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PubMed1984

Kilham rat virus DNA replication in subcellular fractions.

A subcellular system actively replicating Kilham rat virus DNA in vitro was developed. Cellular lysates and isolated nuclei from infected cells showed an amplification of replicative forms in vitro. Solubilized replicative complexes, either partly purified or in the form of a crude extract, were able to synthesize replicative forms and single-stranded DNA. DNA polymerase alpha played a major role in Kilham rat virus DNA synthesis in vivo and in vitro. Furthermore, a factor present in the cytosol of infected cells increased the polymerizing activity of viral replicative complexes.

Animals↗

PubMed1984

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 E1A, E1B, 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.

Adenoviruses, Human↗

PubMed1999

Mapping of protein domains of hepatitis A virus 3AB essential for interaction with 3CD and viral RNA.

The small hydrophobic protein 3AB of the picornaviruses, encompassing the replication primer 3B, has been suggested to anchor the viral replication complex to membranes. For hepatitis A virus (HAV) 3AB, we have previously demonstrated its ability to form stable homodimers, to bind to membranes, and to interact specifically with RNA, implicating its multiple involvement in viral replication. In the present report, we show that HAV 3AB additionally interacts with HAV protein 3CD, a feature also described for the corresponding polypeptide of poliovirus. By assessing the interactions of three deletion mutants, distinct domains of HAV 3AB were mapped. The hydrophobic domain and the 3B moiety were found to be essential for the 3AB interaction with 3CD. Both electrostatic and hydrophobic forces are involved in this interaction. The cluster of charged amino acid residues at the C terminus of 3A seems to determine the specificity of 3AB interaction with RNA structures formed at either terminus of the HAV genome. Furthermore, our data implicate that 3A can interact with HAV RNA. Compared with poliovirus 3AB, which by itself is a nonspecific RNA-binding protein, HAV 3AB specifically recognizes HAV RNA structures that might be of relevance for initiation of viral RNA replication.

3C Viral Proteases↗

PubMed1975

The viral DNA replication machinery of adenovirus 12.

The viral DNA replication complex was found in the M band after fractionation. However, it did not associate with the nuclear membrane, but was found in association with types II and IV inclusions. It is suggested, therefore, that the viral DNA replication complex is the inclusions types II and IV. The analysis with Ad12 ts mutants showed the involvement of three viral genes in initiation of viral DNA replication, in formation of the viral DNA replication complex and the inclusions types II and IV. Temperature sensitivity of the gene products involved in viral DNA replication was also examined. From the data presented, the following sequence of viral DNA replication in Ad12-infected cells is suggested: (1) Synthesis of a protein (gene A product), which may be an acceptor or may modify the nuclear constituent to be an acceptor for parental viral DNA, to form the viral DNA replication complex; (2) synthesis of a protein (gene B product), which may be required for the parental viral DNA to associate with the acceptor and to form a type II inclusion-like structure (precursor of type II inclusion?); (3) synthesis of a protein (gene C product), which may be required for activation of the complex or type II inclusion and to alter type II inclusion to type IV inclusion. Further studies are necessary to substantiate the suggestion.

Adenoviridae↗

PubMed2003

Identification of the nuclear localization signal of human immunodeficiency virus type 2 Vpx.

The Vpx protein of human immunodeficiency virus type 2 (HIV-2) is a viral accessory protein related to, but distinct from, the Vpr protein of HIV-1. Vpx is packaged into virions and, as a component of the viral preintegration complex (PIC), Vpx is required for efficient virus replication in nondividing cells. Therefore, the localization of Vpx in cells is dynamic and dependent upon discrete domains of the protein. Expressed in the absence of other viral proteins, Vpx localizes to the nucleus of cells. However, if expressed with the Gag protein of HIV-2, Vpx localizes to the plasma membrane of cells. To further understand the regulation of Vpx localization, we fused regions of Vpx to beta-galactosidase to identify regions of the protein sufficient to mediate nuclear localization. The minimal transferable region of Vpx that conferred nuclear localization in these assays was aa 65 to 72. Alanine substitution of K(68) and R(70) in a GFP-Vpx construct abolished nuclear localization, suggesting that the basic residues in this region are important for nuclear import. Analysis of the membrane transport of several GFP-Vpx alanine mutants demonstrated that while separable, the domains of Vpx required for nuclear localization are not distinct from the domains required for membrane transport. The results of heterokaryon shuttling assays indicated that Vpx is not a shuttling protein; however, HIV-2 Vpr did shuttle similar to HIV-1 Vpr.

