Viral Nucleoprotein Complexes Research Articles

Protection of retroviral DNA from autointegration: involvement of a cellular factor.

An essential step in the retrovirus life cycle is integration of a DNA copy of the viral genome into a host chromosome. After reverse transcription, there can be a delay of many hours before the viral DNA is integrated. It is important for the retrovirus to ensure that the viral DNA does not integrate into itself during this period; such autointegration is a suicidal process that would result in destruction of the viral genome. Understanding of the mechanism that blocks autointegration of the viral DNA may lead to insights into how to inhibit viral replication by inducing the viral DNA to autointegrate. Evidence is presented in this report that viral nucleoprotein complexes isolated from cells infected with Moloney murine leukemia virus exhibit a barrier to autointegration. The barrier can be disrupted by high salt treatment and, subsequently, restored by addition of factors provided by a host cell extract. Our data indicate an involvement of host machinery in protecting retroviral DNA from autointegration.

Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection.

We have examined components of the preintegration complex of human immunodeficiency virus type 1 (HIV-1) and have analyzed features which govern the association of these components. HIV-1 nucleoprotein complexes, isolated from nuclear and cytoplasmic extracts of CD4+ cells after acute virus infection, contained viral RNA and DNA in association with viral matrix (MA), integrase (IN), and reverse transcriptase (RT) antigens but not capsid (CA) antigens and possessed integration activity in vitro. Association of IN but not RT or MA antigens with viral DNA was detergent-stable. Analysis of viral DNA synthesis and nuclear import of viral nucleoprotein complexes in the presence of a reversible RT inhibitor demonstrated that reverse transcription of viral RNA could be completed entirely in the host cell nucleus. Our studies demonstrate structural and functional features of the nucleoprotein (preintegration) complex of HIV-1 which are pertinent to the understanding of early events in the lentiviral life cycle.

Integration of murine leukemia virus DNA depends on mitosis.

In synchronized rat or mouse cells infected with Moloney murine leukemia virus (MLV), integration of viral DNA and production of viral proteins occur only after the cells traverse mitosis. Integration is blocked when cells are prevented from progressing through mitosis. Viral nucleoprotein complexes isolated from arrested cells contain full-length viral DNA and can integrate this viral DNA in vitro, showing that the block to integration in arrested cells is not due to a lack of mature integration machinery. When infected cells traverse mitosis, there is a sharp increase in nuclear accumulation of viral DNA. The dependence of integration on mitosis may therefore be due to a requirement for mitosis and nuclear envelope breakdown for entry of the viral integration complex into the nucleus.

Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection

Integration of retroviral DNA can serve as a paradigm for cellular functions that are affected by the packaging of DNA into chromatin. We have used a novel polymerase chain reaction-based assay to survey DNA and chromatin for the precise distribution of many integration sites. Integration into naked DNA targets is nonuniform, implying a nucleotide sequence bias. In chromatin, integration occurs preferentially at postitions where the major groove is on the exposed face of the nucleosomal DNA helix, generating a 10 bp periodic spacing of preferred sites. Chromatin assembly enhances the reactivity of many sites, so that integration occurs most frequently at sites in nucleosomal, rather than nucleosome-free, regions of minichromosomes. In contrast, integration is prevented in a region occupied by a site-specific DNA-binding protein. Comparisons of integration events mediated by viral nucleoprotein complexes or by two different retroviral integrases show that the integration machinery also affects target site selection.

Purification and characterization of vaccinia virus structural protein VP8

A major vaccinia virus core protein, designated VP8, has been purified from virions to homogeneity through DEAE-cellulose, CM-cellulose, and hydroxyapatite chromatography. VP8 migrates as a 25-kDa band in SDS-polyacrylamide gel electrophoresis and sediments as a monomeric species in neutral sucrose gradient centrifugation. This protein is a signficant constitutent of the virion, comprising about 6.5% of the total viral polypeptides by mass. Analysis by filter binding and by sucrose gradient centrifugation shows that VP8 binds to double-stranded as well as to single-stranded DNA at low salt concentrations (25 m M NaCl). At higher salt concentrations (100 m M NaCI), the protein binds with a relatively greater affinity to single-stranded DNA. The results from sucrose gradient centrifugation indicate that VP8 probably binds noncooperatively to all structural forms of DNA. The protein is likely to be a component of the viral nucleoprotein complex.

