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Results for “viral complexity”

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PubMed2004

Tracking fluorescence-labeled rabies virus: enhanced green fluorescent protein-tagged phosphoprotein P supports virus gene expression and formation of infectious particles.

Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD DeltaP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.

Animals↗

PubMed1977

Immunopathologic processes in chronic viral diseases.

Immune complex disease is frequently associated with chronic virus infections of both animals and man. The relative pathogenetic significance of tissue injury caused by deposition of immune complexes is only partly understood. Further study should be made of these problems through use of recently developed techniques for immune complex identification.

Animals↗

PubMedDate not supplied

[An analysis of the possible routes of viral macroevolution].

A complex analysis of virus groups was carried out in order to find out possible ways of their macroevolution. The analysis was based on the detection of similar groups of viruses which could be considered to be evolutionally close. The similarity was evaluated both by a complex of virus properties (physicochemical, morphological, etc.) and using systemic analysis of means of genetic information transmission (MGIT) describing the features of replication and transmission properties. The developed systemic approach using the analysis of the MGIT system and integral differences between viruses by the totality of their properties helped to form models of virus evolution taking into account, in particular, their mono- or polyphyletic origin, more definite knowledge on pravirus(es) MGIT, etc. A monophyletic model of macroevolution of viruses was constructed.

Biological Evolution↗

PubMed1979

Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.

Animals↗

PubMed1996

The presentation of a hepatitis C viral peptide by distinct major histocompatibility complex class I allotypes from two chimpanzee species.

A cytotoxic T lymphocyte (CTL) line, derived from the liver of a common chimpanzee (Pan troglodytes) with hepatitis C, specifically recognized a hepatitis C viral 9-mer peptide (KHP-DATYSR in single-letter amino acid code) bound by the major histocompatibility complex (MHC) class I molecule, Patr-A04. This same CTL line also recognized the identical peptide bound by a structurally different class I molecule, Papa-A06, derived from the separate chimpanzee species, Pan paniscus or pygmy chimpanzee. These class I allotypes differ by six amino acids but, in spite of the structural differences, share the same antigen-presenting function. This is the first observation of antigen presentation to a given T cell receptor by different MHC class I allotypes from separate species.

Animals↗

PubMed2001

Analysis of hepatitis B viral load decline under potent therapy: complex decay profiles observed.

We used a new real-time polymerase chain reaction (PCR)-based assay that is sensitive, has a wide dynamic linear range, and is highly reproducible to quantify hepatitis B virus (HBV) DNA in the serum of infected individuals undergoing potent antiviral therapy. In addition, we made frequent measurements of viral load after initiation of treatment and maintained follow-up to about 12 weeks. To analyze the data we used a new model of HBV decay, which takes into account that existing drug treatments do not completely block de novo infection and the possibility of noncytolytic loss of infected cells. On initiation of therapy, there was a mean delay of 1.6 days followed by a biphasic or muliphasic decay of plasma HBV DNA. The slope of the first phase varied considerably, with one individual having rapid decay, corresponding to a virion half-life of 1 hour, but others showing half-lives of up to 92 hours. Individuals either had a slow second-phase decline (t((1/2)) = 7.2 +/- 1.2 days) or a flat second phase. Some individuals exhibited a complex "staircase pattern" of decay, with further phases of viral DNA decline and phases with little change in viral load.

2-Aminopurine↗

PubMed1993

Coexpression of intercellular adhesion molecule-1 and class I major histocompatibility complex antigens on hepatocyte membrane in chronic viral hepatitis.

AIMS: To evaluate the role of hepatocyte expression of leucocyte adhesion molecules and major histocompatibility complex (MHC) antigens in the pathogenesis of chronic viral hepatitis. METHODS: The expression of intercellular adhesion molecule 1 (ICAM-1), lymphocyte function associated antigen 3 (LFA-3), and MHC class I and II antigens on hepatocyte membrane in relation to the histological and biochemical activities was studied in patients with chronic B hepatitis, chronic persistent hepatitis (CPH) n = 23; chronic active hepatitis (CAH) n = 20; chronic D hepatitis (CAH) n = 6; and chronic non-A, non-B hepatitis (CPH n = 4, CAM n = 6). Six of the latter were hepatitis C virus antibody positive. RESULTS: In chronic B hepatitis ICAM-1 and MHC-I were expressed significantly more in patients with CAH than in those with CPH (p < 0.001), while the expression of LFA-3 and MHC-II showed no significant difference, irrespective of serum HBeAg or hepatitis B virus DNA. Similar findings were noted in non-A, non-B hepatitis. Regardless of the viral aetiology, patients with CAH had a significantly higher degree of ICAM-1 and MHC-I expression than LFA-3 (p < 0.001 v ICAM-1 and MHC-I, respectively) and MHC-II (p < 0.001 v ICAM-1 and MHC-I, respectively) expression. Those with CPH showed little or no difference in the expression of these four molecules. Furthermore, serum ALT values positively correlated with the hepatocyte expression of ICAM-1 (p < 0.001) and MHC-I (p < 0.001), but not LFA-3 (p > 0.05) and MHC-II (p > 0.05). CONCLUSIONS: In chronic viral hepatitis hepatocyte expression of ICAM-1 and MHC-I might be important for immunosurveillance against virally infected hepatocytes, while the expression of LFA-3 and MHC-II does not seem to have a role in the pathogenesis of chronic viral hepatitis.

