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At least 757 records · Page 42Linked to original sources
PubMed2001

High numbers of viral RNA copies in the central nervous system of mice during persistent infection with Theiler's virus.

The low-neurovirulence Theiler's murine encephalomyelitis viruses (TMEV), such as BeAn virus, cause a persistent infection of the central nervous system (CNS) in susceptible mouse strains that results in inflammatory demyelination. The ability of TMEV to persist in the mouse CNS has traditionally been demonstrated by recovering infectious virus from the spinal cord. Results of infectivity assays led to the notion that TMEV persists at low levels. In the present study, we analyzed the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length species in the spinal cords of infected mice and infected tissue culture cells by using Northern hybridization. Considering the low levels of infectious virus in the spinal cord, a surprisingly large number of viral genomes (mean of 3.0 x 10(9)) was detected in persistently infected mice. In the transition from the acute (approximately postinfection [p.i.] day 7) to the persistent (beginning on p.i. day 28) phase of infection, viral RNA copy numbers steadily increased, indicating that TMEV persistence involves active viral RNA replication. Further, BeAn viral genomes were full-length in size; i.e., no subgenomic species were detected and the ratio of BeAn virus plus- to minus-strand RNA indicated that viral RNA replication is unperturbed in the mouse spinal cord. Analysis of cultured macrophages and oligodendrocytes suggests that either of these cell types can potentially synthesize high numbers of viral RNA copies if infected in the spinal cord and therefore account for the heavy viral load. A scheme is presented for the direct isolation of both cell types directly from infected spinal cords for further viral analyses.

Animals↗
PubMed1999

Carboxy-terminal five amino acids of the nucleocapsid protein of vesicular stomatitis virus are required for encapsidation and replication of genome RNA.

The encapsidation of vesicular stomatitis virus (VSV) genome RNA, a prerequisite step to the replication process by the nucleocapsid protein (N) was studied by its ability to package VSV leader RNA in vitro in a RNase-resistant form. The VSV leader RNA was derived from the SP6 transcription vector while the N protein was made in rabbit reticulocyte lysate. The in vitro encapsidation was carried out by translating N mRNA in the presence of 32P-labeled presynthesized leader RNA. The RNA encapsidation property of the N protein was completely abrogated when the C-terminal five amino acids (VEFDK-COOH) were deleted. Systematic mutational analyses within the C-terminal five amino acid regions reveal that the RNA encapsidation activity was lost in all mutants except K --> A and K --> R, indicating that C-terminal five amino acids, in particular the lysine residue play critical role in genome RNA encapsidation. To correlate the in vitro encapsidation abilities of these mutant N proteins with genome RNA replication, we have used a full-length cDNA clone of VSV genome RNA to rescue infectious virions from cells expressing L, P, and wt or mutant N proteins and measured the recovery of plaque forming units. The results indicate that the N mutants that are defective in in vitro encapsidation of leader RNA do not support replication, establishing the requirement of C-terminal five amino acids of the N protein in viral replication.

Amino Acids↗
PubMed1982

Globular proteins, GU wobbling, and the evolution of the genetic code.

It has previously been shown that the formation of GU base pairs in RNA copying processes leads to an accumulation of G and U in both strands of the replicating RNA, which results in a non-random distribution of base triplets. In the present paper, this distribution is calculated, and, using the X2-test, a correlation between the distribution of triplets and the amino acid composition of the evolutionarily conservative interior regions of selected globular proteins is established. It is suggested that GU wobbling in early replication of RNA could have led to the observed amino acid composition of present-day protein interiors. If this hypothesis is correct, then GU wobbling must have been very extensive in the imprecisely replicating RNA, even reaching values close to the critical for stability of its double-helical structure. Implications of the hypothesis both for the evolution of the genetic code and of proteins are discussed.

Biological Evolution↗
PubMedDate not supplied

[The morphogenesis of tick-borne encephalitis in light of the new data on virus replication].

The analysis of the RNA virus spread of tick encephalitis in the course of infection has been performed. The virus RNA is detectable first, (as early as one day after the infection) in the lymph nodes. Later it is retained in the cells of T-dependent areas. Viral RNA is also the first to be detected in the vascular structures and ventricular system of the brain. The correlation between the time of the viral RNA replication and specific initial damage in the lymphoid tissue and central nervous system is established. The question is raised on the role of the lymphocyte recirculation in the spread of the virus in the body.

