Simian virus 40 chromatin interaction with the capsid proteins

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.     

Altered Protein Metabolism in Infection by the Late tsB11 Mutant of Simian Virus 40

The DNA of the temperature-sensitive mutant tsB11 is replicated at the same rate as the DNA of wild-type virus in infection at the restrictive temperature. The progeny mutant DNA cannot be distinguished from wild-type DNA by gel electrophoresis and is assembled into a nucleoprotein complex with the same velocity sedimentation characteristics as the wild-type complex. Analysis of in vivo protein synthesis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoprecipitation techniques demonstrated that the capsid components VP1, VP2, and VP3 of the mutant and wild-type virus are synthesized at a similar rate, but VP1 fails to accumulate within cells infected by tsB11. Furthermore, VP1 is located predominantly in the cytoplasmic rather than in the nuclear fraction of extracts from cells infected by the mutant. Immunofluorescent studies localized virion antigen within the nucleolus as well as the cytoplasm. The altered intracellular distribution and stability of VP1 suggest that it may be the mutant protein of tsB11. The synthesis of a 72,000 dalton protein is consistently induced in significant quantity in cells infected by tsB11 at the restrictive temperature. A protein of the same apparent molecular weight is present in smaller quantities in uninfected cells and is only slightly increased in quantity in cells infected by wild-type virus.     

Altered restriction endonuclease cleavage pattern of simian virus 40 DNA.

Three different groups of temperature-sensitive mutants of simian virus 40, isolated and characterized by Chou and Martin (J. Virol. 13:1101--1109, 1974), have been analyzed by using restriction endonucleases. Differences between the restriction endonuclease cleavage pattern of these mutants and that of the standard simian virus 40 strain have been mapped. These include the following observations: (i) tsD202 carries a defective HaeIII cleavage site at position 0.9 map units; (ii) tsB204 exhibits a defective HaIII site at position 0.21 and a defective HinIII site at 0.655 map units, and (iii) tsC219 carries a new HinIII site at position 0.15. We have isolated a few wild-type revertants from each of the temperature-sensitive mutant strains; each displays the endonuclease cleavage pattern of its parental temperature-sensitive strain.     

Simian Virus 40 Chromatin Interaction with the Capsid Proteins

Abstract

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40°C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100–160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100–160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40°C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.     

Resolution of simian virus 40 proteins in whole cell extracts by two-dimensional electrophoresis: heterogeneity of the major capsid protein.

The major capsid protein (VP1) of simian virus 40 (SV40) has been analyzed by two-dimensional electrophoresis. This system separates protein according to isoelectric point by isoelectric-focusing, and according to molecular weight by sodium dodecylsulphate electrophoresis (O'Farrell, 1975). VP1 synthesis in infected CV-1 cells can be monitored directly by analysis of unfractionated whole cell extracts; the resolution of VP1 from cellular proteins allows its detection as early as 13 hr after infection. The two-dimensional separation of VP1 reveals that it is heterogeneous, consisting of one major protein (molecular weight 47,000 daltons and isoelectric point of approximately pH 6.8) and five minor protein components. The minor forms of VP1 are 10% of the total VP1 and differ from the major form of VP1 both in molecular weight (by approximately 500 daltons) and isoelectric point (ranging from approximately pH 6.7 to pH 6.9). Evidence is presented to show that two of the minor forms are phosphorylated derivatives of VP1, and it is further suggested that all the different forms of VP1 are the result of modifications of the primary product of translation. A temperature-sensitive mutant of the BC complementation group (BC11) of SV40 results in the synthesis of VP1 with an altered electrophoretic mobility; both the major form of VP1 and the minor forms are shifted in their isoelectric points. In addition to the specific case of SV40, two aspects of these studies should be generally significant to investigators studying eucaryotic gene expression by two-dimensional gel electrophoresis: first, the genetic origin of a protein can be determined by a temperature-sensitive mutation which causes a charge change in the resultant protein; and second, two or more protein spots on a two-dimensional separation may be the products of a single gene.     

