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PubMed1994

A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.

Retroviral replication depends on integration of the viral genome into a chromosome of the host cell. The steps in this process are orchestrated in vivo by a large nucleoprotein complex and are catalyzed by the retroviral enzyme integrase. Several biochemical properties of the in vivo nucleoprotein complex were reproduced in vitro with purified integrase of human immunodeficiency virus type 1 and model viral DNA substrates. A stable complex between integrase and viral DNA was detected as an early intermediate in the integration reaction. After formation of this initial complex, the enzyme processively catalyzed the 3' end processing and strand transfer steps in the reaction. Complexes containing only purified integrase and the model viral DNA end were stable under a variety of conditions and efficiently used nonviral DNA molecules as integration targets. These complexes required a divalent cation for their formation, and their stability was highly dependent on the 5'-terminal dinucleotide of the viral DNA, for which no functional role has previously been defined. Thus, interactions between integrase and the extreme ends of the viral DNA molecule may be sufficient to account for the stability of the in vivo integration complex.

Base Sequence↗
PubMed1983

Encephalomyocarditis virus replication complexes that prefer nucleoside diphosphates as substrates for viral RNA synthesis.

Replication complexes (RC) containing endogenous viral RNA templates and the viral RNA polymerase were isolated from encephalomyocarditis (EMC) virus-infected Krebs II cells by two different procedures one of which included removal of the bulk of the associated proteins by treatment with 2 M LiCl. Replacement of one or all the four nucleoside triphosphate (NTP) substrates by corresponding nucleoside diphosphates (NDPs) did not eliminate the ability of the RC to synthesize viral RNA products. Moreover, the complexes were shown to accept as substrates even nucleoside monophosphates (NMPs), provided at least one NTP was present in the system. These results suggested that nucleotide kinases, NMP kinase and NDP kinase, were associated with the RC, and this suggestion was confirmed directly. The RC could be resolved, by sucrose gradient centrifugation, into distinct components that exhibited marked preference for either NDPs or NTPs as substrates for RNA synthesis. It is suggested that, in the NDP-preferring components, the NTP molecules generated by the built-in nucleotide kinase system are channeled directly to the vicinity of the replication fork.

Adenosine Diphosphate↗
PubMed1997

Release of superoxide anion from polymorphonuclear leukocytes stimulated by rubella viral antigen-antibody complex in vitro.

Using a highly sensitive and specific chemiluminescence (CL) method, we detected the release of superoxide anion (O2-.) from human polymorphonuclear leukocytes (PMNLs) stimulated by rubella viral antigen-antibody complex (VAAC) adsorbed on latex particles. The amount of superoxide anion produced by PMNLs was proportional to the amount of VAAC. Neither rubella virus alone nor antibody alone, adsorbed on latex particles, stimulated production of superoxide anion by PMNLs. It is likely that rubella virus requires the presence of antibody in order to be recognized by PMNLs, and that the superoxide anion is somehow involved in the disease process of rubella.

Antibodies, Viral↗
PubMed2006

Association of functional influenza viral proteins and RNAs with nuclear chromatin and sub-chromatin structure.

Transcription and replication of the influenza virus genome occur in the nucleus. However, the intra-nuclear localization of viral RNP complexes and the function of nuclear domains involved in viral transcription and replication, if any, are not well known. In the present study, we determined the intra-nuclear localization of viral proteins and viral RNAs and the in vitro RNA synthesis activity of viral RNP complexes associated with distinct nuclear fractions prepared from infected nuclei. A majority of viral RNA polymerases and M1 were recovered in DNase-sensitive fractions, whereas some portion of RNA polymerases and approximately 25% of NP were tightly associated with so-called nuclear matrix fractions. The amount of vRNA associated with the nuclear matrix was significantly more than that of cRNA. The in vitro viral RNA synthesis activity was detected in DNase-insensitive fractions, including the nuclear matrix. In contrast, newly synthesized viral RNAs were recovered in the DNase-sensitive fraction. These observations suggest that vRNP complexes are, at least partially, associated with densely packed chromatin, where viral transcription and replication occur, and the newly synthesized vRNP complexes to be transported into the cytoplasm are released into the nucleoplasm.

