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PubMed1980

Integration of viral DNA sequences in cells transformed by adenovirus 2 or SV40.

We have cloned and propagated in prokaryotic vectors the viral DNA sequences that are integrated in a variety of cells transformed by adenovirus 2 or SV40. Analysis of the clones reveals that the viral DNA sequences sometimes are arranged in a simple fashion, collinear with the viral genome; in other cell lines there are complex arrangements of viral sequences in which tracts of the viral genome are inverted with respect to each other. In several cases the nucleotide sequences at the joints between cell and viral sequences have been determined: usually there is a sharp transition between cellular and viral DNAs. The viral sequences are integrated at different locations within the genomes of different cell lines; likewise there is no specific site on the viral genomes at which integration occurs. Sometimes the viral sequences are integrated within repetitive cellular DNA, and sometimes within unique sequences. In some cases there is evidence that the viral sequences along with the flanking cell DNA have been amplified after integration. The sequences that flank the viral insertion in the line of SV40-transformed rat cells known as 14B have been used as probes to isolate, from untransformed rat cells, clones that carry the region of the chromosome in which integration occurred. Analysis of the structure of these clones by restriction endonculease digestion and heteroduplex formation shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration.

Adenoviruses, Human↗
PubMed1996

Two 21-kilodalton components of the Epstein-Barr virus capsid antigen complex and their relationship to ZEBRA-associated protein p21 (ZAP21).

The viral capsid antigen complex of Epstein-Barr virus (EBV), an important serodiagnostic marker of infection with the virus, consists of at least four components, with molecular masses of 150, 110, 40, and 21 kDa. Here we show that the 21-kDa component of the viral capsid antigen consists of products of two EBV genes, BFRF3 and BLRF2. Both products were expressed from late transcripts, were recognized by human antisera, and were present in virions. The BFRF3 product, but not that of BLRF2, fulfilled the definition of ZEBRA-associated protein p21 (ZAP21). In cells in which EBV was lytically replicating, BFRF3 protein was coimmunoprecipitated together with ZEBRA by a rabbit antiserum directed against amino acids 197 to 245 of BZLF1. In EBV-negative cells cotransfected with BZLF1 and BFRF3 expression vectors, BFRF3 was also coimmunoprecipitated with this antiserum. Although this antiserum could not detect BFRF3 on an immunoblot, it was able to immunoprecipitate BFRF3 in the absence of ZEBRA expression. The rabbit antiserum to amino acids 197 to 245 of BZLF1 was found to detect the same epitope at the carboxy end of BFRF3 as was recognized by rabbit antiserum to BFRF3 itself. Thus, coimmunoprecipitation of BFRF3 p21 with ZEBRA appeared to be due to cross-reactivity of the immunoprecipitating antiserum rather than to direct association of ZEBRA and BFRF3 p21.

Animals↗
PubMed1992

Transgenic mice lacking class I major histocompatibility complex-restricted T cells have delayed viral clearance and increased mortality after influenza virus challenge.

To investigate the role of CD8+ T lymphocytes in recovery from influenza pneumonia, we used transgenic mice either homozygous (-/-) or heterozygous (+/-) for beta 2-microglobulin (beta 2-M) gene disruption. These mice lack major histocompatibility complex-restricted class I (CD8+) T cells. We found that after challenge with a nonlethal influenza virus, the beta 2-M (-/-) mice had significantly delayed pulmonary viral clearance. Furthermore, after challenge with a more virulent influenza virus, the beta 2-M (-/-) mice had a significantly higher mortality rate than did control mice. Thus, CD8+ T cells are important in recovery from virulent influenza infections, but other host defense mechanisms can clear the respiratory tract of more benign infections.

Animals↗
PubMed1979

The DNA polymerases associated with the adenovirus type 2 replication complex: effect of 2'-3'-dideoxythymidine-5'-triphosphate on viral DNA synthesis.

An adenovirus (Ad) DNA replication complex extracted from infected HeLa nuclei could be purified free of the bulk of intracellular DNA polymerase activity by sedimetation in neutral sucrose gradients. However, the replication complex still retained some alpha and gamma DNA-polymerase activity. Since this complex is inhibited by 2', 3' dideoxythymidine-5'-triphosphate (ddTTP), an inhibitor of DNA polymerase gamma, a functional role for this enzyme in Ad DNA replication is suggested. Similar inhibition by ddTTP in intact Ad infected nuclei and comparable inhibition of Ad DNA synthesis in whole cells by dideoxythymidine (ddThy) are consistent with a role for DNA polymerase gamma. Uninfected HeLa nuclei or whole cells are not similarly inhibited by ddTTP or DDThy respectively. Such data does not rule out an additional functional role for other DNA polymerases, and recent experiments from this laboratory (1) suggest that DNA polymerase alpha is also involved in Ad DNA synthesis.

