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PubMed1998

Uncoupled expression of p33 and p92 permits amplification of tomato bushy stunt virus RNAs.

Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5'-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal approximately 6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3'-located segment, region 3.5, was tested. However, when 5'-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5' cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.

Gene Amplification↗

PubMed2005

Replicon cell culture system as a valuable tool in antiviral drug discovery against hepatitis C virus.

Discovery of potential therapeutics against hepatitis C virus (HCV) infection has been hampered in the past decade by the inability to grow this virus in tissue culture and by the lack of robust small animal models. This situation has been improved by the recent development of a selectable HCV replicon cell culture system. For the first time, drug discovery scientists are able to screen large compound collections using the replicon cell culture system to identify small molecules with the potential to inhibit HCV RNA replication. The replicon system has also been used to elucidate inhibitors' antiviral mechanism of action and to optimize antiviral potency. In this review, we will summarize the recent development of HCV replicon cell culture system and its use in anti-HCV drug discovery. The antiviral activities of promising lead compounds are also reviewed.

Antiviral Agents↗

PubMed2026

Japanese encephalitis virus hijacks the host purine biosynthetic network to promote viral replication in neurons.

Japanese encephalitis virus (JEV) is an important neurotropic orthoflavivirus that poses a threat to both human and animal health. However, the mechanism underlying its rapid replication in the central nervous system (CNS) remains poorly understood. In this study, we conducted metabolomic profiling of JEV-infected mouse brains and neurons, revealing a profound reprogramming of central carbon metabolism, particularly an enhancement in nucleotide synthesis. Integrated multi-omics analyses confirmed that JEV infection transcriptionally upregulates key enzymes involved in de novo purine biosynthesis (DNPB), one-carbon (1C) metabolism, and the pentose phosphate pathway (PPP) in neurons. Pharmacological inhibition of the core DNPB enzymes potently suppressed JEV replication in neurons and reduced both viral loads and neuroinflammation in JEV-infected mice, suggesting the essential role of DNPB in JEV replication within CNS. Mechanistically, we delineated the critical functions of both the non-oxidative PPP and MTHFD2-mediated 1C metabolism, which jointly supply essential precursors, such as ribose-5-phosphate and formyl groups, for the de novo biosynthesis of purines required for viral RNA replication. These findings unveil a strategy by which JEV co-opts the host's purine biosynthetic machinery to fulfill the nucleotide demands for its genomic replication, establishing DNPB and its supporting pathways as promising therapeutic targets for infections caused by JEV and other neurotropic viruses.

Animals↗

PubMed1992

Inhibition of HIV-1 RNA production by the diphtheria toxin-related IL-2 fusion proteins DAB486IL-2 and DAB389IL-2.

Productive infection of cells by human immunodeficiency virus type 1 (HIV-1) is associated with the activation state of the cell and its obligatory expression of the interleukin-2 receptor (IL-2R), the latter providing a new target for antiviral therapy. A quantitative RNA-RNA hybridization assay is employed to detect production of HIV-1 RNA and to show that two IL-2 diphtheria toxin-related fusion proteins (DAB486IL-2 and its more potent, truncated form DAB389IL-2) inhibit HIV-1 RNA production in infected cells. A mutant form of DAB486IL-2 containing a single point mutation that inactivates the adenosine diphosphate ribosyltransferase activity of the toxin does not inhibit HIV RNA production, even though the molecule binds to the IL-2R. The active fusion proteins inhibit viral RNA replication in cells infected with HIV-1 clinical isolates as well as with a ZDV-resistant strain of HIV-1. These results indicate that IL-2 receptor-targeted fusion proteins can be utilized to inhibit HIV-1 replication effectively in infected human lymphocytes.

Antiviral Agents↗

PubMed1994

Detection of hepatitis C virus RNA in peripheral blood mononuclear cells of infected patients by in situ hybridization.