Acute Disease↗
PubMed2002

Complementation analysis of the flavivirus Kunjin NS3 and NS5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of NS3 in cis for virus assembly.

We have previously reported successful trans-complementation of defective Kunjin virus genomic RNAs with a range of large lethal deletions in the nonstructural genes NS1, NS3, and NS5 (A. A. Khromykh et al., J. Virol. 74:3253-3263, 2000). In this study we have mapped further the minimal region in the NS5 gene essential for efficient trans-complementation of genome-length RNAs in repBHK cells to the first 316 of the 905 codons. To allow amplification and easy detection of complemented defective RNAs with deletions apparently affecting virus assembly, we have developed a dual replicon complementation system. In this system defective replicon RNAs with a deletion(s) in the nonstructural genes also encoded the puromycin resistance gene (PAC gene) and the reporter gene for beta-galactosidase (beta-Gal). Complementation of these defective replicon RNAs in repBHK cells resulted in expression of PAC and beta-Gal which allowed establishment of cell lines stably producing replicating defective RNAs by selection with puromycin and comparison of replication efficiencies of complemented defective RNAs by beta-Gal assay. Using this system we demonstrated that deletions in the C-terminal 434 codons of NS3 (codons 178 to 611) were complemented for RNA replication, while any deletions in the first 178 codons were not. None of the genome-length RNAs containing deletions in NS3 shown to be complementable for RNA replication produced secreted defective viruses during complementation in repBHK cells. In contrast, structural proteins produced from these complemented defective RNAs were able to package helper replicon RNA. The results define minimal regions in the NS3 and NS5 genes essential for the formation of complementable replication complex and show a requirement of NS3 in cis for virus assembly.

Amino Acids↗
PubMed1995

Detection of Cellular Proteins and Viral Core Protein Interacting with the 5' Untranslated Region of Hepatitis C Virus RNA.

Hepatitis C virus (HCV) is a pesti- and flavi-like virus, which contains a highly conserved 5'-untranslated region (UTR). This region is implicated in the regulation of both translation and RNA replication. To examine the possible cellular factors involved in HCV replication, we performed UV cross-linking experiments to detect cellular protein binding to 5'-UTR of HCV RNA. No cytoplasmic proteins were found to cross-link to 5'-UTR. Surprisingly, when nuclear extracts were used for UV cross-linking, a major protein of 110 kD and several other minor proteins were detected. Competition assays confirmed that the binding of the 110-kD protein was specific to the 5'-UTR. The protein-binding site was mapped within the 78-nt region between nucleotides 199 and 277 from the 5' end of the viral RNA. This protein was present in several different cell lines tested. No cellular proteins specifically bound to the complementary strands of the 5'-UTR. We have also shown by an RNA-protein blotting assay that 5'-UTR bound to the HCV core protein, which can be translocated to the nuclei. These findings suggest that HCV RNA may enter nuclei by complexing with the viral core protein and interact with nuclear proteins that are involved in the regulation of RNA replication or translation. It is thus possible that HCV employs a replication strategy distinct from its related pestiviruses or flaviviruses. Copyright 1995 S. Karger AG, Basel

Journal Article↗
PubMed2003

Inhibition of gammaherpesvirus replication by RNA interference.

RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.

Animals↗
PubMed2004

Interference of hepatitis A virus replication by small interfering RNAs.

The rate of acute liver failure due to hepatitis A virus (HAV) has not decreased, and therapy of severe infections is still of major interest. Using a DNA-based HAV replicon cell culture system, we demonstrate that small interfering RNAs (siRNAs) targeted against viral sequences or a reporter gene contained in the viral genome specifically inhibit HAV RNA replication in HuhT7 cells. Combinations of siRNAs were more effective suppressors of HAV RNA replication. Also, siRNAs targeted against HAV 2C and 3D inhibited the expression of the respective protein. Expressions of endogenous beta-actin and double-stranded-specific RNA-activated serin/threonine kinase (PKR) were unaltered, demonstrating that the siRNA inhibitory effect was not connected to interferon inhibition, but rather was specifically targeted against HAV RNA. These results suggest that RNA interference might ultimately be useful in treatment of severe HAV infection with or without chronic liver diseases.

