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At least 127 records · Page 7Linked to original sources
PubMedDate not supplied

Distribution of viral RNA molecules during the adenovirus type 5 infectious cycle in HeLa cells.

Viral RNA was localized ultrastructurally by in situ hybridization with a biotinylated viral DNA probe and colloidal gold label in HeLa cells during infection with adenovirus type 5. Transcription was monitored by high-resolution autoradiography after short pulses with tritiated uridine. At the earliest stage of virus-induced nuclear transformation, viral RNA was restricted to the small compact fibrillar "early replicative sites" which we had previously demonstrated to be the site of viral DNA replication (Puvion-Dutilleul and Puvion, 1990b). Protease-DNase-treated sections revealed that these fibrillar masses rapidly enlarged and gave rise to a juxtaposed loose fibrillogranular structure devoid of viral RNA. Subsequently, the function of the compact fibrillar zones changed to become the sites of accumulation of single-stranded (ss) viral DNA molecules, and the contiguous new fibrillogranular zones (previously named the peripheral replicative zones, Puvion-Dutilleul and Puvion, 1990a) became not only the centers of replicating viral double-stranded DNA but also the only sites of viral RNA molecules including the elongating RNA transcripts. Pulse-chase experiments revealed an unexpected accumulation of RNA molecules in these extranucleolar regions of infected nuclei.

Adenovirus Infections, Human↗
PubMed2005

Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses.

The high error rates of viral RNA-dependent RNA polymerases create heterogeneous viral populations whose disparate RNA genomes affect each other's survival. We systematically screened the poliovirus genome and identified four sets of dominant mutations. Mutated alleles in capsid- and polymerase-coding regions resulted in dominant negative phenotypes, probably due to the proteins' oligomeric properties. We also identified dominant mutations in an RNA element required for priming RNA synthesis (CRE) and in the protein primer (VPg), suggesting that nonproductive priming intermediates are inhibitory. Mutations that inhibit the activity of viral proteinase 2A were dominant, arguing that inhibition of its known intramolecular activity creates a toxic product. Viral products that, when defective, dominantly interfere with growth of nondefective viruses will probably be excellent drug targets because drug-sensitive viruses should be dominant over drug-resistant variants. Accordingly, a virus sensitive to anticapsid compound WIN51711 dominantly inhibited the intracellular growth of a drug-resistant virus. Therefore, dominant inhibitor screening should validate or predict targets for antiviral therapy with reduced risk for drug resistance.

Alleles↗
PubMed1979

Nuclear accumulation of influenza viral RNA transcripts and the effects of cycloheximide, actinomycin D, and alpha-amanitin.

The use of virus-specific (32)P-labeled complementary DNA and (125)I-labeled virion RNA as hybridization probes has allowed us to quantitate the number of molecules of complementary RNA (cRNA) and progeny virion RNA in MDCK cells infected with influenza virus. We compared the distribution of cRNA between the nucleus and the cytoplasm in cycloheximide-treated cells to that found in untreated cells, beginning 1 h after infection. A greater percentage of the total cRNA was detected in the nucleus of the drug-treated cells at all times investigated. For the first 2 h after infection about 50% of the cRNA synthesized in the cycloheximide-treated cells was found in the nucleus. These nuclear cRNA molecules were characterized and shown to be polyadenylated transcripts of each of the genome virion RNA segments. Viral cRNA synthesis was not completely inhibited by the addition of actinomycin D at the beginning of infection, with or without the concomitant addition of cycloheximide. A large fraction (about 90%) of these cRNA sequences were detected in the nucleus. Characterization of these nuclear cRNA molecules showed that they contained polyadenylic acid and represented transcripts of both those segments coding for proteins synthesized predominantly early after infection ("early" proteins) and those virion RNA segments coding for "late" proteins. Also, in vitro translation of these cRNA molecules showed that they were functional virus mRNA's. In contrast to actinomycin D, alpha-amanitin completely inhibited cRNA synthesis when added at the beginning of infection, and addition of this drug after 1.5 h had no effect on further cRNA synthesis.

Amanitins↗
PubMed2002

Level and pattern of HIV-1-RNA viral load over age: differences between girls and boys?

OBJECTIVE: To estimate RNA viral load patterns over age in vertically infected children that account for between- and within-individual variation, treatment and assay cut-off detection level. To investigate possible sex-based differences. DESIGN: A total of 118 infected children with 894 RNA viral load measurements enrolled in the European Collaborative Study were prospectively followed from birth for up to 15 years. METHODS: Fractional polynomial and mixed effects models with censored data to assess the non-linear pattern of viral load over age, allowing for repeated measures. RESULTS: The RNA viral load peaked at approximately 3 months of age, and gradually declined thereafter. The sex by age interaction was significant (chi2 = 19.7, P < 0.001); viral load peaked higher for girls than boys, but after 4 years the RNA load was consistently 0.25-0.5 log10 lower for girls than boys. The effects of sex and treatment on viral load vary over age (chi2 = 6.31, P = 0.043). Sex differences in RNA viral load relating to measurement without treatment were more pronounced than those under treatment. Disease progression was more rapid for girls than for boys up to the age of 4 years, and less rapid thereafter; the overall difference was not statistically significant. CONCLUSION: Differences in RNA viral load over age between untreated boys and girls may have implications for policies for the initiation of antiretroviral therapy, but do not seem to translate into differences in progression to serious disease. The findings would suggest underlying biological explanations, which need further investigation.