Genetic Analysis of Simian Virus 40 III. Characterization of a Temperature-Sensitive Mutant Blocked at an Early Stage of Productive Infection in Monkey Cells

A temperature-sensitive mutant of simian virus 40 (SV40), ts(*)101, has been characterized during productive infection in monkey kidney cells. The mutant virion can adsorb to and penetrate the cell normally at the restrictive temperature, but cannot induce the synthesis of cellular deoxyribonucleic acid (DNA) nor initiate the synthesis of SV40-specific tumor, virion, or U antigens or viral DNA. First-cycle infection with purified ts(*)101 DNA is normal at the restrictive temperature, but the resulting progeny virions are still temperature-sensitive. The mutant neither complements nor inhibits other temperature-sensitive SV40 mutants or wild-type virions. The affected protein in the ts(*)101 mutant may be a regulatory structural protein, possibly a core protein, that is interacting with the viral DNA.     

Membrane proteins specified by herpes simplex viruses. III. Role of glycoprotein VP7(B2) in virion infectivity.

Experiments done with a temperature"sensitive mutant of herpes simplex virus type 1 (HSV-1) have revealed that one of the virisn glycoproteins, designated VP7(B2), is apparently not required for the production of enveloped virus particles, whereas it does play a critical role in virion infectivity. The mutant, designated HSV-1[HFEM]tsB5, fails to accumulate VP7(B2) at nonpermissive temperature and produces virions that lack detectable quantities of this glycoprotein and that have very low specific infectivity. The poor infectivity of the virions is most readily explained by failure of penetration into the host cell rather than by failure of adsorption to cells because it was shown that the VP7(B2)-deficient virions can bind to cells and that polyethylene glycol, an agent known to promote membrane fusion, can significantly enhance infectivity of the adsorbed virions.     

The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication.

In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.     

Variation in p30-related proteins in gross virus-induced tumor cell lines derived from H-2 congenic mice

The distribution of DNA topoisomers in intracellular simian virus 40 DNA was analyzed by gel electrophoresis. The results suggested that DNA extracted from 70S chromatin had a different superhelical density distribution as compared with the DNA obtained from virions or virion assembly intermediates. The heterogeneity of simian virus 40 viral DNA superhelical density at a late time after infection was partly due to increased virion production and partly due to the intrinsic heterogeneity of the superhelical density of DNA extracted from virions. Using two-dimensional gel electrophoretic analysis we also showed that simian virus 40 DNA templates used for DNA replication have a higher average superhelical density than the bulk of intracellular viral DNA.     

Stable association of viral protein VP1 with simian virus 40 DNA.

Mild dissociation of simian virus 40 particles releases a 110S virion core nucleoprotein complex containing histones and the three viral proteins VP1, VP2, and VP3. The association of viral protein VP1 within this nucleoprotein complex is mediated at least partially through a strong interaction with the viral DNA. Treatment of the virion-derived 110S nucleoprotein complex with 0.25% Sarkosyl dissociated VP2, VP3, and histones, leaving a stable VP1-DNA complex. The VP1-DNA complex had a sedimentation value of 30S and a density of 1.460 g/cm3. The calculated molecular weight of the complex was 7.9 x 10(6), with an average of 100 VP1 molecules per DNA. Agarose gel electrophoresis of the VP1-DNA complex demonstrated that VP1 is associated not only with form I and form II simian virus 40 DNAs but also with form III simian virus 40 DNA generated by cleavage with EcoRI.     

Assembly of infectious Herpes simplex virus type 1 virions in the absence of full-length VP22

