A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells

Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes.     

Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product

We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral El A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant d1312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1 B genes.     

Identification of proteins and protein domains that contact DNA within adenovirus nucleoprotein cores by ultraviolet light crosslinking of oligonucleotides 32P-labelled in vivo

A new approach to the identification of DNA binding proteins has been developed and used to study the DNA-protein interactions within the nucleoprotein core of subgroup C adenoviruses. Virions labelled in vivo with [32P]orthophosphate were exposed to ultraviolet light and the DNA digested by chemical or enzymatic methods. Labelled phosphoamino acids of the virion phosphoproteins were selectively hydrolysed by alkali, permitting proteins crosslinked to DNA to be identified by virtue of their covalently attached, 32P-labelled nucleotides. In parallel experiments, [3H]arginine-labelled virions were crosslinked by exposure to ultraviolet light and analysed by more conventional methods. The results indicate that proteins VII and V lie in close contact with viral DNA within the core. The compact arrangement of the nucleoprotein core appears to be capable of trapping protein VII molecules that are not covalently attached to DNA after exposure to ultraviolet light, suggesting that viral DNA might be wrapped around clusters of protein VII molecules. The domains of protein VII that lie in contact with DNA were identified by partial proteolytic mapping of the sites of covalent-attachment of the 32P-labelled oligonucleotides. The implications of these data for the nature of the interactions that mediate the packaging of viral DNA within the nucleoprotein core of adenovirions are discussed.     

Adenovirus protein VII functions throughout early phase and interacts with cellular proteins SET and pp32

Adenovirus protein VII is the major component of the viral nucleoprotein core. It is a highly basic nonspecific DNA-binding protein that condenses viral DNA inside the capsid. We have investigated the fate and function of protein VII during infection. "Input" protein VII persisted in the nucleus throughout early phase and the beginning of DNA replication. Chromatin immunoprecipitation revealed that input protein VII remained associated with viral DNA during this period. Two cellular proteins, SET and pp32, also associated with viral DNA during early phase. They are components of two multiprotein complexes, the SET and INHAT complexes, implicated in chromatin-related activities. Protein VII associated with SET and pp32 in vitro and distinct domains of protein VII were responsible for binding to the two proteins. Interestingly, protein VII was found in novel nuclear dot structures as visualized by immunofluorescence. The dots likely represent individual infectious genomes in association with protein VII. They appeared within 30 min after infection and localized in the nucleus with a peak of intensity between 4 and 10 h postinfection. After this, their intensity decreased and they disappeared between 16 and 24 h postinfection. Interestingly, disappearance of the dots required ongoing RNA synthesis but not DNA synthesis. Taken together these data indicate that protein VII has an ongoing role during early phase and the beginning of DNA replication.     

Comparison of the interactions of the adenovirus type 2 major core protein and its precursor with DNA.

The interactions of the major core protein of adenovirus type 2 (Ad2) protein VII, and its precursor, protein pre-VII, with viral DNA, were studied using UV light induced crosslinking of 32P-labelled oligonucleotides to the proteins. Proteolytic fragments of these two proteins that contain DNA-binding domains were identified by virtue of their covalently attached, alkali-resistant 32P-radioactivity. The overall efficiency of crosslinking of protein pre-VII to DNA, in H2ts1 virions assembled at 39 degrees C, was comparable to that of the crosslinking of protein VII to DNA in Ad2 virions. However, a protease V8 fragment comprising the N-terminal half of protein pre-VII crosslinked to DNA at least ten times more efficiently than the corresponding N-terminal fragment of protein VII, which is truncated by the removal of 23 amino acids from the N-terminus of protein pre-VII during virion maturation.     

Human immunodeficiency virus infection of cells arrested in the cell cycle.

Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non-dividing macrophages. This ability to grow in non-dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV-1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV-1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non-dividing cells is due to one of the HIV-1 core virion proteins. HIV infection of non-dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.     