Alanine↗

PubMed2003

Herpes simplex virus 1 gene expression is accelerated by inhibitors of histone deacetylases in rabbit skin cells infected with a mutant carrying a cDNA copy of the infected-cell protein no. 0.

An earlier report showed that the expression of viral genes by a herpes simplex virus 1 mutant [HSV-1(vCPc0)] in which the wild-type, spliced gene encoding infected-cell protein no. 0 (ICP0) was replaced by a cDNA copy is dependent on both the cell type and multiplicity of infection. At low multiplicities of infection, viral gene expression in rabbit skin cells was delayed by many hours, although ultimately virus yield was comparable to that of the wild-type virus. This defect was rescued by replacement of the cDNA copy with the wild-type gene. To test the hypothesis that the delay reflected a dysfunction of ICP0 in altering the structure of host protein-viral DNA complexes, we examined the state of histone deacetylases (HDACs) (HDAC1, HDAC2, and HDAC3). We report the following. (i) HDAC1 and HDAC2, but not HDAC3, were modified in infected cells. The modification was mediated by the viral protein kinase U(S)3 and occurred between 3 and 6 h after infection with wild-type virus but was delayed in rabbit skin cells infected with HSV-1(vCPc0) mutant, concordant with a delay in the expression of viral genes. (ii) Pretreatment of rabbit skin cells with inhibitors of HDAC activity (e.g., sodium butyrate, Helminthosporium carbonum toxin, or trichostatin A) accelerated the expression of HSV-1(vCPc0) but not that of wild-type virus. We conclude the following. (i) In the interval in which HSV-1(vCPc0) DNA is silent, its DNA is in chromatin-like structures amenable to modification by inhibitors of histone deacetylases. (ii) Expression of wild-type virus genes in these cells precluded the formation of DNA-protein structures that would be affected by either the HDACs or their inhibitors. (iii) Since the defect in HSV-1(vCPc0) maps to ICP0, the results suggest that this protein initiates the process of divestiture of viral DNA from tight chromatin structures but could be replaced by other viral proteins in cells infected with a large number of virions.

Animals↗

PubMed1997

Anchoring of CREB binding protein to the human T-cell leukemia virus type 1 promoter: a molecular mechanism of Tax transactivation.

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates viral transcription through interaction with the cellular transcription factor CREB (cyclic AMP response element [CRE] binding protein). Although Tax stabilizes the binding of CREB to the Tax-responsive viral CREs in the HTLV-1 promoter, the precise molecular mechanism by which Tax mediates strong transcriptional activation through CREB remains unclear. In this report, we show that Tax promotes high-affinity binding of the KIX domain of CREB binding protein (CBP) to CREB-viral CRE complexes, increasing the stability of KIX in these nucleoprotein complexes by up to 4.4 kcal/mol. Comparable KIX binding affinities were measured for both phosphorylated and unphosphorylated forms of CREB, and in all cases high-affinity binding was dependent upon both Tax and the viral CRE. Tax also promoted association of KIX to a truncated form of CREB containing only the 73-amino-acid basic leucine zipper (bZIP) domain, indicating that the entire amino-terminal CBP-interacting domain of CREB is nonessential in the presence of Tax. Functional studies upheld the binding studies, as expression of the bZIP domain of CREB was sufficient to support Tax transactivation of HTLV-1 transcription in vivo. Finally, we show that transfection of a KIX expression plasmid, which lacks activation properties, inhibited Tax transactivation in vivo. This suggests that KIX occupies the CBP binding site on Tax, and therefore CBP is likely a cofactor in mediating Tax stimulation of HTLV-1 transcription. Together, these data support a model in which Tax anchors CBP to the HTLV-1 promoter, with strong transcriptional activation resulting from the CBP-associated activities of nucleosome remodeling and recruitment of the general transcription machinery.

Activating Transcription Factor 1↗

PubMed1996

Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis.

