Viral DNA-protein Complex Research Articles

Cellular and viral determinants of retroviral nuclear entry

Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1- and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.

Engagement of the Lysine-Specific Demethylase/HDAC1/CoREST/REST Complex by Herpes Simplex Virus 1

Among the early events in herpes simplex virus 1 replication are localization of ICP0 in ND10 bodies and accumulation of viral DNA-protein complexes in structures abutting ND10. ICP0 degrades components of ND10 and blocks silencing of viral DNA, achieving the latter by dislodging HDAC1 or -2 from the lysine-specific demethylase 1 (LSD1)/CoREST/REST repressor complex. The role of this process is apparent from the observation that a dominant-negative CoREST protein compensates for the absence of ICP0 in a cell-dependent fashion. HDAC1 or -2 and the CoREST/REST complex are independently translocated to the nucleus once viral DNA synthesis begins. The focus of this report is twofold. First, we report that in infected cells, LSD1, a key component of the repressor complex, is partially degraded or remains stably associated with CoREST and is ultimately also translocated, in part, to the cytoplasm. Second, we examined the distribution of the components of the repressor complex and ICP8 early in infection in wild-type-virus- and ICP0 mutant virus-infected cells. The repressor component and ultimately ICP8 localize in structures that abut the ND10 nuclear bodies. There is no evidence that the two compartments fuse. We propose that ICP0 must dynamically interact with both compartments in order to accomplish its functions of degrading PML and SP100 and suppressing silencing of viral DNA through its interactions with CoREST. In turn, the remodeling of the viral DNA-protein complex enables recruitment of ICP8 and initiation of formation of replication compartments.

Default assembly of early adenovirus chromatin

In adenovirus particles, the viral nucleoprotein is organized into a highly compacted core structure. Upon delivery to the nucleus, the viral nucleoprotein is very likely to be remodeled to a form accessible to the transcription and replication machinery. Viral protein VII binds to intra-nuclear viral DNA, as do at least two cellular proteins, SET/TAF-Iβ and pp32, components of a chromatin assembly complex that is implicated in template remodeling. We showed previously that viral DNA–protein complexes released from infecting particles were sensitive to shearing after cross-linking with formaldehyde, presumably after transport of the genome into the nucleus. We report here the application of equilibrium-density gradient centrifugation to the analysis of the fate of these complexes. Most of the incoming protein VII was recovered in a form that was not cross-linked to viral DNA. This release of protein VII, as well as the binding of SET/TAF-Iβ and cellular transcription factors to the viral chromatin, did not require de novo viral gene expression. The distinct density profiles of viral DNA complexes containing protein VII, compared to those containing SET/TAF-Iβ or transcription factors, were consistent with the notion that the assembly of early viral chromatin requires both the association of SET/TAF-1β and the release of protein VII.

Adenovirus type 5 DNA–protein complexes from formaldehyde cross-linked cells early after infection

We report here the properties of viral DNA–protein complexes that purify with cellular chromatin following formaldehyde cross-linking of intact cells early after infection. The cross-linked viral DNA fractionated into shear-sensitive (S) and shear- resistant (R) components that were separable by sedimentation, which allowed independent characterization. The R component had the density and sedimentation properties expected for DNA–protein complexes and contained intact viral DNA. It accounted for about 50% of the viral DNA recovered at 1.5 h after infection but less than 20% by 4.5 h. The proportion of R component was independent of multiplicity of infection, even at less than one particle per cell. Viral hexon and protein VII, but not protein VI, were detected in the fractions containing the R component. These properties are consistent with those of partially uncoated virions associated with the nuclear envelope. A substantial proportion of the S component viral DNA had the same density as cellular chromatin. Protein VII was the most abundant viral protein present in gradient fractions that contained the S component. Complexes containing USF transcription factor cross-linked to the adenovirus major late promoter were detected by viral chromatin immunoprecipitation of the fractions containing S component. The S component probably contained uncoated nuclear viral DNA that assembles into early viral transcription complexes.

Mouse cytomegalovirus immediate-early protein 1 binds with host cell repressors to relieve suppressive effects on viral transcription and replication during lytic infection.

