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PubMed1980

Reduced capacity to repair irradiated adenovirus in fibroblasts from xeroderma pigmentosum heterozygotes.

Xeroderma pigmentosum (XP) is one of a number of autosomal recessive syndromes in humans characterized by a marked predisposition to cancer. Fibroblasts from these patients show a defect in DNA repair. The XP heterozygotes also show elevated skin cancer incidence, but reports concerning their DNA repair capacity are conflicting. In this study, the DNA repair capacity of four XP heterozygotes was examined using a sensitive host cell reactivation technique. Unirradiated and irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens in fibroblasts from XP heterozygotes, XP homozygotes, and normals. A reduced host cell reactivation (of viral structural antigen production) for both ultraviolet- and gamma-irradiated Ad 2 was detected in four XP heterozygotes representing three different complementation groups as well as their XP homozygous children. The doses necessary to reduce the survival of viral structural antigen production by irradiated AD 2 to 37% in the XP heterozygous strains were expressed as a percentage of that obtained in normal strains and ranged from 55 to 82% for ultraviolet-irradiated Ad 2 and 71 to 79% for gamma-irradiated Ad 2. These results add further support to a direct relationship between cancer proneness and DNA repair defects and show the merits of this host cell reactivation technique in identifying XP heterozygotes. Identification of XP heterozygotes is of considerable public health interest not only in genetic counseling but also in the prevention of cancer.

Adenoviruses, Human↗

PubMed2000

Plat-E: an efficient and stable system for transient packaging of retroviruses.

A potent retrovirus packaging cell line named Platinum-E (Plat-E) was generated based on the 293T cell line. Plat-E is superior to existing packaging cell lines regarding efficiency, stability and safety. The novel packaging constructs utilized in establishment of Plat-E ensure high and stable expression of viral structural proteins. Conventional packaging constructs made use of the promoter of MuLV-LTR for expression of viral structural genes gag-pol and env, while our packaging constructs utilized the EF1alpha promoter, which is 100-fold more potent than the MuLV-LTR in 293T cells in combination with the Kozak's consensus sequence upstream of the initiation codon resulting in high expression of virus structural proteins in Plat-E cells. To maintain the high titers of retroviruses under drug selection pressure, we inserted the IRES (internal ribosome entry site) sequence between the gene encoding gag-pol or env, and the gene encoding a selectable marker in the packaging constructs. Plat-E cells can stably produce retroviruses with an average titer of 1 x 107/ml for at least 4 months. In addition, as we used only the coding sequences of viral structural genes to avoid inclusion of unnecessary retrovirus sequences in the packaging constructs, the probability of generating the replication competent retroviruses (RCR) by recombination can virtually be ruled out.

Cell Line↗

PubMed1989

Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species.

The known primate lentiviruses can be divided into two subgroups consisting of the human immunodeficiency virus type 1 (HIV-1) isolates and the related HIV type 2 (HIV-2) and simian immunodeficiency virus (SIV) isolates. HIV-1 has been shown to encode a post-transcriptional trans-activator of viral structural gene expression, termed Rev, that is essential for viral replication in culture. Here, we demonstrate that HIV-2 and SIVmac also encode functional Rev proteins. As in the case of HIV-1, these Rev trans-activators are shown to induce the cytoplasmic expression of the unspliced viral transcripts that encode the viral structural proteins. Unexpectedly, the Rev proteins of HIV-2 and SIVmac proved incapable of activating the cytoplasmic expression of unspliced HIV-1 transcripts, whereas HIV-1 Rev was fully functional in the HIV-2/SIV system. This nonreciprocal complementation may imply a direct role for Rev in mediating the recognition of its viral RNA target sequence.

Animals↗

PubMed1980

Characterization of a small, nonstructural viral polypeptide present late during infection of BHK cells by Semliki Forest virus.

BHK cells, late in infection with Semliki Forest virus, were found to contain a small virus-specific polypeptide not found in the mature virion. This polypeptide had an apparent molecular weight of 6,000 and is referred to here as the 6K protein. No [2-3H]mannose was incorporated into 6K, and hence it does not appear to be a glycoprotein. This protein appears to be a primary translation product of the subgenomic 26S mRNA, which encodes the viral structural proteins. The genes encoding the viral structural proteins are arranged on the message in the order of 5'-C-E3-E2-E1-3'. We have found that the gene coding for 6K is located to the 3' side of the gene encoding E2. Subcellular fractionation of pulse-labeled cells infected with Semliki Forest virus demonstrated that 6K, like the viral glycoproteins p62 and E1, was present predominantly in the rough microsomal membrane fraction. 6K appears to be analogous, therefore, to the nonstructural 4.2K protein present in cells infected with Sindbis virus.