Adenoviruses, Human↗
PubMed1997

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.

The mechanisms that direct positive-stranded RNA virus replication complexes to plant and animal cellular membranes are poorly understood. We describe a specific interaction between a replication protein of an RNA plant virus and membranes in vitro and in live cells. The tobacco etch virus (TEV) 6 kDa protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. In the presence or absence of other viral proteins, fluorescent fusion proteins containing the 6 kDa protein associated with large vesicular compartments derived from the endoplasmic reticulum (ER). Infection by TEV was associated with a collapse of the ER network into a series of discrete aggregated structures. Viral RNA replication complexes from infected cells were also associated with ER-like membranes. Targeting of TEV RNA replication complexes to membranous sites of replication is proposed to involve post-translational interactions between the 6 kDa protein and the ER.

Amino Acid Sequence↗
PubMed1996

Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIV(SM).

The vpr genes of human and simian immunodeficiency viruses (HIV/SIV) encode proteins which are packaged in the virus particle. HIV-1 Vpr has been shown to mediate the nuclear import of viral reverse transcription complexes in non-dividing target cells (e.g. terminally differentiated macrophages), and to alter the cell cycle and proliferation status of the infected host cell. Members of the HIV-2/SIV(SM) group encode, in addition to Vpr, a related protein called Vpx. Because these two proteins share considerable sequence similarity, it has been assumed that they also exhibit similar functions. Here, we report that the functions of Vpr and Vpx are distinct and non-redundant, although both proteins are components of the HIV-2/SIV(SM) virion and reverse transcription complex. Characterizing SIV(SM) proviruses defective in one or both genes, we found that Vpx is both necessary and sufficient for the nuclear import of the viral reverse transcription complex. In contrast, Vpr, but not Vpx, inhibited the progression of infected host cells from the G2 to the M phase of the cell cycle. Thus, two independent functions of the HIV-1 Vpr protein are encoded by separate genes in HIV-2/SIV(SM). This segregation is consistent with the conservation of these genes in HIV-2/SIV(SM) evolution, and underscores the importance of both nuclear transport and cell cycle arrest functions in primate lentivirus biology.

Animals↗
PubMed2001

PML mediates the interferon-induced antiviral state against a complex retrovirus via its association with the viral transactivator.

The promyelocytic leukaemia (PML) protein localizes in the nucleus both in the nucleoplasm and in matrix-associated multiprotein complexes known as nuclear bodies (NBs). The number and the intensity of PML NBs increase in response to interferon (IFN). Overexpression of PML affects the replication of vesicular stomatitis virus and influenza virus. However, PML has a less powerful antiviral activity against these viruses than the IFN mediator MxA. Here, we show that overexpression of PML, but not that of Mx1 or MxA, leads to a drastic decrease of a complex retrovirus, the human foamy virus (HFV), gene expression. PML represses HFV transcription by complexing the HFV transactivator, Tas, preventing its direct binding to viral DNA. This physical interaction requires the N-terminal region of Tas and the RING finger of PML, but does not necessitate PML localization in NBs. Finally, we show that IFN treatment inhibits HFV replication in wild-type but not in PML-/- cells. These findings point to a role for PML in transcriptional repression and suggest that PML could play a key role in mediating an IFN-induced antiviral state against a complex retrovirus.

Animals↗
PubMed1995

RNA-protein interactions directed by the 3' end of human rhinovirus genomic RNA.

