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PubMed1999

[Micronutrients and tropical viral infections: one aspect of pathogenic complexity in tropical medicine].

In tropical zones, uncertain living conditions, inadequate food intake, and poor medical facilities enhance unnecessary morbidity and mortality especially involving infants and young children. In addition to protein-caloric malnutrition, deficiencies in essential micronutrients have a specific health impact. Such deficiencies can be the direct cause of disease such as vitamin A deficiency and blindness or have a promoting effect by compromising immune status and increasing susceptibility to and severity of infectious diseases especially of viral origin. The promoting effect of micronutrient deficiency plays a significant role in measles, rotavirus-related diarrhea, and, to a certain extent, progression of HIV infection. Several examples are described to illustrate the relationship between tropical viral infection and micronutrients including vitamin A, selenium, and various other antioxidants. These examples highlight the effect of infectious disease on micronutritional status (vitamin A and measles) and the need to develop reliable, practical tools to evaluate the relevance and effectiveness of dietary supplementation. In any case, improving living conditions and health programs such as the Expanded Vaccination Program are required and illustrate a transverse approach for prevention of infectious and non-infectious tropical disease. The relationship between micronutrients and infection is only one aspect of the multifactorial reality that must be dealt with in tropical medicine.

Child↗

PubMed1973

Specificity of the DNA product of RNA-dependent DNA polymerase in type C viruses. II. Quantitative analysis.

A number of mammalian Type C viruses were analyzed for relatedness by the technique of DNA.RNA hybridization. Viral DNAs were prepared in single-stranded form from complexes with 70S viral RNA formed during endogenous polymerase reactions. Extent of hybridization was assayed with the single-strand nuclease (S-1) from Aspergillus oryzae. Results obtained indicated a high degree of viral specificity, with significant cross-reactions being observed only with viruses obtained from within a species, as in the case of mouse and cat viruses, or in the special case of woolly monkey-gibbon comparisons. Comparisons of RD-114 virus, recently determined to be of feline origin, and conventional feline Type C viruses (FeLV), revealed minimal relatedness, especially when feline virus was grown on human cells, thus indicating the possibility of coexistence of greatly disparate Type C viruses within one species. A rat-specific virus, recovered from tumors induced by murine sarcoma virus, was found to contain genetic material common to both the original mouse virus and viruses indigenous to the rat, even though only rat-specific proteins have been detected during infection by this virus.

Animals↗

PubMed2000

[Laboratory diagnosis of hepatitis C virus infection].

In Austria, the prevalence of hepatitis C virus infections is 0.7% (17). Exclusion of a putative infection as well as diagnosis and continuous monitoring of HCV-disease produce considerable costs for the health system. How many and which patients with HCV infection will acquire life-threatening complications is by far not clear. Also, the causes for viral persistence and liver-complications remain obscure. For certain, complex interactions of viral and immunological mechanisms will determine the individual outcome of the disease (1). These considerations pose decisive demands on clinical diagnostics for HCV infections to be dealt with in detail: methods for qualitative detection of an infection as well as for analysis of subtypes and for quantitative determination of viral copies; monitoring of therapy; estimation of the progress of the disease and/or efficacy of therapy.

Antiviral Agents↗

PubMed1995

Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer).

Reverse transcription of human immunodeficiency virus type-1 (HIV-1) genomic RNA is primed by tRNA(3Lys), whose 3' end 18 nucleotides are complementary to the viral primer binding site (PBS). We used chemical and enzymatic probes to test the conformation of the viral RNA and tRNA(3Lys), in their free form and in the HIV-1 RNA/tRNA(3Lys) binary complex. Extensive reactivity changes were observed in both molecules upon formation of the binary complex. In the viral RNA, reactivity changes occurred up to 69 nucleotides upstream and 72 nucleotides downstream of the PBS. A secondary structure model of the HIV-1 RNA/tRNA(3Lys) complex accounting for all probing data has been constructed. It reveals an unexpectedly complex and compact pseudoknot-like structure in which most of the anticodon loop, the 3' strand of the anticodon stem and the 5' part of the variable loop of tRNA(3Lys) interact with viral sequences 12 to 39 nucleotides upstream of the PBS. The core of the binary complex is a complex junction formed by two single-stranded sequences of tRNA(3Lys), an intramolecular viral helix, an intramolecular tRNA helix, and two intermolecular helices formed by the template/primer interaction. This junction probably highly constrains the tertiary structure of the HIV-1 RNA/tRNA(3Lys) complex. Compared to the structure of the free molecules, only the D arm of tRNA(3Lys) and a small viral stem-loop downstream of the PBS are unaffected in the binary complex. Sequence comparison reveals that the main characteristics of the binary complex model are conserved among all HIV-1 isolates.