Antigens, CD↗

PubMed2006

Human leucocyte antigen-identical haematopoietic stem cell transplantation in major histocompatiblity complex class II immunodeficiency: reduced survival correlates with an increased incidence of acute graft-versus-host disease and pre-existing viral infections.

Major histocompatibility complex class II deficiency, a rare autosomal recessive primary immunodeficiency, is caused by the defective expression of human leucocyte antigen (HLA) class II molecules due to mutated trans-acting elements of any one of four regulatory genes (CIITA, RFXANK, RFX5, RFXAP). The impaired CD4 T-cell differentiation and antigen presentation in the periphery results in a severe defect of cellular and humoral response consistent with severe recurrent infections, leading to a poor prognosis. Currently, allogeneic haematopoietic stem cell transplantation (HSCT) is the only curative approach, but the overall cure rate is lower than in other immunodeficiencies. We report a single centre experience of 17 HSCTs with 15 HLA-identical donors between 1981 and 2004. Eight patients survived, while the occurrence of acute graft-versus-host disease (GVHD) was 50%. This study aimed to identify potential risk factors for GVHD and outcome within pre-HSCT complications related to the immunodeficiency. Five of seven patients with pre-existing viral infections developed acute GVHD > or = grade II, of whom four died. Two of seven patients without detectable pre-existing viral infection developed GVHD > or = grade II, and one died. The difference was significant (P < 0.05). A plausible link with other factors potentially associated with the development of GVHD could not be found. We suggest that the reduced survival after HLA-identical HSCT may be caused by the high incidence of pre-existing viral infections and associated with the onset of severe acute GVHD.

Acute Disease↗

PubMed2005

A redox-sensitive cysteine in Zta is required for Epstein-Barr virus lytic cycle DNA replication.

Epstein-Barr virus (EBV) reactivation from latency is known to be sensitive to redox regulation. The immediate-early protein Zta is a member of the basic-leucine zipper (bZIP) family of DNA binding proteins that stimulates viral and cellular transcription and nucleates a replication complex at the viral lytic origin. Zta shares with several members of the bZIP family a conserved cysteine residue (C189) that confers redox regulation of DNA binding. In this work, we show that replacement of C189 with serine (C189S) eliminated lytic cycle DNA replication function of Zta. The mechanistic basis for this replication defect was investigated. We show that C189S was not significantly altered for DNA binding activity in vitro or in vivo. We also show that C189S was not defective for transcription activation of EBV early gene promoters. C189S was deficient for transcription activation of several viral late genes that depend on lytic replication and therefore was consistent with a primary defect of C189S in activating lytic replication. C189S was not defective in binding methylated DNA binding sites and was capable of activating Rta from endogenous latent viral genomes, in contrast to the previously characterized S186A mutation. C189S was slightly impaired for its ability to form a stable complex with Rta, although this did not prevent Rta recruitment to OriLyt. C189S did provide some resistance to oxidation and nitrosylation, which potently inhibit Zta DNA binding activity in vitro. Interestingly, this redox sensitivity was not strictly dependent on C189S but involved additional cysteine residues in Zta. These results provide evidence that the conserved cysteine in the bZIP domain of Zta plays a primary role in EBV lytic cycle DNA replication.

Amino Acid Sequence↗

PubMed1976

Poliovirus proteins associated with the replication complex in infected cells.