Membrane-bound replication complexes containing transcriptionally active replicative intermediates (RI) can be isolated from poliovirus-infected HEp-2 cells and consist of rosette-like structures of virus-induced vesicles surrounding the replicating viral RNA. At low ionic strength and low temperature, the rosettes reversibly dissociate into individual tubulated vesicles. As determined by immunoelectron microscopy and immunoprecipitation, the vesicles carry a set of viral structural and nonstructural proteins as well as RI RNA. At 30 degrees C, the vesicles reassociate into rosettes synthesizing plus-strand RNA in the RI. The in vitro transcriptional activities of rosettes and vesicles kept separated by high dilution were assessed by an RNase protection assay. The synthesis of the first 178 nucleotides at the 5' end of the plus strand was considered to reflect initiation, and the detection of a 530-nucleotide fragment in the P2 genomic region was considered to reflect elongation. It could be shown that the initiation and elongation of plus strands on individual vesicles are comparable to those in rosettes, with initiation proceeding in de novo-assembled initiation complexes. By use of detergent treatment it was found that initiation, but not elongation, is dependent on vesicular membranes.

Humans↗

PubMed1987

Phosphonoformate (foscarnet): a pilot study in AIDS and AIDS related complex.

Phosphonoformate (PFA; a pyrophosphate analogue) is an effective inhibitor of the reverse transcriptase enzyme in many animal retroviruses. In vitro studies have shown that PFA is also an effective inhibitor of HIV (HTLV III/LAV) at doses readily attainable in vitro. A pilot study was therefore performed with a 3-week intravenous infusion of PFA in 11 patients with AIDS and AIDS-related complex (ARC). Viral isolations were performed before and at regular intervals up to 3 months post-infusion on treated patients, as well as on four untreated control patients. Virus isolation was negative after therapy in eight patients, six of whom were negative throughout the follow-up period. Virus was isolated on 70% of attempts from the four control subjects and on 20% of attempts from treated subjects. Three patients showed an improvement in delayed hypersensitivity responses. No obvious improvement was seen in patients' OKT4 positive lymphocyte counts. Treatment was not limited by side-effects with the exception of one patient who developed an axillary vein thrombosis within 4 days of treatment via a subclavian line. Treatment was therefore discontinued following administration of only one dose and the patient was excluded from further study. A further patient had reversible renal dysfunction. Other side-effects were minor, consisting of headache or thrombophlebitis at the site of infusion. These results suggest that a further trial with PFA administered over a longer period and with a longer follow-up period in AIDS and ARC patients may be warranted, particularly if an oral preparation becomes available.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS-Related Complex↗

PubMed2000

MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes.

Human MxA protein accumulates in the cytoplasm of interferon-treated cells and inhibits the multiplication of several RNA viruses, including Thogoto virus (THOV), a tick-borne orthomyxovirus that transcribes and replicates its genome in the cell nucleus. The antiviral mechanism of MxA was investigated by using two alternative minireplicon systems in which recombinant viral ribonucleoprotein complexes (vRNPs) of THOV were reconstituted from cloned cDNAs. A chloramphenicol acetyltransferase reporter minigenome RNA was expressed either by T7 RNA polymerase in the cytoplasm of transfected cells or, alternatively, by RNA polymerase I in the nucleus. The inhibitory effect of MxA was studied in both cellular compartments by coexpressing wild-type MxA or TMxA, an artificial nuclear form of MxA. Our results indicate that both MxA proteins recognize the assembled vRNP rather than the newly synthesized unassembled components. The present findings are consistent with previous data which indicated that cytoplasmic MxA prevents transport of vRNPs into the nucleus, whereas nuclear MxA directly inhibits the viral polymerase activity in the nucleus.

Animals↗

PubMed1995

Monoclonal antibodies raised against covalently crosslinked complexes of human immunodeficiency virus type 1 gp120 and CD4 receptor identify a novel complex-dependent epitope on gp 120.

The binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cell surface receptor, CD4, represents a molecular interaction involving distinct alterations in protein structure. Consequently, the pattern of epitopes presented on the gp120-CD4 complex should differ from those on free gp120. To investigate this concept, mice were immunized with covalently crosslinked complexes of viral HIV-1IIIBgp120 and soluble CD4. Two monoclonal antibodies (MoAbs) obtained from the immunized mice exhibited a novel epitope specificity. The MoAbs were marginally reactive with HIV-1IIIBgp120, highly reactive with gp120-CD4 complexes, and unreactive with soluble CD4. The same pattern of reactivity was seen in solid-phase assays using HIV-1(451)gp120. A similar specificity for complexes was evident in flow cytometry experiments, in which MoAb reactivity was dependent upon the attachment of gp120 to CD4-positive cells. In addition, MoAb reactivity was detected upon the interaction of CD4 receptors with purified HIV-1IIIB virions. Notably, seroantibodies from HIV-positive individuals competed for MoAb binding, indicating that the epitope is immunogenic in humans. The results demonstrated that crosslinked gp120-CD4 complexes elicit antibodies to cryptic gp120 epitopes that are exposed during infection in response to receptor binding. These findings may have important implications for the consideration of HIV envelope-receptor complexes as targets for virus neutralization.