Base Sequence↗

PubMed2003

Adenovirus type 5 DNA-protein complexes from formaldehyde cross-linked cells early after infection.

We report here the properties of viral DNA-protein complexes that purify with cellular chromatin following formaldehyde cross-linking of intact cells early after infection. The cross-linked viral DNA fractionated into shear-sensitive (S) and shear- resistant (R) components that were separable by sedimentation, which allowed independent characterization. The R component had the density and sedimentation properties expected for DNA-protein complexes and contained intact viral DNA. It accounted for about 50% of the viral DNA recovered at 1.5 h after infection but less than 20% by 4.5 h. The proportion of R component was independent of multiplicity of infection, even at less than one particle per cell. Viral hexon and protein VII, but not protein VI, were detected in the fractions containing the R component. These properties are consistent with those of partially uncoated virions associated with the nuclear envelope. A substantial proportion of the S component viral DNA had the same density as cellular chromatin. Protein VII was the most abundant viral protein present in gradient fractions that contained the S component. Complexes containing USF transcription factor cross-linked to the adenovirus major late promoter were detected by viral chromatin immunoprecipitation of the fractions containing S component. The S component probably contained uncoated nuclear viral DNA that assembles into early viral transcription complexes.

Adenoviridae↗

PubMed1997

Mapping features of HIV-1 integrase near selected sites on viral and target DNA molecules in an active enzyme-DNA complex by photo-cross-linking.

The virally encoded integrase protein carries out retroviral integration, and to do so, it must make specific interactions with both viral and target DNA sequences. The retroviral integrase has three domains: an amino-terminal region of about 50 amino acids that contains a zinc finger-like motif, a tightly folded, phylogenetically conserved core domain that contains the active site, and a carboxy-terminal domain that can bind DNA in a nonspecific manner. The complete roles of the amino- and carboxyl-terminal domains have not yet been determined, but they appear to participate in multimerization and nonspecific or target DNA binding, respectively. The number and identity of integrase's DNA binding sites have been difficult to determine by conventional mutagenesis studies. In this report, we describe a photo-cross-linking approach to address these issues. Our findings suggest that HIV-1 integrase contacts with conserved features of the viral DNA end are likely to be mediated by residues in two peptides within the conserved core domain. Additional cross-links were seen between viral DNA and the carboxy-terminal DNA binding domain. Numerous sites in integrase, including peptides in each of the three domains, could be cross-linked to target DNA features. Integrase is known to function as a multimer, and it remains to be determined which specific contacts are in cis or trans with respect to the active site.

Azides↗

PubMed2003

Replication complex of human parechovirus 1.

The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.

Amino Acid Sequence↗

PubMed1998

Modulation of HIV-1 infectivity by MAPK, a virion-associated kinase.

Infection of a cell by human immunodeficiency virus type 1 (HIV-1) results in the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation require phosphorylation of reverse transcription complex components by a virion-associated kinase. In this study, we identify the virion-associated kinase as mitogen-activated protein kinase (ERK/MAPK). Upon density gradient fractionation, MAPK, but not its activating kinase MEK, co-sedimented with viral particles. Expression of a constitutively active, but not kinase-inactive, MEK1 in virus producer cells was able to activate virion-associated MAPK in trans. Stimulation of virion-associated MAPK activity in trans by the mitogen phorbol myristate acetate (PMA) increased viral infectivity. Conversely, suppression of virion-associated MAPK by specific inhibitors of the MAPK cascade markedly impaired viral infectivity. These studies demonstrate regulation of an early step in HIV-1 infection by the host cell MAPK signal transduction pathway.

CD4 Antigens↗

PubMed1979

Adenovirus DNA template for late transcription is not a replicative intermediate.