Psoralen-cross-linking study of the organization of intracellular adenovirus nucleoprotein complexes

We used the psoralen-cross-linking technique to investigate the structures of adenovirus nucleoprotein complexes during infection. At late times after infection, three types of psoralen cross-linking patterns were observed. A high cross-linking pattern (type I), with about one cross-link in every 10 to 17 base pairs, was found for the newly synthesized and the bulk of the adenovirus late chromatin. Viral templates involved in replication, transcription, and recombination were all found to exhibit this cross-linking pattern. These results suggest that there is no nucleosome-like organization in the unpackaged late adenovirus nucleoprotein complexes. The second type of cross-linking pattern (type II) had a low cross-linking density of about one cross-link in every 700 to 1,000 base pairs. This cross-linking pattern was found to be associated with the viral DNA in the mature virus particles. The sequences at the termini of the virion DNAs, however, were found to have higher cross-linking densities, as shown by electron microscopy. The third type of cross-linking pattern (type III) was composed of a mixture of various proportions of type I and type II patterns in a single molecule. This mixed cross-linking pattern suggests that these molecules are virion assembly intermediates, with viral DNA being partially packaged in the virus particles. The organization of adenovirus nucleoprotein complexes at early times after infection was analyzed by the gel electrophoresis technique following digestion of the DNA with a restriction enzyme that was inhibited by cross-links. Our data suggest that the viral nucleoprotein complexes at early times after infection have accessibility to psoralen cross-linking between the virion DNA and the late viral nucleoprotein complexes. The observed cross-linking density of the early nucleoprotein complex DNA, however, was inconsistent with the nucleosomelike organization suggested by previous investigators.

The mode of assembly of alphavirus cores implies a mechanism for the disassembly of the cores in the early stages of infection. Brief review.

Probably many different strategies will be used by viruses to disassemble viral nucleoprotein complexes during the early stages of infection and the detailed model described aboved may only apply to alphaviruses. On the other hand, the existence of a cellular binding site which has to be saturated to a certain extent with the protein components of viral nucleoproteins before viral nucleoprotein assembly can occur and which binds the protein of the incoming viral nucleoprotein may be a feature which may also be used by other viruses.

Nucleoprotein complexes of minute virus of mice have a distinct structure different from that of chromatin.

We studied the structure of viral nucleoprotein complexes extracted from the nuclei of mouse cells infected with the immunosuppressive strain of the minute virus of mice (MVMi). Two types of complex were detected, with sedimentation coefficients of about 110 and 40S. The complexes sedimenting at 110S contained single-stranded MVMi DNA as well as a second form of viral DNA which apparently had a heat-sensitive secondary structure. The 110S peak also contained proteins which coelectrophoresed with the MVMi capsid proteins. Complexes sedimenting at 40S contained the double-stranded replicative form of MVMi DNA. These complexes sedimented faster than did the pure replicative form DNA (15S), but more slowly than cellular chromatin fragments containing DNA of the same length. They incorporated labeled deoxynucleoside triphosphate in vitro into the replicative form DNA. We investigated the structure of MVMi nucleoprotein complexes in the following ways. Nuclei of MVMi-infected cells were digested with staphylococcal nuclease, and the resulting DNA fragments were electrophoresed, transferred to nitrocellulose, and hybridized first with labeled MVMi DNA and then with cellular DNA. A nucleosomal repeat pattern was seen with the cellular DNA probe but not with the MVMi DNA probe. The DNA in MVMi nucleoprotein complexes was cross-linked with psoralen, purified, denatured, and examined with an electron microscope. Bubbles, indicating the presence of proteins, were seen in the MVMi DNA. The length of the DNA in the bubbles was 90 +/- 29 nucleotides. On the other hand, nucleosomes protected 160 base pairs from cross-linking by psoralen. The MVMi nucleoprotein complexes thus have a distinct structure which is different from that of chromatin.