VP22, the 301-amino-acid phosphoprotein product of the herpes simplex virus type 1 (HSV-1) U(L)49 gene, is incorporated into the tegument during virus assembly. We previously showed that highly modified forms of VP22 are restricted to infected cell nuclei (L. E. Pomeranz and J. A. Blaho, J. Virol. 73:6769-6781, 1999). VP22 packaged into infectious virions appears undermodified, and nuclear- and virion-associated forms are easily differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994). As VP22 packaging-associated undermodification is unique among HSV-1 tegument proteins, we sought to determine the role of VP22 during viral replication. We now show the following. (i) VP22 modification occurs in the absence of other viral factors in cell lines which stably express its gene. (ii) RF177, a recombinant HSV-1 strain generated for this study, synthesizes only the amino-terminal 212 amino acids of VP22 (Delta212). (iii) Delta212 localizes to the nucleus and incorporates into virions during RF177 infection of Vero cells. Thus, the carboxy-terminal region is not required for nuclear localization of VP22. (iv) RF177 synthesizes the tegument proteins VP13/14, VP16, and VHS (virus host shutoff) and incorporates them into infectious virions as efficiently as wild-type virus. However, (v) the loss of VP22 in RF177 virus particles is compensated for by a redistribution of minor virion components. (vi) Mature RF177 virions are identical to wild-type particles based on electron microscopic analyses. (vii) Single-step growth kinetics of RF177 in Vero cells are essentially identical to those of wild-type virus. (viii) RF177 plaque size is reduced by nearly 40% compared to wild-type virus. Based on these results, we conclude that VP22 is not required for tegument formation, virion assembly/maturation, or productive HSV-1 replication, while the presence of full-length VP22 in the tegument is needed for efficient virus spread in Vero cell monolayers.     

Simian Virus 40-Host Cell Interactions II. Cytoplasmic and Nucleolar Accumulation of Simian Virus 40 Virion Protein

We have used immunofluorescence in parallel with transmission and scanning electron microscopy to characterize the unusual cytoplasmic and nucleolar accumulation of Simian virus 40 (SV40) virion protein (C antigen) at restrictive temperatures (39 to 41 C) in monkey cells infected with a temperature-sensitive mutant of SV40 defective in virion assembly, tsB11. Cytoplasmic and nucleolar accumulation of C antigen did not occur in wild-type-infected cells at any temperature. Wild-type- and tsBll-infected cells were not distinguishable at 33 C by immunofluorescence or electron microscopy. Temperature-shift experiments using metabolic inhibitors of DNA (cytosine arabinonucleoside, 20 mug/ml), RNA (actinomycin D, 5 mug/ml), and protein synthesis (cycloheximide, 2 x 10(-4) to 10 x 10(-4) M) were used to investigate the requirements for ongoing DNA, RNA, and protein synthesis in the distribution of virion protein between the nucleus, nucleolus, and cytoplasm. The transport of C antigen from the nucleolus and cytoplasm into the nucleus was complete after a temperature shift-down (41 and 39 to 33 C). Limited virus particle formation occurred after the shift-down in the presence of actinomycin D and cycloheximide, indicating some of the 39 to 41 C synthesized virion protein could be used for capsid assembly at 33 C in the absence of further virion protein synthesis. Nucleolar and cytoplasmic accumulations of C antigen occurred in the absence of drugs after a shift-up (33 to 39 C and 41 C) indicating a continuous requirement for the tsB11 mutant function. Furthermore, the virion protein synthesized at 33 C remained confined to the nucleus when the cells were shifted to 39 and 41 C in the presence of actinomycin D or cycloheximide. In the presence of cytosine arabinonucleoside, however, the virion protein accumulated in large aggregates in the nucleus and nucleolus after the shift-up, but did not migrate into the cytoplasm as it did in drug-free tsB11-infected control cells. Colchicine (10(-3) M) had no effect on the abnormal accumulation of C antigen during shift-up or shift-down experiments suggesting that microtubular transport plays little if any role in the abnormal transport of tsB11 virion protein from cytoplasm to nucleus. Although virus particles were never observed by electron microscopy and V antigen was not detected by immunofluorescence at 39 or 41 C in tsB11-infected cells, dense amorphous accumulations were formed in the nucleoli and cytoplasm. We suggest that the tsB11 function is continuously required for the normal transport of SV40 virion protein between the cytoplasm, nucleolus, and nucleus and for the assembly of capsids and virions. Several possible mechanisms for the altered tsB11 function or protein are discussed. One of the virion proteins may also be involved in some presently undetermined nucleolar function during SV40 productive infection.     

Intracellular forms of simian virus 40 nucleoprotein complexes. II. Biochemical and electron microscopic analysis of simian virus 40 virion assembly.

The simian virus 40 virion assembly process was studied with pulse-labeling kinetics of virion proteins, CsCl gradient analysis, electron microscopy, and low-salt gel electrophoresis. The results obtained are consistent with the model of gradual addition and organization of capsid proteins around simian virus 40 chromatin. Empty virions, as observed in the CsCl gradient by previous workers, were found to be the dissociation product of immature virus. Histone H1 was found in simian virus 40 chromatin and virion assembly intermediates but not in the mature virion banding at 1.34 g/ml in the CsCl gradient.     