The relationship between adenovirus replication and late transcription has been investigated using viral replication and transcription complexes isolated from infected HeLa cell nuclei. These two types of complexes extracted from adenovirus type 2-infected cell nuclei did not sediment at the same rate on sucrose gradients. Viral replicative intermediates were quantitatively precipitated by immunoglobulins raised against purified 72,000-dalton DNA-binding protein, whereas viral transcription complexes remained in the supernatant. These results show that late transcription does not occur on active replication complexes or on 72,000-dalton DNA-binding protein-containing replicative intermediates inactive in DNA synthesis. Additional evidence is presented indicating that it is very unlikely that replicative intermediates lacking the 72,000-dalton DNA-binding protein could be the template for late transcription.

Adenoviruses, Human↗

PubMed2005

Human N-myristoyltransferases form stable complexes with lentiviral nef and other viral and cellular substrate proteins.

Nef is a multifunctional virulence factor of primate lentiviruses that facilitates viral replication in the infected host. All known functions of Nef require that it be myristoylated at its N terminus. This reaction is catalyzed by N-myristoyltransferases (NMTs), which transfer myristate from myristoyl coenzyme A (myristoyl-CoA) to the N-terminal glycine of substrate proteins. Two NMT isoforms (NMT-1 and NMT-2) are expressed in mammalian cells. To provide a better mechanistic understanding of Nef function, we used biochemical and microsequencing techniques to isolate and identify Nef-associated proteins. Through these studies, NMT-1 was identified as an abundant Nef-associated protein. The Nef-NMT-1 complex is most likely a transient intermediate of the myristoylation reaction of Nef and is modulated by agents which affect the size of the myristoyl-CoA pool in the cell. We also examined two other proteins that bear an N-terminal myristoylation signal, human immunodeficiency virus type 1 Gag and Hck protein tyrosine kinase, and found that Gag bound preferentially the NMT-2 isoform, while Hck bound mostly to NMT-1. Recognition of different NMT isoforms by these viral and cellular substrate proteins suggests nonoverlapping roles for these enzymes in vivo and reveals a potential for the development of inhibitors that target the myristoylation of specific viral substrates more selectively.

Acyl Coenzyme A↗

PubMed1998

Footprints on the viral DNA ends in moloney murine leukemia virus preintegration complexes reflect a specific association with integrase.

Retroviral DNA integration is mediated by the preintegration complex, a large nucleoprotein complex derived from the core of the infecting virion. We previously have used Mu-mediated PCR to probe the nucleoprotein organization of Moloney murine leukemia virus preintegration complexes. A region of protection spans several hundred base pairs at each end of the viral DNA, and strong enhancements are present near the termini. Here, we show that these footprints reflect a specific association between integrase and the viral DNA ends in functional preintegration complexes. Barrier-to-autointegration factor, a cellular protein that blocks autointegration of Moloney murine leukemia virus DNA, also plays an indirect role in generating the footprints at the ends of the viral DNA. We have exploited Mu-mediated PCR to examine the effect of mutations at the viral DNA termini on complex formation. We find that a replication competent mutant with a deletion at one end of the viral DNA still exhibits a strong enhancement about 20 bp from the terminus of the mutant DNA end. The site of the enhancement therefore appears to be at a fixed distance from the ends of the viral DNA. We also find that a mutation at one end of the viral DNA, which renders the virus incompetent for replication, abolishes the enhancements and protection at both the U3 and U5 ends. A pair of functional viral DNA ends therefore are required to interact before the chemical step of 3' end processing.

3T3 Cells↗

PubMed1997

Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids.

Assembly of hepadnaviruses depends on the formation of a ribonucleoprotein (RNP) complex comprising the viral polymerase polypeptide and an RNA segment, epsilon, present on pregenomic RNA. This interaction, in turn, activates the reverse transcription reaction, which is primed by a tyrosine residue on the polymerase. We have shown recently that the formation of this RNP complex in an avian hepadnavirus, the duck hepatitis B virus, depends on cellular factors that include the heat shock protein 90 (Hsp90). We now report that RNP formation also requires ATP hydrolysis and the function of p23, a recently identified chaperone partner for Hsp90. Furthermore, we also provide evidence that the chaperone complex is incorporated into the viral nucleocapsids in a polymerase-dependent reaction. Based on these findings, we propose a model for hepadnavirus assembly and priming of viral DNA synthesis where a dynamic, energy-driven process, mediated by a multi-component chaperone complex consisting of Hsp90, p23 and, potentially, additional factors, maintains the reverse transcriptase in a specific conformation that is competent for RNA packaging and protein priming of viral DNA synthesis.