Transcription of adenovirus cores in vitro

Transcription in whole HeLa cell extracts of the nucleoprotein core complexes released from adenovirus type 2 or type 5 virions has been examined. The average length of transcripts from deproteinized DNA templates increased steadily during a 90-min reaction in vitro, exhibiting an elongation rate of approximately 70 nucleotides per minute. On the other hand, transcripts made from viral core templates were restricted to a length of less than 2000 nucleotides. Accordingly, efficient transcription of cores (50 nucleotides elongated/min) ceased after 10–20 min of incubation in whole-cell extracts. Deproteinized viral DNA and viral nucleoprotein complexes appeared to support the initiation of a similar number of transcripts per template molecule, but the rate of initiation was faster when cores were provided as templates. Deproteinized viral DNA supported the synthesis of VA-RNA and of transcripts that hybridized to the region of the viral genome containing the 5′ portion of the major late transcriptional. Viral cores also directed the synthesis of RNA products which hybridized to fragments of the viral genome containing ElA, ElB, and E4 regions. The results of nuclease protection experiments indicated that the presence of core proteins did not preclude accurate initiation of transcription from the E4 region.

Protein composition of adenovirus nucleoprotein complexes extracted from infected cells

A viral nucleoprotein complex was extracted from the nuclei of human cells 20 hr after infection with adenovirus type 2 or several of its temperature-sensitive mutants. In its sedimentation property, density in CsCl, and digestion pattern with micrococcal nuclease, the complex resembled viral cores. The polypeptides V, PVII, IIK, and 36K were found associated with this complex which is formed prior to or in the absence of virus assembly. The results suggest that this nucleoprotein complex is a direct precursor to virus assembly.

Viral proteins and RNAs in BHK cells persistently infected by lymphocytic choriomeningitis virus.

Some Syrian hamster cell lines persistently infected with lymphocytic choriomeningitis virus (LCMV) do not produce extracellular virus particles but do contain intracytoplasmic infectious material. The proteins of these cells were labeled with [35S]methionine or with [3H]glucosamine and [3H]mannose, and immunoprecipitates were prepared with anti-LCMV sera. A substantial amount of the LCMV nucleocapsid protein (molecular weight about 58,000) was detected, along with GP-C, the precursor of the virion glycoproteins GP-1 and GP-2. GP-1 and GP-2 themselves were not detected. A new method of transferring proteins electrophoretically from sodium dodecyl sulfate-polyacrylamide gels to diazotized paper in high yield revealed several additional LCMV proteins present specifically in the persistently infected cells, at apparent molecular weights (X10(3] of 112, 107, 103, 89, 71 (probably GP-C), 58 (nucleocapsid protein), 42 to 47 (probably GP-1), and 40 (possibly GP-2). By iodinating intact cells with I3, GP-1 but not GP-2 or GP-C was revealed on the surfaces of the persistently infected cells, whereas both GP-1 and GP-C were found on the surfaces of acutely infected cells. The absence of GP-C from the plasma membrane of the persistently infected cells might be related to defective maturation of the virus in these cells. Cytoplasmic viral nucleoprotein complexes were labeled with [3H]uridine in the presence or absence of actinomycin D, purified partially by sedimentation in D2O-sucrose gradients, and adsorbed to fixed Staphylococus aureus cells in the presence of anti-LCMV immunoglobulin G. Several discrete species of viral RNA were released from the immune complexes with sodium dodecyl sulfate. Some were appreciably smaller than the 31S and 23S species of standard LCMV virions, indicating that defective interfering viral RNAs are probably present in the persistently infected cells. Ribosomal 28S and 18S RNAs, labeled only in the absence of actinomycin D, were coprecipitated with anti-LCMV serum but not with control serum, indicating their association with LCMV nucleoproteins in the cells.

