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Inhibition of host cell ribosomal ribonucleic acid methylation by foot-and-mouth disease virus.

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.

Animals↗

Interfering passages of Sindbis virus: concomitant appearance of interference, morphological variants, and trucated viral RNA.

Serial passage of Sindbis at high multiplicities of infection resulted in cyclical variations in virus titer. Decreases in virus titer were correlated with the appearance of smaller-sized virions, interference and truncated viral RNA. The smaller particles were 37 nm in diameter, exclusive of the hemagglutinin spikes as compared with a diameter of 50 nm for standard virions. Passages which contained 37-nm partilces also interfered with infectious center formation by standard, plaque-purified virus. Polyacrylamide gel analysis of RNA isolated from virions present in interfering passages demonstrated the sequential appearance of three RNA species smaller than standard RNA with approximate molecular weights of 3.3 X 106, 2.7 X 106, and 2.2 X 106. The 3.3 X 106 RNA was evident in passage 5, by passage 8 both the 3.3 X 106 and 2.7 X 106 RNAs were present, and by passage 13 all three were present with the 2.2 X 106 RNA predominating.

Cell Line↗

Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome.

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.

5' Untranslated Regions↗

Mycophenolic acid inhibits dengue virus infection by preventing replication of viral RNA.

Dengue fever is a mosquito-borne viral disease of global importance with no available antiviral therapy. We assessed the ability of mycophenolic acid (MPA), a drug currently used as an immunosuppressive agent, to inhibit dengue virus (DV) antigen expression, RNA replication, and virus production. Pharmacological concentrations of MPA effectively blocked DV infection, decreasing the percentage of infected cells by 99% and the levels of secreted virus by up to a millionfold. Results were reproduced with four hepatoma cell lines and different flaviviruses, including a recent West Nile virus isolate. Experiments were performed to define the stage in the viral lifecycle at which MPA abrogates infection. Early steps in viral infection, such as viral entry and nucleocapsid uncoating, were not the primary targets of MPA action since its inhibitory effect was retained when naked DV RNA was transfected directly into cells. Biosynthetic labeling experiments showed that MPA did not block the initial phase of viral translation but did interfere with viral protein synthesis in the amplification phase. Quantitative RT-PCR demonstrated that MPA prevented the accumulation of viral positive- and negative-strand RNA as the infection proceeded. We conclude that MPA inhibits flavivirus infection by preventing synthesis and accumulation of viral RNA.

Dengue Virus↗

Effect of rimantadine on cytotoxic T lymphocyte responses and immunity to reinfection in mice infected with influenza A virus.

Administration of rimantadine to mice via drinking water, following a prophylactic dose, reduced lung virus titers by greater than 3 log10 plague-forming units (pfu)/ml but caused only marginal reductions in lung virus titers when therapy was started 8 h after exposure to virus. Mice given rimantadine prophylactically plus therapeutically were resistant to rechallenge with virus at a dose equivalent to that used for the primary infection (50 pfu/mouse) but not to a high dose (1 x 10(5) pfu/mouse). Virus-neutralizing-antibody titers were reduced only by rimantadine treatment, which included prophylaxis, whereas the cytotoxic T lymphocyte (CTL) response was depressed by treatment given with or without prophylaxis. Mice infected with rimantadine-resistant virus had no decrease in CTL or antibody responses when treated with rimantadine. Therefore, the depression in CTL and antibody responses associated with rimantadine treatment appears to be due to a decrease in the amount of viral antigen available or interference with viral antigen processing and not to nonspecific immunosuppressive effects.

Adamantane↗

N-methylisatin-beta-4',4'-diethylthiosemicarbazone, an inhibitor of Moloney leukemia virus protein production: characterization and in vitro translation of viral mRNA.

The mode of inhibition of N-methylisatin-beta-4',4'-diethylthiosemicarbazone (M-IBDET) on Moloney leukemia virus production was studied. Drug treatment of infected cells did not alter the amounts or sizes of the 35S and 22S subgenomic viral RNAs. The translation abilities of poly(A)+ RNA derived from M-IBDET-treated cells was also unaffected, as judged by cell-free translation analysis. Poly(A)+ RNA derived from M-IBDET-treated cells directed translation of equal amounts of viral gag precursors, gPr-80gag and Pr-65gag, as did poly(A)+ RNA prepared from untreated cells. The addition of M-IBDET to a cell-free translation system programmed with either total poly(A)+ RNA extracted from infected cells or hybrid-selected viral RNA inhibited the synthesis of viral protein precursors. An examination of the effect of M-IBDET on polysomes engaged in the translation of viral proteins revealed a fourfold accumulation of polysomal virus-specific RNA in drug-treated cells. These results suggest that the inhibition of Moloney leukemia virus by M-IBDET involves a block in the translation of viral RNA rather than interference with viral RNA transcription.

Gene Products, gag↗

Viral determinants of the variable sensitivity of herpes simplex virus strains to gD-mediated interference.

Cells that express glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) resist infection by HSV-1 and HSV-2 because of interference with viral penetration. The results presented here show that both HSV-1 and HSV-2 gD can mediate interference and that various HSV-1 and HSV-2 strains differ in sensitivity to this interference. The relative degree of sensitivity was not necessarily dependent on whether the cell expressed the heterologous or homologous form of gD but rather on the properties of the virus. Marker transfer experiments revealed that the allele of gD expressed by the virus was a major determinant of sensitivity to interference. Amino acid substitutions in the most distal part of the gD ectodomain had a major effect, but substitutions solely in the cytoplasmic domain also influenced sensitivity to interference. In addition, evidence was obtained that another viral gene(s) in addition to the one encoding gD can influence sensitivity to interference. The results indicate that HSV-1 and HSV-2 gD share determinants required to mediate interference with infection by HSV of either serotype and that the pathway of HSV entry that is blocked by expression of cell-associated gD can be cleared or bypassed through subtle alterations in virion-associated proteins, particularly gD.