We used in situ hybridization to detect hepatitis C virus (HCV) infection of peripheral blood mononuclear cells (PBMNC) from 11 patients with chronic active hepatitis. Using 35S-labeled HCV-RNA probe, HCV-RNA-positive and -negative strands were observed in unstimulated PBMNC from three patients, all of whom were receiving immunosuppressive drugs after orthotopic liver transplantation (OLT). HCV-RNA sequences were also identified in PBMNC from three patients who were not undergoing immunosuppression, after stimulation with either phytohemagglutinin (PHA) or pokeweed mitogen (PWM). In contrast, HCV-RNA was not found in the remaining five patients, who had not undergone OLT and whose cells were not stimulated with mitogens. These results show that mononuclear cells can be infected by HCV and that mitogenic stimulation of infected cells increases HCV-RNA replication.

Adult↗

PubMed1982

Poliovirus-induced inhibition of host RNA synthesis studied in isolated HEp-2 cell nuclei.

Nuclei isolated from uninfected HEp-2 cells synthesized RNA for 60 to 90 min. The individual RNA polymerase activities were determined by alpha-amanitin differential inhibition and the RNA products characterized by electron microscope (EM) autoradiography and sucrose gradient centrifugation. In nuclei prepared from poliovirus-infected cells, the capacity to synthesize RNA in vitro decreased with time after infection. RNA polymerase II activity (hnRNA synthesis) was preferentially inhibited more than was the polymerase I activity (rRNA synthesis). Poliovirus-infected cytoplasm (S-30) inhibited in vitro RNA synthesis in uninfected nuclei by selectively affecting the polymerase II activity. Selective inhibition of hnRNA synthesis by the crude extracts could be monitored by EM autoradiography directly. Determinations of individual RNA polymerase activities by differential alpha-amanitin inhibition were done only after treatment of the infected cytoplasm with micrococcal nuclease to abolish virus RNA replication. Selective inhibition of hnRNA synthesis depended on preincubation of the nuclei together with the infected cytoplasm, indicating that inhibitory substances from the infected cytoplasm entered the nuclei. Isolated nuclei therefore provide a useful system for studying the nature of the inhibitor(s) and of host RNA synthesis inhibition by picornaviruses.

Cell Line↗

PubMed1999

Mutagenic analysis of the 3' cis-acting elements of the rubella virus genome.

Thermodynamically predicted secondary structure analysis of the 3'-terminal 305 nucleotides (nt) of the rubella virus (RUB) genome, a region conserved in all RUB defective interfering RNAs, revealed four stem-loop (SL) structures; SL1 and SL2 are both located in the E1 coding region, while SL3 and SL4 are within the 59-nt 3' untranslated region (UTR) preceding the poly(A) tract. SL2 is a structure shown to interact with human calreticulin (CAL), an autoantigen potentially involved in RUB RNA replication and pathogenesis. RNase mapping indicated that SL2 and SL3 are in equilibrium between two conformations, in the second of which the previously proposed CAL binding site in SL2, a U-U bulge, is not formed. Site-directed mutagenesis of the 3' UTR with a RUB infectious clone, Robo302, revealed that most of the 3' UTR is required for viral viability except for the 3'-terminal 5 nt and the poly(A) tract, although poly(A) was rapidly regenerated during subsequent replication. Maintenance of the overall SL3 structure, the 11-nt single-stranded sequence between SL3 and SL4, and the sequences forming SL4 were all important for viral viability. Studies on the interaction between host factors and the 3' UTR showed the formation of three RNA-protein complexes by gel mobility shift assay, and UV-induced cross-linking detected six host protein species, with molecular masses of 120, 80, 66, 55, 48, and 36 kDa, interacting with the 3' UTR. Site-directed mutagenesis of SL2 by nucleotide substitutions showed that maintenance of SL2 stem rather than the U-U bulge was critical in CAL binding since mutants having the U-U bulge base paired had a similar binding activity for CAL as the native structure whereas mutants having the SL2 stem destabilized had much lower binding activity. However, all of these mutations gave rise to viable viruses when introduced into Robo302, indicating that binding of CAL to SL2 is independent of viral viability.