Animals↗

PubMed1994

Temporal patterns of human immunodeficiency virus type 1 transcripts in human fetal astrocytes.

Human immunodeficiency virus type 1 (HIV-1) infection of the developing central nervous system results in a dementing process in children, termed HIV-1-associated encephalopathy. Infection of astroglial elements of the pediatric nervous system has been demonstrated and suggests that direct infection of some astrocytes may contribute to the neurologic deficit. In this model, HIV-1 establishes a persistent state of infection in astrocytes, which can be reactivated by the cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta). To better understand the natural history of viral persistence in astroglial cells, we characterized infection at the transcriptional level. The most abundant viral transcript during the establishment of persistence was the subgenomic multiply spliced 2-kb message, similar to mononuclear cell models of HIV-1 latency. Following reactivation with TNF-alpha or IL-1 beta the multiply spliced 2-kb message remained the most abundant viral transcript, in contrast to infected mononuclear cells in which reactivation leads to the reemergence of the 9- and 4-kb transcripts. Further characterization of the persistent 2-kb transcript by PCR amplification of in vitro-synthesized viral cDNA showed that, in the absence of cytokine stimulation, the most abundant multiply spliced transcripts were the Nef- and Rev-specific messages. However, following cytokine stimulation, double- and triple-spliced Tat-, Rev-, and Nef-specific messages could be identified. Immunohistochemical staining demonstrated that, during viral persistence, astrocytes expressed Nef protein but few or no viral structural proteins. These results demonstrate that viral persistence in astrocytes at the transcriptional level is fundamentally different from that seen in mononuclear cells and could account for the virtual absence of astroglial expression of viral structural antigens in vivo.

Astrocytes↗

PubMed1984

Hepatitis B viral antigenic structure: signature analysis by monoclonal radioimmunoassays.

An approach has been developed for the analysis of hepatitis B viral (HBV) antigenic structure that creates numerical "signatures" of HBV strains. This technique employs high-affinity IgM and IgG monoclonal antibodies (anti-HBsAg) directed toward distinct and separate determinants on hepatitis B surface antigen (HBsAg). Such antibodies have been used to develop sensitive and specific radioimmuno-assays for measurement of HBsAg-associated determinants in serum. In performing "signature" analysis separate binding curves for each monoclonal anti-HBsAg are generated by measuring immunoreactivity in serial dilutions of HBsAg-positive serum. Since the HBsAg concentration in serum is unknown, the binding profiles of groups of samples from the same "classic" HBV subtype are aligned by an iterative maximum likelihood procedure to give the numerical signature of that HBV subtype. By using this approach, HBsAg shows far more antigenic heterogeneity than previously recognized by polyvalent anti-HBsAg antibodies. Indeed, there are subgroups within the classic HBsAg subtypes. In addition, the a domain (common to all known subtypes or strains of HBV) has been shown to be multideterminant. Thus, these studies have demonstrated heretofore unrecognized differences in HBV subtypes. This approach also has broader significance for the study of subtle or major antigenic changes among other viral agents since it is not necessary to know the concentration of virus or viral protein in complex protein mixtures.

Antibodies, Monoclonal↗

PubMed1973

Reversible inhibition of herpes simplex virus replication by hydroxyurea.

Hydroxyurea, at a concentration of 5 x 10(-2) M, inhibits the replication of herpes simplex deoxyribonucleic acid (DNA) in the nuclei of infected cells. As a result, the synthesis of infectious virus progeny was prevented. The presence of parental viral DNA genomes in inhibited cells led to the synthesis of the viral structural peptides. The inhibitory effect of hydroxyurea was reversible; after washing the cells free from hydroxyurea, virus progeny appeared after a lag of 3 h. Upon resumption of viral DNA replication, the content of radioactive viral structural peptides gradually increased in parallel with the increase in mature virions. It is concluded that the information for the synthesis of viral structural peptides is transcribed from the parental DNA genomes.

DNA, Viral↗

PubMed1993

The structure of viral coat protein and its in disease.

Because the structure of the tobacco mosaic virus capsid protein is known, mutations that alter the phenotype of the virus-plant interaction can be correlated with structural changes in this protein. These mutations affect the disease symptoms caused by the virus and recognition of the virus by a plant resistance gene product. Recognition-host interaction is analogous to the gene-for-gene interactions of bacteria and fungi.

Capsid↗

PubMed1965

Characterization of a Tumorlike Antigen in Type 12 and Type 18 Adenovirus-Infected Cells.

