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PubMed2002

Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus.

The cricket paralysis virus (CrPV), a member of the CrPV-like virus family, contains a single positive-stranded RNA genome that encodes two non-overlapping open reading frames separated by a short intergenic region (IGR). The CrPV IGR contains an internal ribosomal entry site (IRES) that directs the expression of structural proteins. Unlike previously described IRESs, the IGR IRES initiates translation by recruiting 80S ribosomes in the absence of initiator Met-tRNA(i) or any canonical initiation factors, from a GCU alanine codon located in the A-site of the ribosome. Here, we have shown that a variety of mutations, designed to disrupt individually three pseudoknot (PK) structures and alter highly conserved nucleotides among the CrPV-like viruses, inhibit IGR IRES-mediated translation. By separating the steps of translational initiation into ribosomal recruitment, ribosomal positioning and ribosomal translocation, we found that the mutated IRES elements could be grouped into two classes. One class, represented by mutations in PKII and PKIII, bound 40S subunits with significantly reduced affinity, suggesting that PKIII and PKII are involved in the initial recruitment of the ribosome. A second class of mutations, exemplified by alterations in PKI, did not affect 40S binding but altered the positioning of the ribosome on the IRES, indicating that PKI is involved in the correct positioning of IRES-associated ribosomes. These results suggest that the IGR IRES has distinct pseudoknot-like structures that make multiple contacts with the ribosome resulting in initiation factor-independent recruitment and correct positioning of the ribosome on the mRNA.

Animals↗

PubMed1975

Natural and experimentally induced antibodies to defined mammalian type-C virus proteins in primates.

Using sensitive radiommunoprecipitation assays for highly purified type-C RNA tumor virus proteins, we found that 5 of 16 clinically normal gibbons (including 4 of 5 normal animals from a colony with 2 cases of lymphoma) and 4 of 4 experimentally inoculated gibbons formed antibodies to the major structural protein (p30) of gibbon ape leukemia virus (GaLV). An additional woolly monkey immunized with the closely related simian sarcoma virus also formed antibodies detectable with GaLV p30. Of 20 patients immunized with formalin-inactivated Rauscher murine leukemia virus (R-MuLV), 10 were previously reported to have antibodies to MuLV as determined by an internally labeled banded virus radioimmunoprecipitation assay. In comparison studies with purified R-MuLV proteins, 7 of 20 patients formed antibodies: 3/20 to R-MuLV p30 only, 1/20 to R-MuLV glycoprotein (gp) 70 only, and 3/20 to both p30 and gp70. Most responders were melanoma patients receiving immunotherapy with BCG. Additionally, rhesus monkeys produced antibodies to the endogenous cat virus RD114 and closely related endogenous baboon leukemia virus p30's. Thus these studies demonstrated the ability of primates (including humans) to form antibodies to well-characterized proteins from endogenous and exogenous type-C viruses and the potential utility of these assays for seroepidemiologic studies.

Animals↗

PubMed2001

Translocation and accumulation of exogeneous hepatitis B virus preS surface proteins in the cell nucleus.

Recurrent reports about protease-sensitive sites in the junction of the preS and S region of the hepatitis B virus large surface protein have raised the question about a possible biological role of S protein-depleted, independent preS protein fragments in the virus life cycle. In the present study, this question was addressed by exogenous introduction of fluorescence-labeled recombinant preS proteins into permeabilized HepG2 cells. While maltose-binding proteins (MBP) were evenly distributed throughout the cytoplasm, MBP-preS fusion proteins selectively accumulated in the nucleus. Using truncated preS proteins, the effective domain for this nuclear accumulation was localized around the preS2 region. The mode of this action differs from conventional nuclear translocation mechanism in its energy- and mediator-independency and in that it is not saturated regardless of the increase of preS protein concentration. The biological meaning of this phenomenon has to be further studied. However, in regard to hepatitis B virus infection, this observation might provide a clue for unveiling the still poorly characterized events after initial internalization of the virus, which might make use of the nuclear translocation effect of the preS2 region to facilitate the infection.

Amino Acid Sequence↗

PubMed2000

Translation from the internal ribosome entry site of bovine viral diarrhea virus is independent of the interaction with polypyrimidine tract-binding protein.

Translation of the pestiviral polyprotein is initiated cap independently at an internal site of the viral RNA, the internal ribosome entry site (IRES). We investigated the translation from the IRES of bovine viral diarrhea virus (BVDV) and the possible interaction of the unconventional cellular RNA-binding proteins, particularly of polypyrimidine tract-binding protein (PTB). The BVDV IRES is translationally active in rabbit reticulocyte lysate (RRL), and it is translated most efficiently at low concentrations of Mg(2+)- and K(+)-ions. In the UV cross-link assay, several proteins from RRL bind to the BVDV IRES, including proteins of 50, 65 and 72kDa, but no protein of 57kDa possibly corresponding to PTB, although PTB is endogenously present in RRL. However, the BVDV IRES can bind PTB weakly under certain conditions. Interestingly, in a functional depletion and add-back translation system, PTB does not enhance translation of BVDV, although PTB enhances translation of a picornavirus in this translation stimulation assay. These results indicate that PTB can bind the BVDV IRES RNA, but translation is independent of the action of PTB.