Viral polypeptides associated with the membrane-free replication complex of poliovirus RNA were multiple in nature. The structural protein precursors [VP0, VP1, VP3] predominated, and because they were found in a cytoplasmic component with the same S value and density as the replication complex are likely to be attached to it in vivo. They were not present in the form of empty capsids. The electrophoretic polypeptide pattern of the membrane-bound replication complex was similar but showed a predominance of NCVPX or VP1, unless the cells were slightly depleted in amino acids when the non-structural polypeptide NCVP2 became important. Cystine was the only amino acid capable of reversing this depletion effect on its own.

Amino Acids↗

PubMed1997

Shared promoter elements between a viral superantigen and the major histocompatibility complex class II-associated invariant chain.

Superantigens have the ability to stimulate subsets of T lymphocytes bearing particular T-cell receptor Vbeta chains. The best-known viral superantigen is Mls, a product of the murine mammary tumor virus (MMTV) sag gene. The MMTV superantigen is not displayed by the virus itself; however, after infection of B lymphocytes, the superantigen is expressed. The resulting immune stimulation is essential for viral transmission. We have analyzed the transcriptional elements which control Mls-1 expression. Here we present evidence that a region at the 3' end of Mtv-7 env, Penv2, controls B-cell-specific expression of sag. Penv2 has elements homologous with promoters of immunoglobulin H chain, the invariant chain, and major histocompatibility complex class II, suggesting a coordinate regulation of expression of these various B-cell-specific genes and indicating a possible eukaryotic origin of MMTV sag. We have determined that both an IgH heptamer element and a Y box are essential for Penv2 promoter activity and that tandem octamer motifs in the U3 region of the 3' MMTV long terminal repeat function as enhancers. We propose that Penv2 controls constitutive Mls expression in B lymphocytes.

Animals↗

PubMed2006

Hepatitis C virus (HCV) NS5B nonnucleoside inhibitors specifically block single-stranded viral RNA synthesis catalyzed by HCV replication complexes in vitro.

Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

Benzimidazoles↗

PubMed1998

Mutational analysis of the tRNA3Lys/HIV-1 RNA (primer/template) complex.

Retroviruses use a specific tRNA, whose 3' end is complementary to the 18 nucleotides of the primer binding site (PBS), to prime reverse transcription. Previous work has shown that initiation of HIV-1 reverse transcription is a specific process, in contrast with the subsequent elongation phase. HIV-1 reverse transcriptase (RT) specifically recognizes the complex formed by the viral RNA and tRNA3Lys. We previously proposed a secondary structure model of this complex based on chemical and enzymatic probing. In this model, tRNA3Lysextensively interacts with the genomic RNA. Here, we have combined site-directed mutagenesis and structural probing to test crucial aspects of this model. We found that the complex interactions between tRNA3Lysand HIV-1 RNA, and the intra-molecular rearrangements did not depend on the presence of upstream and downstream viral sequences. Indeed, a short RNA template, encompassing nucleotides 123-217 of the HIV-1 Mal genome, was able, together with the primer tRNA, to adopt the same structure as longer viral RNA fragments. This model primer/template is thus amenable to detailed structural and functional studies. The probing data obtained on the tRNA3Lys/mutant viral RNA complexes support the previously proposed model. Furthermore, they indicate that destroying the complementarity between the anticodon of tRNA3Lysand the so-called viral 'A-rich loop' destabilizes all four helices of the extended tRNA3Lys/HIV-1 RNA interactions.

Anticodon↗

PubMed2003

Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons.

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, and altered cellular membranes, is a characteristic feature of plus-strand RNA viruses. Here, we demonstrate the presence of a specific membrane alteration, designated the membranous web, that contains hepatitis C virus (HCV) nonstructural proteins, as well as viral plus-strand RNA, in Huh-7 cells harboring autonomously replicating subgenomic HCV RNAs. Metabolic labeling with 5-bromouridine 5'-triphosphate in the presence of actinomycin D revealed that the membranous web is the site of viral RNA synthesis and therefore represents the replication complex of HCV.

Cell Membrane↗

PubMed1984

Follow-up of circulating immune complexes in the course of acute viral hepatitis, and correlation with serologically relevant parameters.

Type A, type B and type non-A, non-B hepatitis patients were followed up. Several parameters were checked at ten day intervals. Circulating immune complexes (CIC) were detected in a large percentage of patients by using the PEG test and an assay that makes use of bovine conglutinin (K) as recognition unit, and an enzymatically labelled immune complex as the probe. The decrease in the mean level of CIC in the patients correlated with the decrease in serum transaminases and bilirubinaemia in type A and type B hepatitis. Although the pattern of the mean values of the two assays was similar for type A and type B hepatitis, when the two CIC assays were compared for each patient, no significant correlation was found. In light of these and previous results, the necessity for performing CIC monitoring with more than one assay is also discussed.