Temperature-sensitive poliovirus mutant fails to cleave VP0 and accumulates provirions.

A temperature-sensitive mutant of poliovirus, VP2-103, was isolated and characterized. A single nucleotide change, resulting in the substitution of glutamine for arginine at amino acid 76 of the capsid protein VP2, prevented the maturation of virions at the nonpermissive temperature. Particles indistinguishable from the previously elusive provirions were observed; these particles have been proposed to be penultimate in virion morphogenesis. Cleavage of VP0 into VP2 and VP4, the products found in mature virions, was not observed in VP2-103-infected cells at the nonpermissive temperature. The cleavage of VP0 in wild-type poliovirus-infected cells is dependent on RNA packaging; this reaction has been postulated to be autocatalytic. The existence of RNA-containing provirionlike particles in VP2-103-infected cells shows that RNA packaging can be uncoupled from VP0 cleavage.     

Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus.

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.     

Characterization of components released by alkali disruption of simian virus 40.

Treatment of simian virus 40 (SV40) particles at pH 9.8 in the presence of 1 mM dithiothreitol for 5 min at 37 degrees C disrupted the virions into a 60S DNA-protein complex and DNA-free 7S protein particles. The DNA-protein complex contained approximately equal amounts of DNA and protein, and appeared by electron microscopy to be relaxed circular structures with an average of 21 beads joined by short, thin bridges. The major protein components in the complex were host cell histones, but SV40-specific proteins VP3 and VP2 were also present. The 7S protein particles were almost exclusively VP1 and, in negatively stained samples, resembled the capsomer structures of intact virions.     

Role of JC virus agnoprotein in virion formation

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.     

Human immunodeficiency virus type 1 Nef incorporation into virions does not increase infectivity

The viral protein Nef contributes to the optimal infectivity of human and simian immunodeficiency viruses. The requirement for Nef during viral biogenesis particles suggests that Nef might play a role in this process. Alternatively, because Nef is incorporated into viruses, it might play a role when progeny virions reach target cells. We challenged these hypotheses by manipulating the amounts of Nef incorporated in viruses while keeping its expression level constant in producer cells. This was achieved by forcing the incorporation of Nef into viral particles by fusing a Vpr sequence to the C-terminal end of Nef. A cleavage site for the viral protease was introduced between Nef and Vpr to allow the release of Nef fragments from the fusion protein during virus maturation. We show that the resulting Nef-CS-Vpr fusion partially retains the ability of Nef to downregulate cell surface CD4 and that high amounts of Nef-CS-Vpr are incorporated into viral particles compared with what is seen for wild-type Nef. The fusion protein is processed during virion maturation and releases Nef fragments similar to those found in viruses produced in the presence of wild-type Nef. Unlike viruses produced in the presence of wild-type Nef, viruses produced in the presence of Nef-CS-Vpr do not have an increase in infectivity and are as poorly infectious as viruses produced in the absence of Nef. These findings demonstrate that the presence of Nef in viral particles is not sufficient to increase human immunodeficiency virus type 1 infectivity and suggest that Nef plays a role during the biogenesis of viral particles.     

Induction of an error-prone mode of DNA repair in UV-irradiated monkey kidney cells

The survival of UV-irradiated simian virus 40 (SV40) on UV-irradiated monkey kidney CV-1P cells at 33° was increased over survival on unirradiated cells. During this process — called induced-virus reactivation — the progeny virus yielded by UV-irradiated cells had a much higher mutation frequency than did the progeny from unirradiated cells. Mutation rates were quantified by using phenotypic reversion towards wild-type growth of an early (tsA 58) or a late (tsB 201) temperature-sensitive SV40 mutant. Analysis of SV40 revertant genomes indicated that no detectable deletions or additions were resposible for the reversion process.These results suggest that enzymes from UV-irradiated cells are able to replicate UV-irradiated DNA by an error-prone mode of DNA repair. Induced virus reactivation and error-prone replication are probably one of the expressions of SOS functions in mammalian cells.