Tracking adenovirus genomes identifies morphologically distinct late DNA replication compartments

In adenoviral virions, the genome is organized into a chromatin‐like structure by viral basic core proteins. Consequently viral DNAs must be replicated, chromatinized and packed into progeny virions in infected cells. Although viral DNA replication centers can be visualized by virtue of viral and cellular factors, the spatiotemporal regulation of viral genomes during subsequent steps remains to be elucidated. In this study, we used imaging analyses to examine the fate of adenoviral genomes and to track newly replicated viral DNA as well as replication‐related factors. We show de novo formation of a subnuclear domain, which we termed Virus‐induced Post‐Replication (ViPR) body, that emerges concomitantly with or immediately after disintegration of initial replication centers. Using a nucleoside analogue, we show that viral genomes continue being synthesized in morphologically distinct replication compartments at the periphery of ViPR bodies and are then transported inward. In addition, we identified a nucleolar protein Mybbp1a as a molecular marker for ViPR bodies, which specifically associated with viral core protein VII. In conclusion, our work demonstrates the formation of previously uncharacterized viral DNA replication compartments specific for late phases of infection that produce progeny viral genomes accumulating in ViPR bodies.      

Physical and functional interaction between a nucleolar protein nucleophosmin/B23 and adenovirus basic core proteins

We identified nucleophosmin/B23 as a component of template-activating factor-III that stimulates the DNA replication from the adenovirus DNA complexed with viral basic core proteins. Here, we have studied the functional interaction of B23 with viral core proteins. We found that B23 interacts with viral basic core proteins, core protein V and precursor of core protein VII (pre-VII), in infected cells. Biochemical analyses demonstrated that B23 suppresses formation of aggregates between DNA and core proteins and transfers pre-VII to DNA. These results indicate that B23 functions as a chaperone in the viral chromatin assembly process in infected cells.     

Adenovirus core protein synthesis in the absence of viral DNA synthesis late in infection.

The acid extraction of the adenovirus type 5 core proteins V, VII, and pVII (the precursor to VII) from infected cells and the subsequent electrophoresis on a 15% acrylamide-2.5 M urea-0.9 N acetic acid (pH 2.7) gel, revealed that peptide VII has a similar electrophoretic mobility to that of histone H1. The core proteins, which are coded by late adenovirus mRNA, continued to be synthesized late in infection when viral DNA synthesis was inhibited either by cytosine arabinoside in wild-type infections or by shifting adenovirus H5 ts 125-infected cells to the nonpermissive temperature (40 degree C). Only the initiation, not the continuation, of viral DNA replication was essential for core protein synthesis. The synthesis of viral core proteins continued for over 8 h after the cassation of DNA synthesis. This was in contrast to the rapid shutdown of cellular histone synthesis in the absence of cellular DNA synthesis.     

Detection of a cellular polypeptide associated with adenovirus-coded VA RNA using in vitro labeling of proteins cross-linked to RNA.

Ultraviolet light induced RNA-protein cross-linking for identification of polypeptides interacting with RNA in intact cells (Wagenmakers et al. 1980), is limited by the intensity of the label in the proteins or in residual nucleotides remaining attached to the proteins after RNase treatment of the RNA-protein complexes. Here we report a method, where th cross-linked RNA-protein complexes are treated with RNase T1 and the T1-oligonucleotides covalently linked to the proteins are labeled in the 5' terminus using gamma-32P-ATP and T4 polynucleotide kinase. The cross-linked proteins can then readily be identified owing to the incorporated 32P label. As examples, proteins associated with polyadenylated mRNA, hnRNA and adenoviral VA RNA were identified. A protein with a molecular weight of approximately 50,000 is found associated with adenovirus-coded VA RNA. This was confirmed by binding assays, in which labeled VAI RNA is incubated with proteins from uninfected and adenovirus infected HeLa cells immobilized on nitrocellulose sheets.     

Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus.