Base Sequence↗

PubMedDate not supplied

Mechanism of interferon action. Interferon alpha inhibits vesicular stomatitis virus primary transcript accumulation in P1/eIF-2 alpha protein kinase-deficient human fibroblast cells.

The molecular basis of the inhibition of vesicular stomatitis virus (VSV) replication by purified recombinant alpha interferon (IFN-alpha A/D) in human fibroblast GM2767A cells was examined. A saturating concentration of IFN-alpha A/D inhibited infectious VSV yield by about four to five log10. By use of the VSV mutant tsG41, which is competent in RNA transcription but defective in RNA replication at 40 degrees C, it was shown that IFN-alpha A/D treatment significantly inhibited primary viral protein synthesis. However, the apparent IFN-induced inhibition of VSV protein synthesis was due primarily to a reduction in the accumulation of VSV primary transcripts in IFN-alpha A/D treated GM2767A cells rather than to a direct effect on translation per se. The IFN-induced reduction in VSV primary genome expression was detectable after only 1 hour of IFN treatment; actinomycin D treatment of GM2767A cells prior to IFN-alpha A/D treatment blocked the establishment of the IFN-induced inhibition of VSV. In contrast to the results obtained with GM2767A cells, IFN-alpha A/D produced no detectable effect on the accumulation of VSV primary transcripts in human amnion U cells even though VSV primary protein synthesis and infectious virus yield were significantly reduced. In summary, the principal cause of the IFN-alpha induced inhibition of VSV replication in protein P1/eIF-2 alpha kinase-deficient human fibroblast GM2767A cells appears to be at or prior to primary transcript accumulation; thus, the antiviral mechanisms of IFN-alpha in GM2767A cells is fundamentally different from the IFN-alpha induced translation inhibition observed in kinase-sufficient human amnion U cells.

Amnion↗

PubMed1986

Dominance of temperature-sensitive phenotypes. I. Studies of the mechanism of inhibition of the growth of wild-type vesicular stomatitis virus.

It has been reported previously that temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) with an RNA- phenotype interfere with the growth of wild-type (wt)-VSV at both the permissive (34 degrees) and nonpermissive (39.5 degrees) temperatures (J. S. Youngner and D. O. Quagliana (1976) J. Virol. 19, 102-107). Investigation of the mechanism of this interference has revealed the following information. In double infection with RNA- ts mutants and wt-VSV, the cumulative synthesis of viral RNA is inhibited. By varying the relative multiplicities of the two viruses, it was observed that the level of RNA synthesis reflects the level of interference with wt-VSV growth. Although viral RNA synthesis was severely compromised in double infections, this inhibition was not at the level of primary transcription or the translation of primary transcripts. Rather, secondary transcription and genome RNA replication were drastically reduced. Sequential infection with wt-VSV and the ts mutants revealed that there is an early point in the replication cycle of wt-VSV (1 to 2 hr) after which the ts mutants can no longer interfere with the growth of wt-VSV. Ultraviolet irradiation of ts G 41, a mutant belonging to complementation group IV, was used to determine the target size of the interference function. The calculated value for the target size was very close to the target size of the N gene. Additional experiments showed that RNA+ ts mutants representing complementation groups III and V also were able to interfere with the growth of wt-VSV.

Genes, Dominant↗

PubMed1995

Japanese encephalitis virus nonstructural protein NS3 has RNA binding and ATPase activities.

Sequence data suggest that Japanese encephalitis virus (JEV) protein NS3 is a multifunctional protein with sequence motifs characteristic of a protease and a helicase. To examine the functions of JEV-NS3, a fusion protein of NS3 in Escherichia coli was generated. Analysis by Western blot using monospecific rabbit antisera generated against the fusion protein (anti-MBJEN3) showed that NS3 was localized in the membrane fraction of JEV-infected cells and the particulate fraction of bacteria extracts. The addition of anti-MBJEN3 sera reduced JEV-specific RNA synthesis activity in a in vitro system. In addition, NS3 was shown to exhibit RNA binding and ATPase activities, suggesting this protein has an important role in viral RNA replication in virus-infected cells.