The replication of a picornavirus genomic RNA is a template-specific process involving the recognition of viral RNAs as target replication templates for the membrane-bound viral replication initiation complex. The virus-encoded RNA-dependent RNA polymerase, 3Dpol, is a major component of the replication complex; however, when supplied with a primed template, 3Dpol is capable of copying polyadenylated RNAs which are not of viral origin. Therefore, there must be some other molecular mechanism to direct the specific assembly of the replication initiation complex at the 3' end of viral genomic RNAs, presumably involving cis-acting binding determinants within the 3' noncoding region (3' NCR). This report describes the use of an in vitro UV cross-linking assay to identify proteins which interact with the 3' NCR of human rhinovirus 14 RNA. A cellular protein(s) was identified in cytoplasmic extracts from human rhinovirus 14-infected cells which had a marked binding preference for RNAs containing the rhinovirus 3' NCR sequence. This protein(s) showed reduced cross-linking efficiency for a 3' NCR with an engineered deletion. Virus recovered from RNA transfections with in vitro transcribed RNA containing the same 3' NCR deletion demonstrated a defective replication phenotype in vivo. Cross-linking experiments with RNAs containing the poliovirus 3' NCR and cytoplasmic extracts from poliovirus-infected cells produced an RNA-protein complex with indistinguishable electrophoretic properties, suggesting that the appearance of the cellular protein(s) may be a common phenomenon of picornavirus infection. We suggest that the observed cellular protein(s) is sequestered or modified as a result of rhinovirus or poliovirus infection and is utilized in viral RNA replication, perhaps by binding to the 3' NCR as a prerequisite for replication complex assembly at the 3' end of the viral genomic RNA.

Base Sequence↗
PubMed1995

Structure of the complex between the Fab fragment of a neutralizing antibody for type 1 poliovirus and its viral epitope.

The crystal structure of the complex between the Fab fragment of C3, a neutralizing antibody for poliovirus, and a peptide corresponding to the viral epitope has been determined at 3.0 A resolution. Although this antibody was originally raised to heat inactivated (noninfectious) virus particles, it strongly neutralizes the Mahoney strain of type 1 poliovirus. Eleven peptide residues are well-defined in the electron-density map and form two type I beta-turns in series. At the carboxyl end, the peptide is bound snugly in the antibody-combining site and adopts a conformation that differs significantly from the structure of the corresponding residues in the virus. Structural comparisons between the peptide in the complex and the viral epitope suggests that on binding to infectious virions, this antibody may induce structural changes important for neutralization.

Amino Acid Sequence↗
PubMed2001

Cytoplasmic recruitment of INI1 and PML on incoming HIV preintegration complexes: interference with early steps of viral replication.

During the early phase of the retroviral life cycle, only a fraction of internalized virions end up integrating their genome into the chromosome, even though the resulting proviruses are almost systematically expressed. Here, we reveal that incoming retroviral preintegration complexes trigger the exportin-mediated cytoplasmic export of the SWI/SNF component INI1 and of the nuclear body constituent PML. We further show that the HIV genome associates with these proteins before nuclear migration. In the presence of arsenic, PML is sequestered in the nucleus, and the efficiency of HIV-mediated transduction is markedly increased. These results unveil a so far unsuspected cellular response that interferes with the early steps of HIV replication.

Active Transport, Cell Nucleus↗
PubMed1990

A nucleoprotein peptide of influenza A virus stimulates assembly of HLA-B27 class I heavy chains and beta 2-microglobulin translated in vitro.

Most cytotoxic T lymphocytes (CTL) recognize epitopes of foreign viral proteins in association with class I major histocompatibility complex (MHC) molecules. Viral proteins synthesized in the cytoplasm require intracellular fragmentation and exposure to the class I antigens for the development of CTL responses. Although indirect evidence for binding of peptides to class I antigens has accumulated, direct binding has only been shown recently. The formation of complexes between peptide and class I antigen may occur in the endoplasmic reticulum (ER) and peptides have been shown to induce assembly of the class I complex. We have translated the messenger RNAs encoding HLA-B27 (subtype 2705) and beta 2-microglobulin in a rabbit reticulocyte lysate supplemented with human microsomal membranes (to mimic ER membranes), in the absence and presence of a peptide derived from the nucleoprotein (residues 384-394) of influenza A virus. This peptide induces CTL activity against target cells expressing the HLA-B27 antigen. Here we report direct evidence that the nucleoprotein peptide promotes assembly of the HLA-B27 heavy chain and beta 2-microglobulin, and that this can occur in the ER immediately after synthesis of the two proteins.

Cell Compartmentation↗
PubMed1984

Production of monoclonal antibody to a late intracellular Epstein-Barr virus-induced antigen.

A monoclonal antibody designated L2 was produced against a late intracellular protein induced by Epstein-Barr virus (EBV). This protein was expressed in cells producing virus but not in EBV genome-positive nonproducer cell lines, EBV genome-negative cell lines, or producer cultures cultivated in the presence of phosphonoacetic acid as determined by immunofluorescence. In addition, the antibody did not react with the membranes of infected cells indicating that it was not directed against an EBV-induced membrane antigen component. The monoclonal antibody was shown to recognize a glycoprotein with a molecular weight of approximately 125K by SDS-polyacrylamide gel electrophoresis. This glycoprotein was consistently found to be slightly larger when isolated from the P3HR-1 cell line as opposed to the B-95-8 cell line. A similar difference was also noted by comparison of peptide maps of this protein isolated by immunoaffinity chromatography from the two cell lines. Serological studies indicated that this 125K glycoprotein was a major component of the viral capsid-antigen (VCA) complex as defined by immunofluorescence.