Animals↗

PubMed2000

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus.

Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.

5' Untranslated Regions↗

PubMed2005

Suppression of hepatitis A virus genome translation and replication by siRNAs targeting the internal ribosomal entry site.

Small interfering RNAs (siRNAs) targeting the coding region of hepatitis A virus (HAV) were shown to specifically inhibit viral genome replication. Compared to the coding region, the HAV internal ribosomal entry site (IRES) in the 5' non-coding region is highly sequence-conserved and folds into stable secondary structures. Here, we report efficient and sustained RNA interference mediated by both RNase III-prepared siRNA (esiRNA) and vector-derived short hairpin RNAs (shRNAs) that are targeted to various domains of the HAV IRES. Using reporter constructs, and the DNA-based HAV replicon system, we found that shRNAs targeting the HAV IRES domains IIIc and V sustainably suppressed genome translation and replication whereas the IRES domains IIIa and IV were resistant to RNA interference. Our study suggests that some HAV IRES domains might be used as a universal and effective target for specific inhibition of HAV infection.

Base Sequence↗

PubMed1992

Virus infection blocks the processing and presentation of exogenous antigen with the major histocompatibility complex class II molecules.

Helper T cell recognition of antigen requires that antigen be processed and presented by class II expressing antigen-presenting cells (APC). Many antigens presented by the immune system are part of infectious organisms, for example, bacteria and viruses, which themselves may affect APC function. Here we show that infection of B cell lines as APC with viruses of two different families, namely, influenza A or vaccinia, completely block processing and presentation of an exogenous globular protein antigen pigeon cytochrome c. The block appears to be primarily within the processing pathway, as virus infection has little effect on the presentation of an antigenic peptide of pigeon cytochrome c which does not require processing. It is likely that several steps in the processing pathway are affected. Only live infectious virus, not UV-inactivated virus blocks APC function, indicating that there is no competition of viral particles with cytochrome c for the class II processing machinery. As compared to uninfected cells, virus-infected cells internalize less antigen bound to surface Ig but degrade a similar portion of that which enters the cell. Virus infection results in reduced protein synthesis in APC which may also be a factor in decreasing APC function. Significantly, we show that the processing of a high affinity evolutionary variant of cytochrome c from Drosophila melanogaster is reduced less by virus infection as compared to c. Such knowledge may guide the selection of antigenic epitopes in vaccine design.

Animals↗

PubMedDate not supplied

Isolation of avian influenza virus (subtype H5N2) from chicken eggs during a natural outbreak.

Avian influenza virus (A/Chicken/Pennsylvania/83; H5N2) was recovered from the yolk, albumen, and shell surface of eggs obtained from naturally infected chicken flocks in Pennsylvania and Virginia. These findings represent the first reported isolation of avian influenza virus from the internal contents of eggs from naturally infected flocks. The need for adequate safeguards to prevent spread of the virus during commercial movement of table and hatching eggs, cracked and "checked" eggs, and egg flats and other materials is emphasized.

Animals↗

PubMed2006

Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-PCR with lyophilized reagents.

We developed an internal positive control (IPC) RNA to help ensure the accuracy of the detection of avian influenza virus (AIV) RNA by reverse transcription (RT)-PCR and real-time RT-PCR (RRT-PCR). The IPC was designed to have the same binding sites for the forward and reverse primers of the AIV matrix gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the viral RNA and the IPC by RRT-PCR were monitored with two different fluorescent probes in a multiplex format, one specific for the AIV matrix gene and the other for the IPC. The RRT-PCR test was further simplified with the use of lyophilized bead reagents for the detection of AIV RNA. The RRT-PCR with the bead reagents was more sensitive than the conventional wet reagents for the detection of AIV RNA. The IPC-based RRT-PCR detected inhibitors in blood, kidney, lungs, spleen, intestine, and cloacal swabs, but not allantoic fluid, serum, or tracheal swabs The accuracy of RRT-PCR test results with the lyophilized beads was tested on cloacal and tracheal swabs from experimental birds inoculated with AIV and compared with virus isolation (VI) on embryonating chicken eggs. There was 97 to 100% agreement of the RRT-PCR test results with VI for tracheal swabs and 81% agreement with VI for cloacal swabs, indicating a high level of accuracy of the RRT-PCR assay. The same IPC in the form of armored RNA was also used to monitor the extraction of viral RNA and subsequent detection by RRT-PCR.

Animals↗

PubMed1988

Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus.

The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment. Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase. After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M2 requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.