Interferon pretreatment inhibits simian virus 40 infections by blocking the onset of early transcription

CV-1 cells treated with human leukocyte interferon before infection with simian virus 40 (SV40) are protected from virus-induced cytopathic effects. Accumulation of viral mRNA is inhibited markedly. If T antigen is present in interferon-treated cells before infection, viral DNA replication is inhibited only slightly. These results show that many SV40 virions reach the nucleus in a form which can be replicated but not transcribed. Accumulation of SV40 mRNA is not inhibited in cells treated with interferon after infection. Thus the block mediated by interferon is specific for the onset of transcription. Infections with SV40 DNA rather than virions are insensitive to treatment with interferon, showing that the inhibition involves an interaction between viral nucleoprotein complexes and cellular factors.

Chromosomal organization of the herpes simplex virus type 2 genome

Nucleoprotein complexes containing herpes simplex virus type 2 DNA were extracted from CV-1 cells with NP-40 and (NH 4) 2SO 4. The viral DNA-protein complex sedimented in neutral sucrose as a relatively homogeneous peak at around 120 S. Pooled material from the 120 S region of sucrose gradients contained DNA which, when deproteinized, sedimented at 55 S and had a density of 1.728 g/cm 3 as expected for herpesvirus DNA. Buoyant density studies with glutaraldehyde-fixed complexes indicate that protein is bound to viral DNA at a ratio of approximately 1.1 to 1. Micrococcal nuclease digests of isolated viral nucleoprotein complexes indicate that these complexes do not have a nucleosomal structure. Chromatin prepared from nuclei following isolation of viral nucleoprotein complexes contained viral DNA. When infected nuclei were treated with micrococcal nuclease, nucleosome-sized fragments were obtained which contained both virus and cell DNA.

Accurate transcription of simian virus 40 chromatin in a HeLa cell extract.

During simian virus 40 viral maturation, a series of modifications occur which alter the composition of viral nucleoprotein complexes. As a consequence, the chromatin that is extracted from extracellular simian virus 40 virions exhibits properties that are similar to those of transcriptionally active eucaryotic chromatin. The influence of this chromatin structure on specific RNA initiation by RNA polymerase II was examined by using the in vitro HeLa cell extract of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980). The 5' ends of RNA transcripts were positioned by the "run-off" assay, in which transcripts extend from the initiation site to termination sites created by restriction cleavage and by S1 nuclease analysis, using DNA probes labeled at their 5' termini. Two major early RNA transcripts, which originated at map positions 5,240 +/- 10 and 5,145 +/- 10, and two major late RNA transcripts, which originated at map positions 325 +/- 10 and 185 +/- 10, were identified. Transcripts were initiated with comparable relative efficiencies at the same 5' site when either purified DNA or chromatin was used as the template. Our results suggest that extracellular simian virus 40 virion chromatin modifications do not regulate simian virus 40 promoter selection but function to increase the accessibility of RNA promoter sequences to RNA polymerase II and allow efficient elongation of the RNA chain after the initiation event.

Visualization of nascent RNA transcripts and simultaneous transcription and replication in viral nucleoprotein complexes from adenovirus 2-infected HeLa cells

The study of potential regulatory events in gene expression in animal cells, such as RNA processing and template specificity for transcription or replication, was approached by visualization of these cellular processes in animal virus-infected cells. Viral nucleoprotein complexes were selectively extracted from purified HeLa cell nuclei 15 to 24 hours following infection with human adenovirus serotype 2. These viral nucleoprotein complexes contained replicative and transcriptional activities as judged by biochemical and structural analysis. Methods were developed that permitted electron microscopic visualization of adenovirus serotype 2 nucleoprotein complexes separated from the mass of cellular chromatin and containing measurable nascent RNA transcripts. Importantly, nascent RNA transcripts were observed on molecules that were intermediate in replication. Quantitation revealed that transcripts were present on replicating molecules with approximately the same frequency with which they were present on non-replicating molecules. This demonstrated that molecules of adenovirus serotype 2 DNA could serve simultaneously as templates for replication and transcription and that there was no obvious preference for replicating molecules as transcriptional templates.

Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species.

The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP1 was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP1 was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP1 appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP1 species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP1 species and three of the capsid VP1 species were found in capsomere preparations enriched for hexon subunits. Two VP1 species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP1 species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP1 species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50.

Intracellular forms of simian virus 40 nucleoprotein complexes. III. Study of histone modifications.

The modification patterns of histones present in various forms of intracellular simian virus 40 nucleoprotein complexes were analyzed by acetic acid-urea-polyacrylamide gel electrophoresis. The results showed that different viral nucleoprotein complexes contain different histone patterns. Simian virus 40 chromatin, which contains the activities for the synthesis of viral RNA and DNA, exhibits a histone modification pattern similar to that of the host chromatin. However, virion assembly intermediates and mature virions contain highly modified histones. Pulse-chase experiments with [3H]lysine showed that the newly incorporated histones in the virion assembly intermediates were already highly modified. The majority of in vivo acetylation activity of histones occurred on the 70S simian virus 40 chromatin as analyzed by pulse-labeling with [3H]acetate. These results and our previous analysis of the virion assembly pathway suggest that three stages are involved in the packaging of simian virus 40 chromatin into the mature virion: (i) modification of histones, (ii) accumulation of capsid protein around the chromatin with highly modified histones, and (iii) organization of capsid proteins into salt-resistant shells. The role of histone modification in virion assembly is discussed.

Replication of polyoma DNA in nuclear extracts and nucleoprotein complexes

Viral nucleoprotein complexes containing radioactive form l DNA or replicative intermediates were extracted from nuclei isolated from polyoma-infected 3T6 fibroblasts, pulse labelled with [ 3H]thymidine. Such extracts incorporated labelled dGTP into viral DNA, similar to intact isolated nuclei, but at a decreased rate and for shorter periods. The two kinds of nucleoprotein complexes containing form l DNA or replicative intermediates were separated and purified. Each complex retained some capacity to incorporate labelled dGTP and this reaction was stimulated by ATP. The new DNA consisted mainly of short strands hydrogen-bonded to the template. With replicative intermediate complexes incorporation occurred at random into different parts of the viral DNA, while form l complexes incorporated dGTP preferentially into a region around the origin of replication. A crude preparation of T-antigen stimulated the incorporation. The amount of synthesis was low and it was not possible to decide with certainty whether some of the incorporation observed with form 1 complexes represented initiation of new rounds of replication or whether it represented elongation of early replicative intermediates.

Histone modifications in simian virus 40 and in nucleoprotein complexes containing supercoiled viral DNA

Simian virus (SV40) nucleoprotein complexes containing circular supercoiled viral DNA were extracted from infected cells and purified by differential centrifugation. The protein content of these complexes was compared by electrophoresis on 15% acrylamide gels with the protein content of purified SV40 virions and with histones from virus-infected cells. The electrophoretic patterns of histones from each of the sources revealed several major differences. SV40 virions contained histones H3, H2B, H2A, and H4 but not H1. Nucleoprotein complexes and host cells contained all five major histone groups. Relative to cellular histones, virion and nucleoprotein complex histones were enriched 15 to 40% in histones H3 and H4. In addition to the major classes of histones, several subfractions of histones H1, H3, and H4 were observed in acrylamide gels of proteins from SV40 virions and viral nucleoprotein complexes. Acetate labeling experiments indicated that each subfraction of histones H3 and H4 had a different level of acetylation. The histones from SV40 virions and nucleoprotein complexes were acetylated to significantly higher levels than those of infected host cells. No apparent differences in phosphorylation of the major histone groups were observed.

Association of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.