Adenosine Triphosphatases↗

PubMed1988

Subcellular localization of tobacco mosaic virus minus strand RNA in infected protoplasts.

Radioactive RNA probes were prepared which specifically hybridize with sequences complementary to 5' and 3' regions of tobacco mosaic virus (TMV) RNA. These probes were used in Northern hybridization to locate TMV-RNA minus strands in the subcellular fractions of infected tobacco protoplasts. When the protoplasts were lysed with Triton X-100, full-length minus strands were present in the cytoplasmic but not in the nuclear fraction. With mechanically broken protoplasts, the crude nuclear fraction (250 g pellet) contained small amount of minus strands which appeared to derive from unbroken protoplasts, but most of minus strands were recovered in a fraction sedimented between 250 and 2500 g, little if any being found in lighter fractions. The results indicate that TMV-RNA replicates in association with an extranuclear structure.

Blotting, Northern↗

PubMedDate not supplied

A gene coding for a monomeric form of cucumber mosaic virus satellite RNA confers tolerance to CMV.

A gene whose transcript bears a monomeric form of cucumber mosaic virus (CMV) satellite RNA was introduced into tobacco (Nicotiana tabacum 'Xanthi' nc) plants by using an Ri plasmid-based vector system. On CMV infection, the transcript of the satellite RNA gene was used as a template to yield unit-length satellite RNA, which was efficiently amplified by the virus. Plants bearing the satellite RNA gene displayed long-term tolerance to CMV infection and were also tolerant to CMV infection by aphids, the natural vector of CMV. Implications of these results concerning the mechanism of satellite RNA replication are discussed.

Animals↗

PubMed2005

Characterization of new recombinant noroviruses.

Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF 1) and ORF 2. The results allowed us to identify several recombinant noroviruses: GGIIb viruses were detected for the first time in France in August 2000 and then spread through France and to Europe during the following winter. Here we present the characterization of three other probable GII recombinants which showed different phylogenetic positions depending on their ORF 1 and ORF 2 sequences. Analysis of the region located between ORF 1 and ORF 2 by a nucleotide identity window search showed a sudden shift in similarities. Moreover, recombination breakpoints were identified upstream and downstream of the beginning of ORF 2 by using a statistical test, thus confirming the involvement of this region in recombination. Unlike GGIIb, the three recombinants described here do not seem to have diffused widely in the community: one was found in a waterborne outbreak, and the other two were found in sporadic cases. Recombination is important for the evolution of RNA viruses and has already been described for noroviruses. Our results suggest that recombination is not a rare phenomenon among noroviruses, but not all these presumed recombinants that formed during RNA replication are able to spread widely.

Base Sequence↗

PubMed2003

Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase.

To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Synthesis and biological evaluation of 5R- and 5S-methyl substituted D- and L-configuration 1,3-dioxolane nucleoside analogs.

1,3-Dioxolane and 1,3-oxathiolane nucleoside analogs play an important role in anti-viral and anti-neoplastic chemotherapy. We report here the synthesis of 2-hydroxymethyl-5-methyl-1,3-dioxolanylpurine nucleosides from 4-acetoxy-2-(benzyloxymethyl)-5-methyldioxolane. Dioxolanes of alpha-D-, beta-D-, alpha-L-, and beta-L-configuration were prepared, that included 5-methyl derivatives of both 5R and 5S configuration. Molecular mechanics calculations indicate that the 5S and 5R diastereoisomeric 1,3-dioxolanes possess distinct conformational bias, suggesting that methyl substitution may alter the conformational preference of 1,3-dioxolanes. The ability of the 1,3-dioxolanes to inhibit HCV RNA replication was evaluated in a cell-based, subgenomic replicon assay. In addition, activity against vaccinia and HIV was evaluated in cell-based assays. The 2-hydroxymethyl-5-methyl-1,3-dioxolanes were found to be inactive.