Herpesviruses start their transcriptional cascade at nuclear domain 10 (ND10). The deposition of virus genomes at these nuclear sites occurs due to the binding of the interferon-inducible repressor protein promyelocytic leukemia protein (PML) and/or Daxx to a viral DNA-protein complex. However, the presence of repressive proteins at the nuclear site of virus transcription has remained unexplained. We investigated the mouse cytomegalovirus (MCMV) immediate-early 1 protein (IE1), which is necessary for productive infection at low multiplicities of infection and therefore likely to be involved in overcoming cellular repression. Temporal analysis of IE1 distribution revealed its initial segregation into ND10 by binding to PML and/or Daxx and IE1-dependent recruitment of the transcriptional repressor histone deacetylase-2 (HDAC-2) to this site. However, these protein aggregates are dissociated in cells producing sufficient IE1 through titration of PML, Daxx, and HDAC-2. Importantly, binding of IE1 to HDAC-2 decreased deacetylation activity. Moreover, inhibition of HDAC by trichostatin-A resulted in an increase in viral protein synthesis, an increase in cells starting the formation of prereplication compartments, and an increase in the total infectious viruses produced. Thus, IE1, like trichostatin-A, reverses the repressive effect of HDAC evident in the presence of acetylated histones in the immediate-early promoter region. Since HDAC also binds to the promoter region of IE1, as determined by the chromatin immunoprecipitation assay, these combined results suggest that IE1 inhibits or reverses HDAC-mediated repression of the infecting viral genomes, possibly by a process akin to activation of heterochromatin. We propose that even permissive cells can repress transcription of infecting viral genomes through repressors, including HDAC, Daxx, and PML, and the segregation of IE1 to ND10 that would inactivate those repressors. The virus can counter this repression by overexpressing IE1 when present in sufficient copy number, thus reducing the availability and effectiveness of these repressors.

Nuclear import of moloney murine leukemia virus DNA mediated by adenovirus preterminal protein is not sufficient for efficient retroviral transduction in nondividing cells.

Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G(1)/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration.

Evidence for a ligation step in the DNA replication of the autonomous parvovirus minute virus of mice.

Newly replicated DNA of the autonomous parvovirus minute virus of mice was pulse-labeled with 32PO4 during the time of maximal viral DNA replication in highly synchronized A9 cells. The subsequent processing of viral DNA-protein complexes was monitored during a chase period with no label. Several distinct classes of duplex replicative-form and progeny single-stranded DNA molecules were characterized and found to accumulate at different times during infection. Analysis of the terminal structures associated with these various forms provided new insights into the mechanism by which viral DNA replicates and, in particular, suggested that interstrand ligation occurs during this process.

The NS-1 polypeptide of minute virus of mice is covalently attached to the 5' termini of duplex replicative-form DNA and progeny single strands.

When A9 cells are infected with minute virus of mice, a small proportion of the virally coded NS-1 polypeptide becomes covalently attached to newly synthesized viral DNA. Antisera directed against NS-1 will specifically precipitate two forms of monomer duplex replicative-form DNA, multimeric duplex intermediates and progeny single strands, and restriction analysis of the duplex forms in these precipitates reveals that NS-1 is exclusively associated with extended-form conformers of the genomic termini. Pulse-labeled viral DNA, harvested at various times in a highly synchronized infection, can be almost quantitatively precipitated with any one of a series of antisera directed against different protein domains distributed throughout the NS-1 molecule but not with antibodies directed against other viral proteins. In each case the interaction with NS-1 can be shown to involve both termini of duplex DNA and single-strand forms, suggesting that in each case a full-length (83-kilodalton) copy of NS-1 is present. Precipitation of the replicating viral DNA with an antibody directed against a synthetic 16-amino-acid peptide containing the sequence at the extreme carboxy terminus of NS-1 can be quantitatively and specifically inhibited with the immunizing peptide in its unconjugated form, showing that the antibodies responsible for precipitating viral DNA are directed against the NS-1 sequence itself and not against a trace contaminant. Exonuclease digestion studies show that the association effectively blocks the 5' ends of the DNA molecules. Very little (less than 0.1%) of the newly synthesized [35S]methionine-labeled NS-1 made in highly synchronized cells during a 15-min pulse early in infection (6.25 to 6.5 h into the S phase) becomes associated with viral DNA immediately. However, pulse-chase experiments show that later in infection (10 to 13 h into the S phase), when viral DNA replication is reaching its peak, a few percent of the molecules in these preexisting pools of NS-1 do become covalently attached to the newly replicated DNA. Isolated viral DNA-protein complexes labeled with [35S]methionine in this way can be obtained by fractionation of the immunoprecipitated complexes on Sepharose CL4B in sodium dodecyl sulfate. Digestion of the purified complexes with nuclease releases an 83-kilodalton molecule which exactly comigrates with authentic NS-1 in sodium dodecyl sulfate-polyacrylamide gels.