Adolescent↗
PubMed1993

Electrotransfection of turnip yellow mosaic virus RNA into Brassica leaf protoplasts and detection of viral RNA products with a non-radioactive probe.

We describe here a convenient and efficient system for studying turnip yellow mosaic virus (TYMV) replication in leaf protoplasts. Inoculation of rapeseed (Brassica napus) or Chinese cabbage (B. sinensis) protoplasts was achieved via electroporation, and sensitive detection of viral RNA products was performed by Northern blot analyses using a non-radioactive digoxigenin-labelled cDNA probe. Virus replication was detected when 1.5 x 10(6) rapeseed protoplasts were inoculated with 20 ng of TYMV RNA. Electrotransfection of TYMV RNA was more efficient in rapeseed than in Chinese cabbage protoplasts, and gave somewhat higher signals than those of TYMV virions. TYMV RNA appeared to replicate equally well whether the protoplasts were incubated in the dark or under constant light.

Blotting, Northern↗
PubMed2003

Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene.

The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U-A replacing G-C at nucleotide positions 12'-11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.

Animals↗
PubMed2006

Crucial role of the 5' conserved structure of bamboo mosaic virus satellite RNA in downregulation of helper viral RNA replication.

Satellite RNA of Bamboo mosaic virus (satBaMV), a single-stranded mRNA type satellite encoding a protein of 20 kDa (P20), depends on the helper BaMV for replication and encapsidation. Two satBaMV isolates, BSF4 and BSL6, exhibit distinctly differential phenotypes in Nicotiana benthamiana plants when coinoculated with BaMV RNA. BSL6 significantly reduces BaMV RNA replication and suppresses the BaMV-induced symptoms, whereas BSF4 does not. By studies with chimeric satBaMVs generated by exchanging the components between BSF4 and BSL6, the genetic determinants responsible for the downregulation of BaMV replication and symptom expression were mapped at the 5' untranslated region (UTR) of BSL6. The 5' UTR of BSL6 alone is sufficient to diminish BaMV RNA replication when the 5' UTR is inserted in cis into the BaMV expression vector or when coinoculation with mutants that block the synthesis of P20 protein takes place. Further, the 5' UTR of natural satBaMV isolates contains one hypervariable (HV) region which folds into a conserved apical hairpin stem-loop (AHSL) structure (W. B. Yeh, Y. H. Hsu, H. C. Chen, and N. S. Lin, Virology 330:105-115, 2004). Interchanges of AHSL segment of HV regions between BSF4 and BSL6 led to the ability of chimeric satBaMV to interfere with BaMV replication and symptom expression. The conserved secondary structure within the HV region is a potent determinant of the downregulation of helper virus replication.

5' Untranslated Regions↗
PubMed1999

Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells.

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.

Animals↗
PubMed2004

Modelling HIV-RNA viral load in vertically infected children.

Human immunodeficiency virus (HIV) ribo-nucleic acid (RNA) viral load is a measure of actively replicating virus and is used as a marker of disease progression. For a thorough understanding of the dynamics of the evolution of the virus in the early life of HIV-1 vertically infected children, it is important to elucidate the pattern of HIV-RNA viral load over age. An aspect of assay systems used in the quantification of RNA viral load is that they measure values above particular cut-off values for detection, below which the assays used are not sufficiently sensitive. In this way, measurements are potentially left-censored. Recent adult studies suggest that to adequately model RNA pattern over age, it is necessary to account for within-subject correlation, due to repeated measures, and censoring. The aim of this study, therefore, was to establish whether it is necessary to use complex methods to allow for repeated measures within individuals and censoring of the HIV-RNA viral load in children enrolled in a cohort study. The approach involved the identification of an appropriate model for the basic pattern of RNA viral load by age and subsequent assessment of various estimation procedures accounting for repeated measures and censoring in different ways. Methods developed by Hughes involving the expectation-maximization (EM) algorithm and the Gibbs sampler were taken as the benchmark for comparison of simpler alternatives. Other approaches considered involve linear mixed-effects and ordinary least squares in which censoring is dealt with informally by taking the cut-off value as absolute or taking the mid-point between cut-off and zero. Fractional polynomials provided a substantially superior approach for modelling the dynamics of viral load over age compared to conventional polynomials or change-point models. Allowing for repeated measures was necessary to improve the power of the likelihood ratio tests required to establish the final model, but methods beyond taking the mid-point for censored values did not further improve the fit. Although Hughes' methodology is the best approach, its implementation is not necessary for the identification of the optimal model.

Algorithms↗
PubMedDate not supplied

[Quantification of T CD4+ lymphocytes and viral RNA in patients with HIV/AIDS].

Quantification of CD4+ T lymphocytes and viral RNA in patients with HIV/AIDS. Levels of progression markers (viral load and CD4+ T lymphocytes) in 410 patients with HIV/AIDS that were in different clinical stages of the disease and under different therapeutic schemes were quantified. The objective was to determine the correlation between values of progression markers and clinical stage of the patients. Commercial methodologies for the quantification of lymphocytes, subpopulations and circulating viral RNA were used. Results indicate that there was a correlation between low CD4+ values and high viral load in patients with antiretroviral treatment but not in patients without treatment. Furthermore, analysis of 1,208 samples processed during 1999 showed that 46% of the patients had less than 200 CD4+ T lymphocytes/mL blood and more than 500 copies of circulating viral RNA. Implications of these results in public health in Mexico are discussed.

CD4 Lymphocyte Count↗