Animals↗
PubMed1991

[Elevated Clq-bearing immune complexes in hemophiliacs with viral infections].

One hundred hemophilia A and 30 hemophilia B patients who had been treated with non-heated and heated factor VIII or prothrombin complex concentrates were examined by immunological tests including Clq-bearing immune complexes assay. Antibodies to human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), hepatitis C virus (HCV) and human parvovirus B19 (B19) were analyzed by Western blotting, enzyme immunoassay, passive hemagglutination or radio-immunoassay. Clq-bearing immune complexes were assayed by a monoclonal anti-Clq ELISA system (Immunomedics). Seropositivity to HIV-1, HBV, HCV, and B19 was 56.9%, 87.7%, 79.2% and 100% respectively. Clq-bearing immune complexes were positive in 109 of the 130 patients (83.8%). The positivity and the levels were extremely higher than those in normal individuals. Clq-bearing immune complex levels in patient positive for HIV-1, HCV, or HBV were higher than those in the negative group (HIV: P less than 0.001, HCV: P less than 0.005, HBV: P less than 0.05). When the patients were divided into four groups according to seropositivity to HIV-1 and/or HCV, Clq-bearing immune complex levels were the highest in the group positive for both antibodies, and the lowest in the group negative for both antibodies. These results suggested that each viral infection influences the formation of immune complexes and repeated viral infection increased the level of Clq-bearing immune complexes in these patients.

Adolescent↗
PubMed1998

Photo-cross-linking studies suggest a model for the architecture of an active human immunodeficiency virus type 1 integrase-DNA complex.

The virally encoded integrase protein carries out retroviral integration, which requires specific interactions with the two ends of the viral DNA, and also with host DNA that is the target of integration. We attached a photo-cross-linking agent to specific viral and target DNA sites to identify regions of the integrase polypeptide that are in close proximity to those substrate features in the active integrase-DNA complex. The active form of integrase is a multimer. The higher-order organization of the active integration complex was therefore investigated by determining whether specific cross-links occurred to the active-site containing protomer. Both viral and target DNA cross-links to human immunodeficiency virus type 1 (HIV-1) integrase mapped predominantly to integrase protomers in trans to the active site, in a multimeric integrase complex. The results provide the basis for a model of the protein-DNA architecture of an active HIV-1 integration complex that suggests specific functions for the N-terminal, core, and C-terminal domains of retroviral integrase. One implication of this model is that the integrase multimer that mediates concerted integration of the viral DNA ends is composed of at least eight integrase protomers.

Catalysis↗
PubMed1990

Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores.

Reconstitution of influenza virus nucleoprotein (NP)-RNA complexes was performed with segment 8 RNA, which was synthesized in vitro from cDNA, and NP purified from virions. Under optimum conditions established using a filter binding assay and a gel retardation assay, NP was found to bind any RNA longer than 15 nucleotides. NP-RNA complexes formed at 30 degrees C are more resistant to high concentrations of NaCl than those formed at 0 degrees C. Treatment of NP with N-ethylmaleimide gave no effect on its RNA binding activity, whereas treatment with alkaline phosphatase enhanced its RNA binding activity. The newly developed "reverse-printing" method of RNase V1-treated complexes revealed that reconstituted NP-RNA complexes carry RNase V1-sensitive sites as do native ribonucleoprotein (RNP) cores (RNA polymerase-NP-RNA complexes), implying that RNA-NP complexes structurally similar to native RNP cores are reconstituted from isolated components.

Alkaline Phosphatase↗
PubMed2005

The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus.