Actins↗
PubMed2003

Inhibition of virus replication by RNA interference.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene function by reverse genetics. Additionally, inhibition of virus replication by means of induced RNAi has now been reported for numerous viruses, including several important human pathogens such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, dengue virus, poliovirus and influenza virus A. In this review, we will summarize the current data on RNAi-mediated inhibition of virus replication and discuss the possibilities for the development of RNAi-based antiviral therapeutics.

Animals↗
PubMed1989

Internal and terminal cis-acting sites are necessary for in vitro replication of the L-A double-stranded RNA virus of yeast.

Empty particles of the L-A dsRNA virus of Saccharomyces cerevisiae bind to added viral (+) strands and convert them to dsRNA (RNA replication) in an in vitro reaction that is dependent on host factors. X dsRNA (530 bp long) is a deletion derivative of L-A dsRNA (4.5 kb). By modifying our cDNA clone of X and testing template activity of T7 RNA polymerase transcripts, we have found that both the 3' end 30 bases and an internal site on the (+) strand are necessary for optimal replication [in vitro (-) strand synthesis]. Changing any one of the 3' terminal three bases eliminates template activity, but the 3' terminal five bases of M1 (a satellite virus of L-A) can replace the 3' terminal four bases of X. A subterminal stem-loop structure is also important for template activity. The internal site that enhances replication is approximately 400 bp from the 3' end and is distinct from the site necessary for binding of (+) strands to the empty viral particles.

Base Sequence↗
PubMed2005

[RNA viruses and mutations].

Actively replicating RNA viruses in nature are continually changing their genetic information by spontaneous mutations. These changes often result in alterations in immune-sensitivity, drug-sensitivity, cell-tropism, and host-range, causing uncontrollability of the pathogen and emerging/re-emerging infections. To better understand the virus changes and develop effective methods to control the moving targets, it is essential to obtain information on changes in viral genomes and proteins. Although information on genetic changes is being accumulated very rapidly, assessment of changes in protein structure and function still requires time-consuming works. In this review, we will overview mutation studies of human immunodeficiency virus and other RNA viruses. In addition, we will introduce recent advances in the computational science and its application on mutation studies and drug development.

Animals↗
PubMed2004

Non-nucleoside benzimidazole-based allosteric inhibitors of the hepatitis C virus NS5B polymerase: inhibition of subgenomic hepatitis C virus RNA replicons in Huh-7 cells.

A previously disclosed series of non-nucleoside allosteric inhibitors of the NS5B polymerase of the hepatitis C virus (HCV) was optimized to yield novel compounds with improved physicochemical properties and activity in cell-based assays. Replacement of ionizable carboxylic acids with neutral substituents in lead compounds produced inhibitors with cellular permeability and antiviral activity in a cell-based assay of subgenomic HCV RNA replication (replicon EC(50) as low as 1.7 microM). The improvement in potency in this ex vivo model of HCV RNA replication validates, in part, the mechanism by which this class of allosteric benzimidazole derivatives inhibits the polymerase and represents a significant step forward in the discovery of novel HCV therapeutics.

Allosteric Regulation↗
PubMed2005

Modulatory effect of mannose-binding lectin on cytokine responses: possible roles in HIV infection.

BACKGROUND: Mannose-binding lectin (MBL) is a soluble receptor of the innate immune system, probably contributing to antimicrobial defence. The possible role of MBL in HIV infection is unclear. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) from 28 HIV-infected patients and 13 healthy controls were stimulated with MBL and costimulated with HIV-1 gp120 or mannan from Saccharomyces cerevisiae before inflammatory responses in PBMC cultures were examined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. HIV-1 RNA replication in vitro was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR) in supernatants from patients with measurable HIV-1 RNA levels. RESULTS: (i) Enhanced TNF-alpha responses were observed when PBMCs from healthy controls and HIV-infected patients were stimulated with MBL and costimulated with HIV-1 gp120 or mannan. (ii) MBL stimulation induced increased HIV RNA replication in culture supernatants when costimulated with mannan. CONCLUSIONS: The present study suggests a modulatory role of MBL on cytokine responses, and HIV replication after stimulation with microbial products. These effects of MBL on inflammatory responses and viral replication may be clinically relevant for HIV infection.

Adult↗