During influenza virus infection, protein synthesis is maintained at high levels and a dramatic switch from cellular to viral protein synthesis occurs despite the presence of high levels of functional cellular mRNAs in the cytoplasm of infected cells (M. G. Katze and R. M. Krug, Mol. Cell. Biol. 4:2198-2206, 1984). To determine the step at which the block in cellular mRNA translation occurs, we compared the polysome association of several representative cellular mRNAs (actin, glyceraldehyde-3-phosphate dehydrogenase, and pHe7 mRNAs) in infected and uninfected HeLa cells. We showed that most of these cellular mRNAs remained polysome associated after influenza viral infection, indicating that the elongation of the proteins encoded by these cellular mRNAs was severely inhibited. Because the polysomes containing these cellular mRNAs did not increase in size but either remained the same size or decreased in size, the initiation step in cellular protein synthesis must also have been defective. Several control experiments established that the cellular mRNAs sedimenting in the polysome region of sucrose gradients were in fact associated with polyribosomes. Most definitively, puromycin treatment of infected cells caused the dissociation of polysomes and the release of cellular, as well as viral, mRNAs from the polysomes, indicating that the cellular mRNAs were associated with polysomes that were capable of forming at least a single peptide bond. A similar analysis was performed with HeLa cells infected by adenovirus, which also dramatically shuts down cellular protein synthesis. Again, it was found that most of the cellular mRNAs, which were translatable in reticulocyte extracts, remained associated with polysomes and that there was a combined initiation-elongation block to cellular protein synthesis. In cells infected by both adenovirus and influenza virus, influenza viral mRNAs were on larger polysomes than were several late adenoviral mRNAs with comparably sized coding regions. In addition, after influenza virus superinfection of cells infected by the adenovirus mutant dl331, a situation in which there is a limitation in the amount of functional initiation factor eIF-2 (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986), influenza viral mRNAs, but not late adenoviral mRNAs, were on polysomes. These results indicate that influenza viral mRNAs are better initiators of translation than are late adenoviral mRNAs.     

Action of Interferon: Kinetics and Differential Effects on Viral Functions

A highly purified rabbit interferon was tested for its capacity to inhibit various manifestations of infection of primary rabbit kidney (RK) cells with vesicular stomatitis (VS) virus. A kinetic analysis of the actinomycin-sensitive phase of interferon-induced cellular resistance revealed that RK cells could transcribe virtually all of the hypothetical antiviral messenger ribonucleic acid (mRNA) within 3 hr. Similar exposure to interferon reduced virus yield by 95 to 99% and markedly inhibited cytopathic effect on RK cells infected at a multiplicity of 10 or less. Interferon was less effective in blocking cytopathic effects when RK cells were infected at a multiplicity of 100. However, RK cells pretreated with the same amount of interferon and infected at a multiplicity of 100 failed to incorporate (3)H-amino acids into structural or nonstructural proteins of VS virus identified by polyacrylamide gel electrophoresis. Despite this inhibition of viral protein synthesis, interferon did not prevent the switch off by VS virus of cellular protein synthesis. The rapidity with which a high multiplicity of VS virus switched off cellular protein synthesis, even in cells rendered resistant to viral infection by interferon, is further evidence that this reaction is caused by an infecting virion component rather than by a newly synthesized viral product.     

Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells.

The proteins that interact with cytoplasmic and nuclear polyadenylated RNA in adenovirus type 5 (Ad5) infection of HeLa cells were examined by UV-induced RNA-protein cross-linking in intact cells. The Ad5 100-kilodalton late nonvirion protein (100K protein) was cross-linked to both host and viral polyadenylated cytoplasmic RNA (mRNA). The cross-linking of the 100K protein to mRNA appears to correlate with productive infection, because the protein is not cross-linked to mRNA in abortive infection of wild-type Ad5 in monkey cells (CV-1) even though normal amounts of it are produced. However, when CV-1 cells are infected with Ad5 hr404, and Ad5 mutant which overcomes the host restriction to wild-type Ad5 infection in these cells, the 100K protein is cross-linked to mRNA. To identify and obtain antibodies to RNA-contacting proteins, a mouse was immunized with oligo(dT)-selected cross-linked RNA-protein complexes from Ad5-infected cells and the serum was used for immunoblotting experiments. It was found that in addition to the 100K protein, the Ad5 72K DNA-binding protein is also associated with RNA in the infected cells. The 72K DNA-binding protein is cross-linked to polyadenylated nuclear RNA sequences. These findings indicate that adenovirus proteins interact with RNAs in the infected cell and suggest possible mechanisms for the effects of the virus on mRNA metabolism.     

Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid.

Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA) equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells. Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro replication system has the potential to probe host DNA synthesis requirements as well as viral factors.     

Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production

We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on these observations, we sought in the current study to identify the precise defect in K30 virion infection of permissive equine dermal (ED) cells. The results of these experiments clearly demonstrated that K30 virions entered target ED cells and produced early (minus-strand strong-stop) and late (Gag) viral DNA products as efficiently as did the replication-competent E32 mutant and parental EIAV(UK) viruses. However, in contrast to the replication-competent E32 mutant and parental viruses, infection with K30 mutant virus failed to produce detectable two-long-terminal-repeat DNA circles, stable integrated provirus, virus-specific Gag mRNA expression, or intracellular viral protein expression. Taken together, these data demonstrate that the K30 mutant is defective in the ability to produce sufficient nuclear viral DNA to establish a productive infection in ED cells. Thus, these observations indicate for the first time that the EIAV Gag p9 protein performs a critical role in viral DNA production and processing to provirus during EIAV infection, in addition to its previously defined role in viral budding mediated by the p9 L domain.     

Early Events of Virus-Cell Interaction in an Adenovirus System

The interaction of (32)P-labeled adenovirus type 2 and HeLa or KB cells has been examined during early infection. The kinetics of virus uncoating to deoxyribonuclease-sensitive products, the partial characterization of three such products by gradient centrifugation, and the distribution of these products in the extranuclear and nuclear portions of infected cells are reported. The results are compatible with the following model. Extracellular virus attaches to a receptor on the plasma membrane. The membrane-bound virus has a half-life of less than 15 min and is transformed to a partly uncoated product which is free inside the cell and about half of which rapidly enters the cell nucleus. This is rapidly transformed, in both cytoplasm and nucleus, to a membrane-bound virion "core." The proteins of the bound "core" are then removed from the intact virus deoxyribonucleic acid (DNA). In the nucleus, viral DNA is the main product and there the overall sequence is completed in about 2 hr.     

Adenovirus capsid proteins interact with HSP70 proteins after penetration in human or rodent cells

Soon after penetration of adenovirus serotype 2 in BHK-21 and HeLa cells, HSP70 and HSC70 proteins become associated with the viral capsid. By analysis with a polyclonal antibody derived from a fusion protein containing the C-terminal domain, 290 amino acids of HSP70, and using both immunological methods and infected cells fractionation we observed that a significant amount of HSP70 proteins moved to the nucleus and colocalized with the adenovirus particles. HSP70 proteins of infected cells were isolated as a complex cross-linked with intracytoplasmic adenovirus type 2. By coprecipitation, using a polyclonal-specific antiserum derived from the fusion protein, or two different monoclonal-specific antisera, we showed that HSP70 and HSC70 proteins were associated with hexon, the major adenovirus capsid protein.     

Entry of herpes simplex virus 1 in BJ cells that constitutively express viral glycoprotein D is by endocytosis and results in degradation of the virus.

The BJ cell line which constitutively expresses herpes simplex virus 1 glycoprotein D is resistant to infection with herpes simplex viruses. Analysis of clonal lines indicated that resistance to superinfecting virus correlates with the expression of glycoprotein D. Resistance was not due to a failure of attachment to cells, since the superinfecting virus absorbed to the BJ cells. Electron microscopic studies showed that the virions are juxtaposed to coated pits and are then taken up into endocytic vesicles. The virus particles contained in the vesicles were in various stages of degradation. Viral DNA that reached the nucleus was present in fewer copies per BJ cell than that in the parental BHKtk- cells infected at the same multiplicity. Moreover, unlike the viral DNA in BHKtk- cells which was amplified, that in BJ cells decreased in copy number. The results suggest that the glycoprotein D expressed in the BJ cell line interfered with fusion of the virion envelope with the plasma membrane but not with the adsorption of the virus to cells and that the viral proteins that mediate adsorption to and fusion of membranes appear to be distinct.