Replication of plus-stranded RNA viruses is performed by the viral replicase complex, which, together with the viral RNA, must be targeted to intracellular membranes, where replication takes place in membraneous vesicles/spherules. Tombusviruses code for two overlapping replication proteins, the p33 auxiliary protein and the p92 polymerase. Using replication-competent fluorescent protein-tagged p33 of Cucumber necrosis virus (CNV), we determined that two domains affected p33 targeting to peroxisomal membranes in yeast: an N-proximal hydrophobic trans-membrane sequence and the C-proximal p33:p33/p92 interaction domain. On the contrary, only the deletion of the p33:p33/p92 interaction domain, but not the trans-membrane sequence, altered the intracellular targeting of p92 protein in the presence of wt p33 and DI-72(+) RNA. Moreover, unlike p33, p92 lacking the trans-membrane sequence was still functional in supporting the replication of a replicon RNA in yeast, whereas the p33:p33/p92 interaction domain in both p33 and p92 was essential for replication. In addition, p33 was also shown to facilitate the recruitment of the viral RNA to peroxisomal membranes and that p33 is colocalized with (+) and (-)-stranded viral RNAs. Also, FRET and pull-down analyses confirmed that p33 interacts with other p33 molecules in yeast cells. Based on these data, we propose that p33 facilitates the formation of multimolecular complexes, including p33, p92, viral RNA, and unidentified host factors, which are then targeted to the peroxisomal membranes, the sites of CNV replication.

Bacterial Proteins↗
PubMed1993

Virus and cytotoxic T lymphocytes: crucial role of viral peptide secondary structure in major histocompatibility complex class I interactions.

Viral antigens are presented to cytotoxic T lymphocytes (CTLs) by H-2-restricted major histocompatibility complex (MHC) glycoproteins. Binding of the endogenously processed viral peptides (epitopes) to their specific MHC molecules is an early intracellular event in the recognition process and is necessary for subsequent killing of virus-infected cells by virus-specific CTLs. It is now well established that interaction between a viral antigenic peptide and MHC is dependent on the primary structure (length and amino acid sequence) of that antigen. Here we show, using the H-2Db-restricted epitope GP277-286 of lymphocytic choriomeningitis virus as a model, that the secondary structure (conformation) of the viral sequence also plays a crucial role in the binding of a viral antigen to MHC glycoprotein and in its subsequent presentation to virus-specific CTLs.

Amino Acid Sequence↗
PubMed1991

Effects of Brefeldin A on the Golgi complex, endoplasmic reticulum and viral envelope glycoproteins in murine erythroleukemia cells.

This report concerns the effects of Brefeldin A (BFA): i) on the Golgi complex and the ER of retrovirus-transformed murine erythroleukemia (MEL) cells and, ii) on the viral proteins these cells express. Golgi complexes were extensively disorganized by BFA. Within 5 min, most stacked cisternae were converted to vesicles scattered throughout the centrosphere region. By 30 min, the Golgi complexes were completely disassembled. Only clusters of small vesicles ("Golgi remnants") persisted in the vicinity of the centrioles and microtubule-organizing centers. Some of these small vesicles had a simple coat structure on their membranes. Over the next 1 to 2 h of BFA treatment, the number of vesicles in the Golgi area decreased concomitantly with the expansion of a predominantly smooth membrane portion of the ER, consisting of a network of dilated tubules in continuity with regular RER cisternae, annulate lamellae and the nuclear envelope. By electron microscopy, viral glycoproteins appeared to accumulate on the membranes of this network, and immature virions were found to bud preferentially into its cisternal space. Viral accumulations increased with time under BFA. The rest of the RER appeared normal, apparently unaffected by the drug. Preferential virion budding suggests that this expanding network is a chemically differentiated part of the ER. By immunofluorescence, antibodies to viral envelope proteins gave a punctate staining at the surface of control cells, presumably in the areas of virion budding, whereas relatively large intracellular masses of antigens were found in BFA-treated cells. We assume that these masses represent the differentiated parts of the ER. Taken together, these findings suggest that BFA blocks intracellular transport of newly synthesized cellular and viral proteins immediately distal to the distinct compartment of the ER in which virion budding preferentially occurs. BFA effects are rapidly and fully reversible. Within 1 min of the removal of the drug, stacks of Golgi cisternae began to reappear in the vicinity of the centrioles, and by 30 min, Golgi complexes regained their normal structural appearance.

