concentrated from transiently transfected cell supernatants were incubated with primary cord blood lymphocytes or with transformed T-cell lines to establish that these particles were infectious. Expression of spliced, viral mRNAs in the T-cell cultures after both primary and secondary infections with cell-free virus revealed that pCS-HTLV encodes an infectious provirus.