Adenosine Triphosphate↗

PubMed2005

Analysis of the interaction of the adenovirus L1 52/55-kilodalton and IVa2 proteins with the packaging sequence in vivo and in vitro.

We previously showed that the adenovirus IVa2 and L1 52/55-kDa proteins interact in infected cells and the IVa2 protein is part of two virus-specific complexes (x and y) formed in vitro with repeated elements of the packaging sequence called the A1-A2 repeats. Here we demonstrate that both the IVa2 and L1 52/55-kDa proteins bind in vivo to the packaging sequence and that each protein-DNA interaction is independent of the other. There is a strong and direct interaction of the IVa2 protein with DNA in vitro. This interaction is observed when probes containing the A1-A2 or A4-A5 repeats are used, but it is not found by using an A5-A6 probe. Furthermore, we show that complex x is likely a heterodimer of IVa2 and an unknown viral protein, while complex y is a monomer or multimer of IVa2. No in vitro interaction of purified L1 52/55-kDa protein with the packaging sequence was found, suggesting that the L1 52/55-kDa protein-DNA interaction may be mediated by an intermediate protein. Results support roles for both the L1 52/55-kDa and IVa2 proteins in DNA encapsidation.

Adenoviruses, Human↗

PubMed1984

Individual cells in tissues of DHBV-infected ducks express antigens crossreactive with those on virus surface antigen particles and immature viral cores.

Double immunofluorescence assays of fixed sections of tissues from Pekin ducks congenitally infected with duck hepatitis B virus were carried out to score cells reactive to antiserum elicited either to viral DNA synthesis complexes (immature cores) purified from liver of infected ducks or to viral surface antigen particles and virions purified from sera of viremic ducks. Subpopulations of doubly fluorescent cells were detectable in liver (hepatocytes and bile duct epithelia), in kidney (proximal tubular epithelia), and in pancreas (acinar-associated cells and endocrine alpha and beta cells); all cells reactive with one antiserum were reactive with the second antiserum. Competition assays established that the cell-associated antigens recognized by the two antisera were mutually non-crossreactive. The cell-associated antigen recognized by the antiserum elicited to the purified immature cores appears to be crossreactive with a 35,000-Da polypeptide fraction associated with immature cores.

Animals↗

PubMed2005

[Pathogenesis of primary systemic vasculitides (II): ANCA-negative vasculitides].

The pathogenesis of different types of systemic vasculitis negative for antineutrophil cytoplasm antibodies (ANCA) and involving small or medium-sized vessels is not very well documented. During polyarteritis nodosa (PAN), which is related to hepatitis B virus (HBV) infection, as well as during cryoglobulinemic vasculitides, associated with hepatitis C virus (HCV), and probably during Henoch Schönlein purpura, histological lesions may result from the deposition of immune complexes formed from viral antigens and from antibodies responsible for the activation of the classic complement pathway and for recruitment of polymorphonuclear neutrophils. Two other mechanisms are discussed for other types of ANCA-negative systemic vasculitis: immune complex deposition and sheer stress at arterial bifurcation points. A bacterial superantigen is suspected in Kawasaki disease but remains unproved.

Animals↗

PubMed2000

Identification of domains of the human papillomavirus type 11 E1 helicase involved in oligomerization and binding to the viral origin.

The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by single-stranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.

Adenosine Triphosphatases↗

PubMed2005

Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA.

BACKGROUND: Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. RESULTS: A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. CONCLUSION: These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

Cells, Cultured↗

PubMed1990

[Enterosorption in the combined treatment of patients with viral hepatitis].

The authors studied the efficacy of using USSR-made adsorbent "Enterosgel" in the complex treatment of viral hepatitis. A scheme of employment of the drug is described. Recommendations on the doses and duration of treatment are given. It was established that the efficacy of "Enterosgel" treatment was optimal in patients with intoxication syndrome due to hepatic insufficiency, cholestasis with marked skin pruritus and allergic manifestations. Use of the named drug permits to restrict the employment of other agents that effected favourably the course and outcome of viral hepatitis.

Adsorption↗