Acute Disease↗

PubMed1997

The HIV-1 associated dementia complex: a metabolic encephalopathy fueled by viral replication in mononuclear phagocytes.

HIV enters the brain soon after virus exposure but elicits profound neurological deficits in infected humans years later usually during progressive immunosuppression and the development of the acquired immune deficiency syndrome. The neurological disease complex associated with virus infection occurs in a large proportion of infected patients and is commonly referred to as HIV-1 associated dementia complex. The neuropathogenesis of central nervous system/viral infection revolves around mononuclear phagocytes (brain macrophage/microglial) infection and immune activation in brain. Macrophages secrete neurotoxic factors that elicit neuronal injury and inevitably death leading to the constellation of cognitive and motor impairments common during progressive disease. Neurotoxic factor production requires virus entry and replication, the evolution/selection of neurovirulent HIV-1 strains and the production of viral and cellular immune factors injurious to human neurons. Interestingly, neurological deficits, the HIV-1 associated neuropathology and viral replication disease are not always associated. This has led to the notion that viral replication induces the autocrine/paracrine production of cellular/viral factors leading to a metabolic encephalopathy. Anti-retroviral and anti-inflammatory therapies should prove increasingly beneficial for treatment and, ultimately, reversal of HIV-1 associated dementia complex in the affected human host.

AIDS Dementia Complex↗

PubMed2001

Inhibition of nuclear export of ribonucleoprotein complexes of influenza virus by leptomycin B.

We have studied nuclear export of influenza virus components using an in vitro transport system with digitonin-treated infected cells. We first monitored the efficiency of export of the viral ribonucleoprotein (vRNP) complex by analyzing viral components with western blotting. We used leptomycin B (LMB), an inhibitor of nuclear export signal (NES)-and its receptor, CRM1/Exportin1-mediated protein export. LMB efficiently inhibited vRNP export, while it did not affect the subcellular localization and export of matrix protein (M) 1 and nonstructural protein (NS) 2. Second, indirect immunofluorescence assays also revealed that vRNP export is sensitive to LMB. NS2 in NS2-transfected cells was not accumulated in nuclei in the presence of LMB, while NS2 in infected cells was found slightly accumulated in nuclei in the presence of LMB. Finally, we performed in vitro RNA synthesis assays using digitonin-treated infected cells and exported fractions. The exported vRNP was RNA synthesis-competent. Analyses using glycerol density gradients showed that a major fraction of M1 and NS2 was not complexed with the exported vRNP. These results suggest that nuclear export of RNA synthesis-competent vRNP is dependent on a LMB-sensitive pathway and that there would be two types of NS2, i.e. LMB-sensitive and -insensitive NS2. The involvement of viral late proteins in vRNP export during late stages of infection is discussed.

Active Transport, Cell Nucleus↗

PubMed1986

Varicella-zoster virus p32/p36 complex is present in both the viral capsid and the nuclear matrix of the infected cell.

Varicella-zoster virus (VZV) directs the synthesis of numerous glycosylated and nonglycosylated infected-cell-specific proteins, many of which are later incorporated into the virion as structural components. In this study, we characterized a nonglycosylated polypeptide complex with the aid of a VZV-specific murine monoclonal antibody clone, 251D9. As detected by indirect immunofluorescence, the antibody bound mainly to antigens located within the nuclei of infected cells and did not attach to an uninfected cell substrate. The polypeptide specificity of the monoclonal antibody was determined by immunoblot analysis of electrophoretically separated infected cell extracts to react with a 32,000-molecular-weight VZV-specific protein (p32); in addition, the antibody also bound to a 36,000-molecular-weight polypeptide. The synthesis of these antigens was unaffected by inhibitors of glycosylation. Nonionic or ionic detergents were only marginally effective in solubilization of the p32-p36 complex, and relatively small amounts were eluted from nuclei by high salt concentrations (2 M NaCl). The same proteins remained associated with the nuclear matrix of VZV-infected cells. We also demonstrated that the protein complex was a major component of purified VZV nucleocapsids; p32 was especially prominent in both full and empty capsids. Immunoblot analysis of the nucleocapsid preparation revealed two additional species (p34 and p38) in the p32-p36 complex. Phosphorylation was a distinctive feature of some of the constituents. In summary, these results indicate that the p32-p36 complex represents a family of structural proteins closely associated with the assembly of VZV nucleocapsids and the encapsidation of viral DNA.

Antibodies, Monoclonal↗