Animals↗
PubMed1993

In vivo viral and cellular Jun complexes exhibit differential interaction with a number of in vitro generated 'AP-1- and CREB-like' target sequences.

A direct comparison of the relative DNA-binding capabilities of in vivo Jun-containing complexes derived from overexpression of the highly transforming viral Jun (VJ-1 CEF), the weakly transforming chicken cellular Jun (CJ-3 CEF) or background endogenous Jun (RCAS CEF) was assessed by gel mobility-shift assays using a synthetic oligonucleotide containing the consensus sequence TGACTCA (consensus AP-1). Chicken embryo fibroblasts (CEFs) expressing background c-Jun levels (RCAS CEF) contain almost undetectable levels of c-Jun but retain significant DNA-binding activity with two distinct complexes capable of binding specifically to the consensus AP-1 site. CEFs overexpressing either v-Jun or c-Jun contain these same two complexes and, while showing marked increases in Jun protein levels, do not exhibit any increase in DNA binding or transcriptional activation activity, suggesting that much of the overexpressed protein is inactive. Gel-shift assays performed in the presence of a Jun-specific antibody revealed a reduction in binding by both complexes, suggesting that each contains Jun or a Jun cross-reactive protein. Antibodies specific for Jun B, c-Fos, Fos B and CREB failed to interact with either complex. However, antibody specific for Fra-2 caused a slight supershift, suggesting that one or both complexes may contain Fra-2. Gel-shift competition assays with 16 'AP-1- and CREB-like' target sequences revealed that, within each cell type, the two protein complexes varied in their ability to recognize the mutant target sequences. These results clearly indicate differences in potential target recognition by each specific in vivo complex, and suggest that each may preferentially bind its own subset of target DNAs. In addition, a comparison of binding by individual complexes derived from CEFs overexpressing v-Jun and c-Jun also revealed differences in target recognition. Thus, in vivo complexes formed by overexpression of v-Jun and c-Jun vary in their ability to recognize and bind to a number of 'AP-1- and CREB-like' target sequences. This has important implications with regard to the mechanisms involved in cell transformation by v-Jun.

Animals↗
PubMed1983

Coat protein binding sites on RNA 1 of alfalfa mosaic virus.

The largest genome segment, RNA 1, of alfalfa mosaic virus forms complexes with viral coat protein. These complexes were subjected to digestion with ribonucleases T1 or A and filtered onto Millipore filters. Specific fragments were collected from the filters by phenol extraction. After electrophoretic separation in denaturing polyacrylamide gels, these fragments were sequenced. Besides extracistronic fragments originating from the 3'-terminal region of RNA 1, fragments were found originating from an intracistronic region of the RNA. A striking phenomenon is that the intracistronic fragments were not found when ribonuclease A was used to degrade RNA/protein complexes. The findings are in agreement with the postulation of Houwing and Jaspars (1978), that a conformational change at the 3' ends of the genome RNAs induced by the coat protein is a prerequisite to start an infection cycle.

Base Sequence↗
PubMed2003

Characterization of the adenoassociated virus Rep protein complex formed on the viral origin of DNA replication.