Gilead, Zvee (University of Pennsylvania, Philadelphia), and Harold S. Ginsberg. Characterization of a tumor-like antigen in type 12 and type 18 adenovirus-infected cells. J. Bacteriol. 90:120-125. 1965.-An antigen that reacts with antibody from type 12 adenovirus tumor-bearing hamsters was identified in extracts of KB cells infected with type 12 or 18 adenovirus. In contrast, viral structural proteins separated by chromatography on diethylaminoethyl-cellulose did not react with the sera from tumorous hamsters. The tumorlike (T) antigen in infected cells was found to be smaller than the viral structural antigens and, therefore, could be separated from them by centrifugation in a linear sucrose gradient. Investigation of the production of the T antigen in virus-infected cells further distinguished it from viral structural proteins by the following properties: (i) the T antigen was first detected 3 to 4 hr after infection, whereas viral antigens were synthesized 17 to 20 hr after infection; and (ii) the T antigen was produced when deoxyribonucleic acid (DNA) biosynthesis was inhibited by 5-fluorodeoxyuridine (10(-6)m), but viral proteins were not synthesized in the absence of viral DNA replication.

Journal Article↗

PubMedDate not supplied

[Characteristics of the etiological structure of viral hepatitis in children determined by radioimmunological analysis].

Determination of the specific markers of acute viral hepatitides A and B (HA and HB) by radioimmunoassay demonstrated that in the structure of acute viral hepatitides in children HA prevails in the interepidemic period (49.4%) followed by HB (19.3%), then by a polyetiological group of acute viral hepatitides of obscure etiology (17.2%), then by HA in the presence of chronic HB (9.7%) and combined acute forms of HA and HB (4.3%).

Adolescent↗

PubMed1988

[Etiologic structure of viral hepatitis in children based on data from an comprehensive serologic study of hospitalized patients].

During a year an overall serological examination of 1,200 children hospitalized with the diagnosis of viral hepatitis was carried out with a view to establish the presence of hepatitis A and hepatitis B markers (anti-HAV IgM, HBsAg, anti-HBs, anti-HBc and anti-HBe IgM) by means of the enzyme immunoassay and radioimmunoassay. The average annual proportion of cases of hepatitis A (61.4%), hepatitis B (20.4%), hepatitis non A, non B (11.2%) and mixed infection (7.0%) among children, as well as seasonal and age-group fluctuations of this proportion, were established. The etiological structure of viral hepatitides, based on commonly accepted clinico-epidemiological diagnostic criteria, was shown to differ from their true etiological structure as determined in accordance with specific serological markers detected in this investigation.

Adolescent↗

PubMed1986

The synthesis and transport of SV40 structural proteins.

The kinetics of the synthesis and transport of viral structural proteins, Vp1 and Vp3, and of actin in SV40 infected TC7 cells were studied. The newly synthesized proteins were found in the NP-40-soluble (Sol) fraction of the cell cytoplasm. The majority of newly synthesized viral structural proteins, destined for the cell nucleus for virion assembly, were transported to the cell nucleus (Nuc) between 10 and 30 min after synthesis, whereas the majority of newly synthesized actin remained in the Sol fraction of the cytoplasm, suggesting that some specific mechanism exists for selecting the proper sites for transport. The synthesis and transport of both Vp1 and Vp3 throughout infected cells were similar. However, there is a difference in the transport properties of these two proteins. Once Vp1 was synthesized, the mature Vp1 was transported to both the cytoskeletal (Csk) and the Nuc fractions in the absence of further protein synthesis, whereas the movement of Vp3 from the Sol to the Csk, but not to the Nuc fraction, was partially inhibited in the absence of protein synthesis. Modification of Vp1 occurred in the cell cytoplasm before transport to the cell nucleus. Its modification pattern suggests that the Csk is the site for the modification of Vp1. The efficiency of viral protein transport to the cell nucleus was diminished after 47 hr of infection. This trend was preceded by a decrease in the ability to incorporate label into actin 12 hr earlier in infection. Thus, some marking event appears to have occurred prior to the actual decrease in transport efficiency and the integrity of the cytoarchitecture appears to be important for viral protein transport.

Actins↗

PubMed1992

[The prevalence and etiological structure of viral hepatitis in a climatic-geographic area at high risk for infection].

The spread of viral hepatitides on the territory of Central Asia has been analyzed, taking into account regional and territorial factors and using the Fergana Valley as an example. The analysis, based on the use of modern immunochemical, clinico-epidemiological and biochemical criteria, has made it possible to determine the etiological structure of viral hepatitides on the territory of the Fergana Valley. The epidemic outbreak on this territory has been proved to be caused by viral hepatitis E. The necessity of organizing constant epidemiological monitoring on the interstate and territorial level has been substantiated.

Climate↗

PubMed1990

Identification of sequences important in the nucleolar localization of human immunodeficiency virus Rev: relevance of nucleolar localization to function.