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.

Viable variants in VA-RNA I gene of an Ad2-Ad5 recombinant

We have developed a simple method for selecting viral mutants whose DNA lack one of the sites of a restriction endonuclease that has more than one site on the viral genome. We have used this method for isolating viable variants of an Ad2-Ad5 recombinant that lack a BamHI cleavage site located at mp 29 spanning the VA-RNA I gene. The viral DNA-protein complex was prepared from a stock of the Ad2-Ad5 recombinant passed under high m.o.i., and cleaved with BamHI. We would expect BamHI to cleave the parental Ad2-Ad5 recombinant DNA at two sites, 29 and 59.5, but to cleave naturally arising mutant DNAs at one or the other site. With mutation at mp 29 site, BamHI cleavage would produce a fusion fragment from mp 0–59.5. Our objective, then, was to recombine the mutant DNA fragment in vivo with an overlapping fragment containing sequences from the right half of the genome, e.g., SalA (mp 45.9–100) to produce an infectious mutant DNA with a single BamHI site at mp 59.5. DNA protein complex was digested with BamHI and cotransfected with the DNA-protein complex cleaved with SalI, and the resulting plaques were screened for DNAs lacking the BamHI site at mp 29. Two such mutants were isolated, one of which is probably a deletion mutant (203) and the other an insertion mutant (204). Mutant 203 made VA-RNA I of slightly higher molecular weight and mutant 204 made VA-RNA I of slightly higher molecular weight. These mutants grew to levels comparable to that of the parental virus in KB cells indicating that these mutations did not alter the growth of the mutants. The method described here should also be valuable in isolating mutants elsewhere in the genome.

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.

An adenovirus from Tupaia (tree shrew): Growth of the virus, characterization of viral DNA, and transforming ability

An adenovirus was isolated from a kidney cell culture of an apparently healthy tree shrew ( Tupaia belangeri) and termed Tupaia adenovirus (TAV). An extensive host range study with different animal and human cells revealed that only Tupaia cells were susceptible to TAV infection. Tupaia embryonic kidney cells are the cells of choice for the plaque assay and for the efficient production of cell-free TAV at a titer of 10 8 PFU/ml within 36 hr post infection. TAV immortalizes skin fibroblasts of the New World monkey Callithrix jacchus. Purified TAV particles had a capsid diameter of 78–80 nm. The molecular weight of the viral DNA was 21.5 × 10 6 as determined by contour length measurements. The buoyant density of TAV DNA was 1.706 g × ml −1 as determined by isopycnic CsCl centrifugation. TAV DNA, when extracted with guanidinium hydrochloride, contains protein bound to its termini, since circles of double-stranded DNA were observed by electron microscopy, and restriction enzyme cleavage analysis of the TAV DNA-protein complex shows that the protein is associated with the two terminal TAV DNA fragments. The infectivity of the viral DNA-protein complex was 200-fold higher than TAV DNA. Cell biological and immunological data indicate that TAV is different from other known adenoviruses.

Isolation and characterization of polyoma uncoating intermediates from the nuclei of infected mouse cells.