Amino Acid Sequence↗

Linker insertion mutants of simian virus 40 large T antigen that show trans-dominant interference with wild-type large T antigen map to multiple sites within the T-antigen gene.

Linker insertion mutants affecting the simian virus 40 (SV40) large tumor (T) antigen were constructed by inserting a 12-base-pair oligonucleotide linker into restriction endonuclease cleavage sites located within the early region of SV40. One mutant, with the insertion at amino acid 5, was viable in CV-1p and BSC-1 cells, indicating that sequences very close to the amino terminus of large T could be altered without affecting the lytic infection cycle of SV40. All other mutants affecting large T were not viable. In complementation assays between the linker insertion mutants and either a late-gene mutant, dlBC865, or a host range/helper function (hr/hf) mutant, dlA2475, delayed complementation was seen with the 6 of the 10 nonviable mutants. Of these 10 mutants, 5 formed plaques 3 to 4 days later than in control complementations, while complementation by one of the mutants, inA2827, with an insertion at amino acid 520, was delayed more than 1 week. Most mutants which showed delayed complementation replicated less well in Cos-1 cells than did a control mutant, dlA1209, which produced no T antigen. The replication of inA2827(aa520) was reduced by more than 90%. Similar interference with viral DNA replication was seen when CV-1, HeLa, or 293 cells were cotransfected with an origin-defective plasmid encoding wild-type large T antigen and with inA2827(aa520). Only one of the mutant T antigens, inA2807(aa303), was unstable. These results indicate that some of the mutant T antigens interfered with functions of wild-type T required for viral DNA replication. However, not all of the mutants which showed delayed complementation also showed interference with viral DNA replication. This indicates that mutant large T antigens may interfere trans dominantly with multiple activities of wild-type large T antigen.

Animals↗

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.

Aedes↗

Sensitivity of brome mosaic virus RNA1 replication to mutations in the 3' tRNA-like structure implies a requirement for sustained synthesis of replicase protein 1a.

The replication competence of a series of brome mosaic virus (BMV) RNA1 variants with defined mutations in the 3' tRNA-like structure, previously characterized in vitro to be defective in minus-strand synthesis and several tRNA-associated functions, was analyzed in barley protoplasts. Inocula containing wild type RNAs2 and 3 and RNA1 bearing either Deltaknob or 5'Psk mutation failed to replicate. Two additional RNA1 variants, each bearing either M4 or 5'AGA mutation, resulted in detectable accumulation of progeny but are inhibitory to overall viral replication when supplied in high concentrations. Another aminoacylation-defective mutation Delta5' supported viral replication but did not interfere with viral replication even at higher concentrations. Coinoculation of replication-incompetent variants of RNAl with wt RNAs2 and 3 to Chenopodium hybridum plants resulted in the delayed development of local necrotic lesions characteristic of a wt infection. Sequence analysis of progeny RNA recovered from these lesions indicated that, in each case, a functional 3' noncoding sequence was restored due to homologous recombination with a corresponding sequence from wt RNA3. Taken together the results suggest that, unlike protein 2a which is required in catalytic amounts, the intrinsic involvement of protein 1a at various stages of virus infection cycle demands its sustained synthesis.

3' Untranslated Regions↗

Autointerference of Marituba Virus (Bunyaviridae) in mouse L cells by defective interfering particles.

The growth characteristics of Marituba virus, a member of the Bunyaviridae family, were studied in L-A9 cells. Virus yield was strictly dependent on the MOI. Quantitation of infectious virus released from the cells revealed a decrease in magnitude with continued serial passage. Specificity of the Marituba virus inhibitory response was investigated in relation to interference within homologous and heterologous viral classes. Virus particles were studied by isopycnic centrifugation in sucrose gradients. Under conditions of multiple viral passages at high multiplicity, two major classes of virus particles were produced, one band at 1.19 g/ml and another at 1.16 g/ml. Particles at 1.16 g/ml were noninfectious. Our results suggest that during the replication of Marituba virus at high MOI a population of defective interfering particles is generated.

Animals↗

Antisense oligonucleotides in solution or encapsulated in immunoliposomes inhibit replication of HIV-1 by several different mechanisms.

Phosphodiester and phosphorothioate oligonucleotides in alpha and beta configurations directed against the initiation codon region of the HIV-1 rev gene were evaluated for their ability to inhibit HIV-1 replication in acutely and chronically infected human CEM cells. Encapsulation in antibody-targeted liposomes (immunoliposomes) permitted intracellular delivery and distinction between oligonucleotide-mediated inhibition of viral entry and intracellular effects on viral RNA. Our results are consistent with four mechanisms of antiviral activity for these antisense oligonucleotides: (i) interference with virus-mediated cell fusion by free but not liposome-encapsulated phosphorothioate oligonucleotides of any sequence; (ii) interference with reverse transcription in a sequence non-specific manner by phosphorothioate oligonucleotides in alpha and beta configurations; (iii) interference with viral reverse transcription in a sequence-specific and RNase-H-independent manner by alpha and beta phosphodiester oligonucleotides; (iv) interference with viral mRNA in a sequence-specific and RNase-H-dependent manner by beta-phosphorothioate oligonucleotides.

Antiviral Agents↗