The human immunodeficiency virus rev gene product regulates the expression of viral structural genes. It was recently shown that Rev regulates the export of viral structural mRNAs from the nucleus to the cytoplasm. Analysis of Rev subcellular localization reveals marked accumulation in the nucleolus, suggesting a role for the nucleolus in this export process. We report here the identification of amino acid residues critical to the nucleolar localization of Rev. Consistent with this finding, a Rev/beta-galactosidase fusion protein, harboring this region of Rev, localized entirely within the nucleolus. Of most significance, mutations that eliminated nucleolar localization markedly diminished Rev function, even though accumulation in the nucleoplasm was retained. These findings support a model whereby Rev-induced export of human immunodeficiency virus structural mRNAs from the nucleus to the cytoplasm is likely to involve nucleolar events.

Amino Acid Sequence↗

PubMed1983

Inhibition of Rous sarcoma virus assembly by treatment with 2',5' adenosine nucleotides.

We have investigated the influence of 2',5' adenosine nucleotides on the replication and transformation of cells by Rous sarcoma virus (RSV). Treatment with the nucleotides ppp2',5'A4 and 2',5'A4 causes a striking reduction (50-fold) in the yield of infectious progeny virus, while ppp2',5A2 and 2',5'A3 had virtually no effect. The reduction in infectivity seen with 2',5'A4 nucleotides is paralleled by a smaller but significant (three- to four-fold) reduction in the amount of particles released as measured by reverse transcriptase activity and levels of viral structural proteins. The reduced infectivity of released particles is not due to viral RNA being missing since the amount of genomic RNA in particles from 2',5'A4-treated cultures was likewise only reduced by a factor of 2-3. Pulse-chase radioactive label experiments showed that processing of both viral group-specific antigens (gag) and viral envelope glycoprotein (env) gene products was completely normal in nucleotide-treated cultures, but that the rate of appearance of viral proteins in mature virus in the culture supernatants was reduced by a factor of about 3-4. Taken together, the data show that assembly of viral structural proteins into virions which can be released into the medium is slowed, and that assembly of virus particles with reduced infectivity follows upon nucleotide treatment. This inhibition of infectious virus production takes place without significant toxic effects on the cell; host protein synthesis is only 20% inhibited. There is also no significant effect on the secretory ability of the cells as measured by total protein release into the medium or release of fibronectin. The transformed cell phenotype was also subtly affected by 2',5'A4, but not by other oligomers. Plasminogen activator protease activity was sharply reduced upon treatment, while other typical features of RSV-transformed cells such as elevated hexose transport, and pp60src-associated protein phosphokinase activity, were little affected.

Adenine Nucleotides↗

PubMed1974

In vitro synthesis of proteins by membrane-bound polyribosomes from vesicular stomatitis virus-infected HeLa cells.

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.

Animals↗

PubMed2002

Structural unity among viral origin binding proteins: crystal structure of the nuclease domain of adeno-associated virus Rep.

Adeno-associated virus (AAV), unique among animal viruses in its ability to integrate into a specific chromosomal location, is a promising vector for human gene therapy. AAV Replication (Rep) protein is essential for viral replication and integration, and its amino terminal domain possesses site-specific DNA binding and endonuclease activities required for replication initiation and integration. This domain displays a novel endonuclease fold and demonstrates an unexpected structural relationship to other viral origin binding proteins such as the papillomavirus E1 protein and the SV40 T antigen. The active site, located at the bottom of a positively charged cleft, is formed by the spatial convergence of a divalent metal ion and two conserved sequence motifs that define the rolling circle replication superfamily.

Amino Acid Sequence↗

PubMed1982

Evolutionary selection for perfect hairpin structures in viral DNAs.

Several recent discoveries have pointed to nucleic acid secondary structure as an additional dimension in gene expression. Further evidence for the formation of hairpins in RNA is the fact that cruciforms exist in negatively supercoiled DNAs. As potential binding sites for proteins, these structures have been proposed to play a part in the regulation of various crucial reactions, such as replication, transcription, or RNA processing. As any random nucleotide sequence can self-anneal with an approximately 50% chance of forming some Watson-Crick-type base pairs, it is difficult to assess which, if any, of all possible hairpin-like secondary structures may be biologically relevant. We have computed the expected distribution of perfectly base-paired structures as a function of loop size and stem length and compared it with the distribution observed in the complete genome of eight DNA viruses from animals, plants and bacteria. We report here that hairpins having six or more consecutive base pairs in the stem are not distributed randomly long the genome, occur much more often than chance would predict, and are particularly over-represented in regions that appear to have regulatory significance. The average loop size was found to decrease with an increase in stem length. These results support our previous hypothesis that these structures are biologically relevant.

Bacteriophages↗