A method was developed which enabled the efficient recovery of polyoma uncoating intermediates from the nuclei of infected cells at early times after infection (15 min to 12 h). Cells were infected with radiolabeled virus and lysed with the detergent Nonidet P-40. The nuclei were then collected and sonicated, and the products were analyzed on sucrose gradients. The uncoating intermediate sedimented at 190S and was a viral DNA-protein complex closely associated with a structure of host origin. The host material associated with the 190S uncoating intermediate was determined by polyacrylamide gel electrophoresis and visualized by electron microscopy. The amount of 190S uncoating intermediate found in the nucleus increased with time after infection. The viral DNA was predominantly for I. All of the viral proteins were present in the 190S uncoating intermediate in amounts similar to those found in viral DNA-protein complex cores.

Distinct nonstructural polypeptides in polyoma and simian virus 40 DNA-protein complexes

As already reported for polyoma virus [Qureshi, A. A., and Bourgaux, P., Virology 74, 377–385 (1976)] , a polypeptide absent from the virion was detected in a viral DNA-protein complex isolated using Triton X-100 from cells productively infected with simian virus 40 (SV40). Migration in sodium dodecyl sulfate-polyacrylamide gels indicated molecular weights of 76,000 and 86,000, respectively, for the SV40 and the polyoma virus polypeptide.

The infectivity of adenovirus 5 DNA-protein complex

Adenovirus 5 DNA-protein complexes were prepared by treating virions with 4 M guanidinium chloride and resolving the faster sedimenting viral DNA from released capsid protein. These complexes were characterized by both gel electrophoresis and electron microscopy. After dialysis into saline buffer, approximately one-half of the viral DNA-protein complex was aggregated by association at its termini into conformations other than linear monomer length duplex molecules. However, the monomer length linear molecules in the viral DNA-protein complex sample also have protein attached to both of their termini: These linear molecules are resistant to digestion by a pressessive nuclease, adenosine triphosphate-dependent deoxyribonuclease, that requires a free terminus for activity, while Pronase-treated adenovirus DNA is susceptible to digestion, and the restriction endonuclease cleavage fragments from both ends of the viral DNA-protein complex migrate at an anomalous rate during electrophoresis in an agarose gel. As the infectivity of Ad5 DNA is exceptionally low, the infectivity of the DNA-protein complex was tested using the calcium technique for transfection. The efficiency of transfection with the Ad5 DNA-protein complex is about 100-fold higher than that of Pronase-treated Ad5 DNA.

Polypeptides of a viral DNA-protein complex from polyoma virus-infected cells

Abstract A nascent viral DNA-protein complex was isolated from cells productively infected with polyoma virus (Py), by a conventional extraction procedure. This complex was purified by velocity sedimentation through neutral sucrose gradients, followed by ion-exchange chromatography. After in vitro labeling with 125 I, the protein moiety of the purified complex was analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Four polypeptides were thus detected: viral structural polypeptides VP1, VP2, and VP3, and a nonstructural polypeptide of a molecular weight of approximately 70,000. The latter represented as much as 48% of the 125 I radioactivity associated with all four polypeptides.

Decapsidation of polyoma virus: Identification of subviral species

Mouse embryo cells that had been exposed to polyoma virus (Py) labeled in its protein and/or DNA moiety were fractionated at various times after infection. Sedimentation analysis of the cytoplasm in 1 M NaCl solution revealed the presence of a labeled viral DNA-protein complex, termed NP, which is absent from virus preparations used for infection. This complex has a sedimentation coefficient of about 135 S and a DNA/protein ratio higher than that of virus, reflecting primarily loss of histone-like polypeptides. Another newly recognized species, a DNA-free complex (DFC) sedimenting at about 185 S, was identified in the cytoplasm. It contains reduced amounts of histone-like polypeptides and little or no DNA, as compared with virus. Unlike the material sedimenting like “full” virus particles which was also found in the cytoplasm, neither NP or DFC was stable in 2.7 M CsCl. Histone-like polypeptides lost from the above-mentioned species were detected in the cytoplasm as slowly sedimenting material. Radioactive material sedimenting as free DNA in 1 M NaCl solution was recovered from the nucleus and, occasionally, from the cytoplasm. It never represented more than 1.5% of the labeled DNA molecules present in virus at the time of infection. We propose a scheme for the decapsidation of Py in the cell. As the viron meanders in the cytoplasm, histone-like polypeptides are lost and NP is generated. The ensuing step is transfer of DNA to the nucleus, with a simultaneous reorganization of the capsid proteins into DFC. The viral DNA in the nucleus presumably is instrumental in directing the infectious process.