Animals↗

PubMed1984

Vesicular stomatitis virus defective interfering particles can contain extensive genomic sequence rearrangements and base substitutions.

We sequenced the 5' and 3' RNA termini of 16 defective interfering (DI) particles of vesicular stomatitis virus (VSV) isolated at intervals from persistent infections and from a series of undiluted lytic passages. All DI RNAs exhibited complementary termini, but sequences internal to these termini were extensively rearranged in a variety of ways. Despite extensive rearrangement, these internal sequences (in addition to the termini) apparently are important for DI particle interference properties. Some of these DI particles are derived from multiple intrastrand and interstrand recombination events, and the generation of each can be explained by current replicase error models. During viral evolution in persistent and acute infections, DI particles with specific termini base substitutions are selected. One DI particle exhibits a remarkable clustering of specific A----G (and complementary U----C) substitutions, apparently as a result of repetitive misincorporations by an error-prone viral polymerase complex.

Animals↗

PubMed1981

Isolation of monoclonal antibodies against avian oncornaviral protein p19.

For the production of monoclonal antibodies against pp60src and the gag precursor protein Pr76gag, the spleens of mice bearing tumors that had been induced by avian sarcoma virus Schmidt-Ruppin D-transformed cells were used. One hybridoma culture produced antibodies that were directed against the p19 portion of the gag precursor. However, no antibodies directed against pp60src could be detected in any of the hybridoma supernatants. The anti-p19-producing hybridoma culture was cloned twice in soft agar, and a stable clone was used for the production of high-titer ascites fluid in mice. The monoclonal antibodies belonged to the immunoglobulin G subclass 2b. The antibodies precipitated Pr76gag and the processed virion-associated p19, as well as the 75,000-molecular-weight gag fusion protein from avian erythroblastosis virus-transformed bone marrow cells. Also, viral ribonucleoprotein complexes were specifically precipitable, indicating that they contain p19 molecules.

Alpharetrovirus↗

PubMed1999

Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication.

The aim of the present study was to define the site of replication of the coronavirus mouse hepatitis virus (MHV). Antibodies directed against several proteins derived from the gene 1 polyprotein, including the 3C-like protease (3CLpro), the putative polymerase (POL), helicase, and a recently described protein (p22) derived from the C terminus of the open reading frame 1a protein (CT1a), were used to probe MHV-infected cells by indirect immunofluorescence (IF) and electron microscopy (EM). At early times of infection, all of these proteins showed a distinct punctate labeling by IF. Antibodies to the nucleocapsid protein also displayed a punctate labeling that largely colocalized with the replicase proteins. When infected cells were metabolically labeled with 5-bromouridine 5'-triphosphate (BrUTP), the site of viral RNA synthesis was shown by IF to colocalize with CT1a and the 3CLpro. As shown by EM, CT1a localized to LAMP-1 positive late endosomes/lysosomes while POL accumulated predominantly in multilayered structures with the appearance of endocytic carrier vesicles. These latter structures were also labeled to some extent with both anti-CT1a and LAMP-1 antibodies and could be filled with fluid phase endocytic tracers. When EM was used to determine sites of BrUTP incorporation into viral RNA at early times of infection, the viral RNA localized to late endosomal membranes as well. These results demonstrate that MHV replication occurs on late endosomal membranes and that several nonstructural proteins derived from the gene 1 polyprotein may participate in the formation and function of the viral replication complexes.

Animals↗

PubMed2003

Monocyte/macrophage-derived CC chemokines and their modulation by HIV-1 and cytokines: a complex network of interactions influencing viral replication and AIDS pathogenesis.