Interaction between the adenoassociated virus (AAV) replication proteins, Rep68 and 78, and the viral terminal repeats (TRs) is mediated by a DNA sequence termed the Rep-binding element (RBE). This element is necessary for Rep-mediated unwinding of duplex DNA substrates, directs Rep catalyzed cleavage of the AAV origin of DNA replication, and is required for viral transcription and proviral integration. Six discrete Rep complexes with the AAV TR substrates have been observed in vitro, and cross-linking studies suggest these complexes contain one to six molecules of Rep. However, the functional relationship between Rep oligomerization and biochemical activity is unclear. Here we have characterized Rep complexes that form on the AAV TR. Both Rep68 and Rep78 appear to form the same six complexes with the AAV TR, and ATP seems to stimulate formation of specific, higher order complexes. When the sizes of these Rep complexes were estimated on native polyacrylamide gels, the four slower migrating complexes were larger than predicted by an amount equivalent to one or two TRs. To resolve this discrepancy, the molar ratio of protein and DNA was calculated for the three largest complexes. Data from these experiments indicated that the larger complexes included multiple TRs in addition to multiple Rep molecules and that the Rep-to-TR ratio was approximately 2. The two largest complexes were also associated with increased Rep-mediated, origin cleavage activity. Finally, we characterized a second, Rep-mediated cleavage event that occurs adjacent to the normal nicking site, but on the opposite strand. This second site nicking event effectively results in double-stranded DNA cleavage at the normal nicking site.

Adenosine Triphosphate↗
PubMed1978

The structure of the Rous sarcoma virus glycoprotein complex.

The viral envelope glycoprotein gp85 is released from purified Rous sarcoma virus by treatment with 2-mercaptoethanol, while the second surface antigen gp35 remains associated with the membrane of intact virus particles. The data represented substantiate and extend previous observations on the Rous sarcoma virus glycoprotein complex.

Avian Sarcoma Viruses↗
PubMed1999

Reduction of viral load and immune complex load on CD4+ lymphocytes as a consequence of highly active antiretroviral treatment (HAART) in HIV-infected hemophilia patients.

BACKGROUND AND OBJECTIVES: Human immunodeficiency virus (HIV)-induced immune complex load on circulating CD4+ blood lymphocytes is associated with dysfunction and depletion of CD4+ lymphocytes and with increased monocyte/macrophage function. It was investigated whether HAART reduces both the viral load in plasma and the number of immune complex-coated CD4+ lymphocytes in the blood, and whether CD4+ counts are associated with viral load and/or immune complex load. MATERIALS AND METHODS: Twelve HIV+ hemophilia patients before and after conversion to HAART (group 1); eight HIV+ hemophilia patients without antiretroviral therapy (group 2). HIV-1 RNA copies in plasma using NASBA/Nuclisens kits; CD4+ lymphocytes coated in-vivo with immune complexes using flowcytometry on whole blood samples; in-vitro responses of immune complex-coated T lymphocytes in cell culture assays. RESULTS: After conversion to HAART there was a significant reduction of viral load, CD4+ gp120+, CD4+ IgM+, and CD4+ IgG+ circulating blood lymphocytes and plasma neopterin, paralleled by a significant increase of CD4+ and CD8+ counts. The percentage of immune complex-coated CD4+ lymphocytes of converted patients was significantly associated with CD4+ counts, in-vitro responses to concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA), anti-CD3 and pooled allogeneic stimulator cells, and with plasma neopterin levels. CONCLUSION: HAART reduces viral load and HIV-induced immune complex load on circulating CD4+ blood lymphocytes. The results of this study can be interpreted to suggest that HAART increases CD4+ lymphocyte counts in part by counteracting HIV-induced autoimmune phenomena.

Anti-HIV Agents↗
PubMed1993

Integration of murine leukemia virus DNA depends on mitosis.

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

Animals↗
PubMed1997

Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo.

The interaction of influenza virus NS1 protein with other viral products in the infected cell was analysed by co-immunoprecipitation studies. The three subunits of the polymerase and the nucleoprotein, but not M1 protein, were co-immunoprecipitated by NS1-specific serum but not when control serum was used. Such co-immunoprecipitation was not sensitive to RNase treatment of the immunoprecipitates. Co-immunoprecipitation was also obtained when the viral transcription-replication system was reconstituted in vivo by transfection of cDNAs and model vRNA template into vaccinia virus-T7-infected cells. Analysis of the RNA pulled-down in the NS1-specific precipitates indicated the presence of both vRNA and mRNA. These results are discussed in the context of the phenotype of virus temperature-sensitive mutants affected in the NS1 gene.

Animals↗