Demonstration of infectious DNA in transformed cells. III. Correlation of detection of infectious DNA-protein complexes with persistence of virus in simian adenovirus SA7-induced tumor cells.

Primary tumors induced in newborn hamsters by simian adenovirus SA7 were investigated in transfection experiments. Infectious DNA-protein complexes were readily detected in both the supernatant and pellet fractions obtained by a modified Hirt extraction procedure; DEAE-dextran was required for infectivity to become manifest. Infectivity could be abolished by exposure to DNase, but it was unaffected by SA7-specific antiserum or RNase, and only partially inactivated by trypsin treatment. SA7 tumor cells serially passaged either in tissue culture or in hamsters yielded infectious DNA complexes much less frequently and appeared to evolve into nonyielder cell lines. When large numbers of cells were lysed, intact virus could be recovered from all the primary tumors and from some of the subcultured cell lines. There was a correlation between the persistence of complete virus and the presence of infectious DNA-protein complexes. When the tumor cells carried very small amounts of intact virus, infectious DNA complexes could still be detected; when virus could no longer be detected in the tumor cells, infectious DNA complexes could no longer be found. The results suggest that a portion of the infectious DNA moieties exists as viral DNA-protein complexes in the tumor cells.

Biochemical and electron microscopic observations of vaccinia virus morphogenesis in HeLa cells after hydroxyurea reversal

Examination of thin sections of S3 HeLa cells propagated in suspension cultures and infected with vaccinia virus in the presence of hydroxyurea (HU) showed accumulation of immature virus particles. Upon removal of the drug the typical cytoplasmic development of virions was observed, confirming the usefulness of HU for separating early and late events in the replication cycle of poxvirions in spinner cultured cells and for studying viral morphogenesis under quasisynchronous conditions. Six hours after HU reversal, cytoplasm prepared by lysis of cells in Nonidet P-40 was fractionated by sucrose density centrifugation. Particles obtained by the fractionation procedure were negatively stained and their morphology characterized as follows: fraction I contained complete virions and cores with lateral bodies; fraction II contained particles 2300 × 3000 Å in size which resembled viral cores but lacked some of the surface layers found on such subviral particles prepared in vitro; fraction III contained spherical particles 2600–2800 Å in diameter and tubular structures of varying lengths and 1300 Å in diameter; fraction IV yielded particles which morphologically resembled viral cores but had irregular surfaces and appeared damaged; their sedimentation behavior during density gradient centrifugation and subsequent isolation suggested that they may be membrane bound in the cytoplasm and were mechanically released during isolation. When cytoplasmic samples were centrifuged in velocity gradients under conditions where subviral and viral particles were pelleted, progeny DNA was found throughout the gradient. In sucrose gradients prepared in 10 −3 M phosphate buffer, pH 7.2, this DNA could be resolved into three classes. A minor fraction (10% or less of the radioactivity) remained at the top of the gradient, major components sedimented at 50–100 S (about 30–40%), the third > 100 S (10–15%). The remainder of the radioactivity was in the pellet. When the 50–100 S fraction was analyzed in alkaline gradients, the denatured DNA sedimented at about 30 S. Early after HU reversal (1–3 hr), before significant assembly of viral DNA into DNase resistant structures had occurred, 85–90% of the newly synthesized viral DNA could be recovered in the form of virosomes. Thin sections of viral DNA protein complexes when examined in the electron microscope were found to be heterogeneous in composition. Irregular ovoid bodies, 0.8–1.0 μm by 0.5 to 0.7 μm in size and filamentous material often in large aggregates composed of fibrils about 20 Å in diameter comprised the major components observed in virosome preparations.