Antibodies, Monoclonal↗
PubMed1973

Comparison of direct and indirect solid-phase microradioimmunoassays for the detection of viral antigens and antiviral antibody.

Viral antigens were fixed to the surface of microtiter wells, and serial dilutions of antiviral antibody were added. The amount of antiviral antibody bound to viral antigens was determined by measuring the extent to which the antiviral antibody either inhibited the specific binding of (125)I-labeled antiviral immunoglobulin G (IgG) (direct technique) or enhanced the specific binding of (125)I-labeled anti-IgG (indirect technique). Immune complexes composed of viral antigens and antiviral antibody (human) could be detected by the binding of (125)I-labeled rheumatoid factor. Specific binding was influenced by the concentration of protein in the diluents used during the different steps of the procedure. A high concentration of protein in the diluent used with the viral antigens decreased specific binding, whereas a high concentration of protein in the diluent used with (125)I-labeled anti-IgG increased specific binding by decreasing nonspecific attachment of the labeled anti-IgG. Under the conditions employed, the titer of a given antiviral serum was several hundredfold greater by the indirect than by the direct technique.

Animals↗
PubMed2004

The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex.

The RNA genome of influenza virus is transcribed and replicated by the viral RNA polymerase complex in the cell nucleus. We have generated green fluorescent protein (GFP)-tagged polymerase subunits to study the assembly of the polymerase complex. Our results show that individually expressed polymerase basic protein 1 (PB1) and polymerase acidic protein (PA) subunits were distributed in both the cytoplasm and the nucleus, while the polymerase basic protein 2 (PB2) subunit accumulated in the nucleus. Although it has been reported that PB1 alone accumulates in the nucleus, we demonstrate that PB1 requires the coexpression of PA for efficient nuclear accumulation. Our results support a model which proposes that PB1 and PA are transported into the nucleus as a complex.

Active Transport, Cell Nucleus↗
PubMed2006

[Efficacy of chophytol in complex treatment of patients with the chronic forms of hepatitis and cirrhosis of viral etiology].

The preparation chophitol in complex treatment significantly improves the life quality of patients with chronic hepatitis and liver cirrhosis of viral etiology. It normalizes the index of cytolysis (ALT, AST) in patients with chronic hepatitis of viral etiology with moderate activity, and also the level of bilirubin in significantly short periods of time. In patients with liver cirrhosis of viral etiology classified as Child-Pugh A and B after the treatment with chophitol has been normalized index of overall protein and albumin, and in patients with liver cirrhosis classified as Child-Pugh class C appeared a tendency to the normalization of overall protein and albumin and prothrombin index, also creatinine in blood, which was accompanied with increased diuresis. The preparation is not expensive, is well tolerated. There has been observed no side effects on the treatment.

Adult↗
PubMed2005

High resolution structures of highly bulged viral epitopes bound to major histocompatibility complex class I. Implications for T-cell receptor engagement and T-cell immunodominance.

Although HLA class I alleles can bind epitopes up to 14 amino acids in length, little is known about the immunogenicity or the responding T-cell repertoire against such determinants. Here, we describe an HLA-B*3508-restricted cytotoxic T lymphocyte response to a 13-mer viral epitope (LPEPLPQGQLTAY). The rigid, centrally bulged epitope generated a biased T-cell response. Only the N-terminal face of the peptide bulge was critical for recognition by the dominant clonotype SB27. The SB27 public T-cell receptor (TcR) associated slowly onto the complex between the bulged peptide and the major histocompatibility complex, suggesting significant remodeling upon engagement. The broad antigen-binding cleft of HLA-B*3508 represents a critical feature for engagement of the public TcR, as the narrower binding cleft of HLA-B*3501(LPEPLPQGQLTAY), which differs from HLA-B*3508 by a single amino acid polymorphism (Arg156 --> Leu), interacted poorly with the dominant TcR. Biased TcR usage in this cytotoxic T lymphocyte response appears to reflect a dominant role of the prominent peptide x major histocompatibility complex class I surface.

Amino Acid Sequence↗