Monocytes/macrophages are cells of the innate arm of the immune system and exert important regulatory effects on adaptive immune response. These cells also represent major targets of HIV infection and one of the main reservoirs. Notably, macrophage-tropic viruses are responsible for the initial infection, predominate in the asymptomatic phase, and persist throughout infection, even after the emergence of dual-tropic and T-tropic variants. Functional impairment of HIV-infected macrophages plays an important role in the immune dysregulation typical of AIDS. Recent studies have underlined the pivotal role of chemokines, cytokines, and their receptors in HIV pathogenesis. It is becoming increasingly apparent that the expression level of chemokine receptors, serving as HIV coreceptors, influences the susceptibility of a CD4+ cell to viral infection and to certain HIV envelope-induced alterations in cellular functions. Numerous pathogens, including HIV, can stimulate the production of chemokines and cytokines, which in turn can modulate coreceptor availability, resulting in differential replication potential for R5 and X4 strains, depending on the microenvironment milieu. Thus, a complex network of interactions involving immune mediators produced by monocytes/macrophages and other cell types as a direct/indirect consequence of HIV infection is operative at all stages of the disease and may profoundly influence the extent of viral replication, dissemination, and pathogenesis.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Human immunodeficiency virus type 1 DNA nuclear import and integration are mitosis independent in cycling cells.

An essential step in human immunodeficiency virus type 1 (HIV-1) replication is the movement of the viral preintegration complex from the cytoplasm into the nucleus. The pathway(s) and timing for HIV-1 DNA nuclear entry in cycling cells have not been established. Here, we show that if cycling cells are infected before S phase, viral DNA can be integrated prior to passage of the host DNA replication fork through the integration site, as indicated by stable inheritance in both daughter cells. We conclude that efficient nuclear entry can occur independently of mitotic nuclear disassembly in cycling cells.

Active Transport, Cell Nucleus↗

PubMed2004

Monomer and dimer of Chandipura virus unphosphorylated P-protein binds leader RNA differently: implications for viral RNA synthesis.

Interaction of the leader RNA with the unphosphorylated P-protein has been proposed to play a key role in the transcription-replication transition of Chandipura virus, a model rhabdovirus. Electrophoretic mobility shift assay with the leader RNA and the unphosphorylated P-protein demonstrated existence of two distinct complexes in vitro. Measurements of stoichiometry indicate the protein monomer/RNA ratio to be 1:1 and 2:1 for faster and slower migrating bands, respectively. We have also observed a concentration-dependent oligomerization of the unphosphorylated P-protein, in sub-micromolar to low micromolar range. Sedimentation velocity, dynamic light scattering and large zone gel filtration experiments suggest a monomer-dimer-tetramer model of association. RNA binding experiments suggest that the two complexes assembled from one molecule of the leader RNA binding to either a protein monomer or a dimer. A truncated RNA consisting of a 3' region of the leader transcript exclusively formed the 1:1 complex, whereas a RNA consisting of only the 5' region forms the 2:1 complex exclusively. RNA binding experiments at different protein concentrations suggest that binding of the RNA comprising the 3' region weakens significantly at higher P(0) concentrations, whereas in contrast the binding of the RNA comprising the 5' region becomes modestly tighter. Implications of two different types of leader RNA-P-protein complexes in viral RNA synthesis are discussed.

Macromolecular Substances↗

PubMed1980

Analysis of herpes simplex virus nucleoprotein complexes extracted from infected cells.

HEp-2 cells were infected with herpes simplex virus type 1 and labeled with [3H]thymidine and 14C-amino acids. Infected cells or nuclei prepared from them were extracted with Triton X-100 and NaCl, utilizing a method recently described, and the low-speed supernatant (extract) was partially purified by sedimentation on sucrose gradients. A nucleoprotein complex which sedimented as a wide peak around 200S was identified. The nucleoprotein complex contained viral DNA, which banded at the expected density in CsCl isopycnic gradients and was intact after measurements taken on electron microscopic photographic enlargements. The autoradiographic pattern of 14C-labeled proteins after electrophoresis showed that only a few of the virus-specific polypeptides were present in the nucleoprotein complexes, in particular, VP5, VP12, VP15.2, VP19, and VP24. Cellular histones were absent. The extracts and the nucleoprotein complexes were centrifuged to equilibrium in metrizamide density gradients without prefixation. Electron microscopic direct visualization of the nucleoprotein complexes after sucrose or metrizamide purification revealed that the proteins were preferentially associated with one end of the DNA molecule and formed large irregular terminal thickenings or capsid-like transparent shells enclosing polyglobular cores. No nucleosomes were observed on herpes simplex virus nucleoprotein complexes. The same type of complex was detected after phosphonoacetic acid addition, and grossly altered nucleocapsids were formed.

Amino Acids↗