Antiviral Agents↗

PubMed1995

The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles.

In alphavirus-infected cells the four virus-specific nonstructural proteins (nsP1-nsP4) are located on modified endosomes and lysosomes known as type I cytopathic vacuoles. In this paper, we show that when nsP1 was expressed alone in HeLa cells with the aid of the recombinant T7 RNA polymerase vaccinia (vTF7-3) virus system, it was tightly associated with intracellular smooth membranes. The membrane association may be due to acylation, since nsP1 could be labeled with [3H]palmitic acid in both Semliki Forest virus-infected and nsP1-transfected HeLa cells. Release of the 3H-label by alkaline methanolysis suggests that the palmitate was associated with nsP1 via an ester bond. Pulse-chase experiments done on nsP1-transfected cells revealed that this protein was rapidly associated with the membranes. After synchronizing the synthesis of the nsP1 gene product in transfected cells, nsP1 appeared first at the plasma membrane and thereafter on vesicles, many of which contained the endosomal transferrin receptor marker. Later, nsP1 appeared on large vacuoles, which contained the lysosome specific h-lamp-1 protein. Membrane association of nsP1, and its affinity to endosomes and lysosomes, suggest a role of this protein in the biogenesis of the alphavirus-specific RNA replication complex.

Acylation↗

PubMed2002

Complete nucleotide sequence of the M RNA segment of Andes virus and analysis of the variability of the termini of the virus S, M and L RNA segments.

Hantavirus pulmonary syndrome (HPS) has been recognized increasingly as a significant public health problem in South America since Andes virus was first discovered in Argentina. Here, the isolation of Andes virus is reported from an infected rodent captured in Argentina in close vicinity to the place of the first HPS case, AH1. The complete nucleotide sequences of the virus M segment, partial L segment and the termini of the S, M and L segment genome RNAs were determined. The Andes virus M RNA segment is 3671 nt in length and is predicted to encode a glycoprotein precursor 1138 aa in length; it generally resembles the other HPS-associated hantaviruses in its organization. Relative to the G1 glycoprotein of other HPS-associated hantaviruses, an additional potential glycosylation site was found but this is located in the predicted cytoplasmic domain and is therefore unlikely to be glycosylated. In phylogenetic analyses, Andes virus, together with the more related hantaviruses, represented a monophyletic lineage. The S-terminal nucleotides were conserved relative to other New World hantaviruses. The M and L segment RNA termini had short deletions in the region believed to contain the sequence and structural features necessary for initiation of virus RNA replication and transcription. Clinical manifestations of Andes virus infections range from fulminant respiratory disease with high lethality to mild course without sequelae. Andes virus has also been associated with person-to-person transmission. Accumulation of Andes virus genetic data will be essential for understanding the factors that regulate virus replication and transmission and to determine the pathogenesis of HPS.

Animals↗

PubMed1994

cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides.

Unencapsidated double-stranded viral RNAs belonging to the genus Hypovirus attenuate virulence of the chestnut blight fungus, Cryphonectria parasitica. A full-length cDNA clone of hypovirus CHV1-713 double-stranded RNA was recently shown to be infectious when introduced into the C. parasitica genome by DNA-mediated transformation. In this study, we show that the viral RNA derived from the chromosomally integrated cDNA copy is trimmed of extraneous vector nucleotide sequences. The cDNA-derived viral RNA was also found to contain a 73-bp deletion located within the 5'-noncoding leader sequence as a result of a pre-mRNA splicing event. Implications of these results are discussed in terms of hypovirus RNA replication and anticipated field studies involving engineered hypovirulent C. parasitica strains.

Base Sequence↗