Animals↗

PubMed1988

Do polyclonal rheumatoid factors carry an 'internal image' of cytomegalovirus, Epstein-Barr virus and nuclear antigens?

Rabbit antisera have been prepared against isolated rheumatoid factors (RF's). It was considered that RFs are anti-idiotype antibodies and that the anti-RF antisera had anti-anti-idiotype specificity. Furthermore, it was considered that the RF's carry an "internal image" of the putative "antigen X" and that the rabbit antisera would have specificity for this "antigen-X". Reactions of the rabbit antisera with the early and late antigens of CMV, EBV antigens and nuclear antigens suggest that there may be an "internal image" of these antigens in rheumatoid factor molecules and that they all may be related to the immunogenesis of RF.

Animals↗

PubMed1992

Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase.

We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA. A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptors. In this system, HIV RT produced strand transfer products. An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently. An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer. HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E. coli enzyme. Evidently, RNase H activity enhances, or is required for, internal strand transfer. Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested. The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor. Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer. When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 microM, increasing RT pausing, the efficiency of strand transfer also increased. A model for RT-catalyzed strand transfer consistent with our results is presented.

Base Sequence↗

PubMed1995

An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region.

Translation of the human hepatitis C virus (HCV) RNA genome occurs by a mechanism known as "internal ribosome entry." This unusual strategy of translation is employed by naturally uncapped picornaviral genomic RNAs and several cellular mRNAs. A common feature of these RNAs is a relatively long 5' noncoding region (NCR) that folds into a complex secondary structure harboring an internal ribosome entry site (IRES). Evidence derived from the use of dicistronic expression systems, combined with an extensive mutational analysis, demonstrated the presence of an IRES within the HCV 5'NCR. The results of our continued mutational analysis to map the critical structural elements of the HCV IRES has led to the identification of a pseudoknot structure upstream of the initiator AUG. The evidence presented in this study is based upon the mutational analysis of the putative pseudoknot structure. This is further substantiated by biochemical and enzymatic probing of the wild-type and mutant 5'NCR. Further, the thermodynamic calculations, based upon a modified RNAKNOT program, are consistent with the presence of a pseudoknot structure located upstream of the initiator AUG. Maintenance of this structural element is critical for internal initiation of translation. The pseudoknot structure in the 5'NCR represents a highly conserved feature of all HCV subtypes and members of the pestivirus family, including hog cholera virus and bovine viral diarrhea virus.

Base Sequence↗

PubMed2002

Interferons specifically suppress the translation from the internal ribosome entry site of hepatitis C virus through a double-stranded RNA-activated protein kinase-independent pathway.

Interferon (IFN) therapy is used worldwide as the best available treatment for hepatitis C virus (HCV) infection; however, little is known about how IFN or other drugs work against liver diseases. The effect of 6 drugs (glycyrrhizin, ursodeoxycholic acid, ribavirin, methylprednisolone, IFN-alpha, and IFN-beta) on HCV RNA translation from the HCV internal ribosome entry site (IRES) was investigated, using a bicistronic reporter containing the HCV IRES. IFNs suppressed both cap-dependent and HCV IRES-dependent translation, with HCV IRES-dependent translation being more significantly suppressed. In contrast to HCV IRES, IFN did not suppress either foot-and-mouth disease virus IRES-dependent or encephalomyocarditis virus IRES-dependent translation more than it suppressed cap-dependent translation. Moreover, dominant inhibition of HCV IRES-dependent over cap-dependent translation depended neither on the double-stranded RNA-activated protein kinase activation nor on La protein function. These results indicate a novel antiviral effect of IFNs against HCV.

Anti-Infective Agents↗

PubMed2001

Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning.

The mutant spectrum of a virus quasispecies in the process of fitness gain of a debilitated foot-and-mouth disease virus (FMDV) clone has been analysed. The mutant spectrum was characterized by nucleotide sequencing of three virus genomic regions (internal ribosome entry site; region between the two AUG initiation codons; VP1-coding region) from 70 biological clones (virus from individual plaques formed on BHK-21 cell monolayers) and 70 molecular clones (RT--PCR products cloned in E. coli). The biological and molecular clones provided statistically indistinguishable definitions of the mutant spectrum with regard to the distribution of mutations among the three genomic regions analysed and with regard to the types of mutations, mutational hot-spots and mutation frequencies. Therefore, the molecular cloning procedure employed provides a simple protocol for the characterization of mutant spectra of viruses that do not grow in cell culture. The number of mutations found repeated among the clones analysed was higher than expected from the mean mutation frequencies. Some components of the mutant spectrum reflected genomes that were dominant in the prior evolutionary history of the virus (previous passages), confirming the presence of memory genomes in virus quasispecies. Other components of the mutant spectrum were genomes that became dominant at a later stage of evolution, suggesting a predictive value of mutant spectrum analysis with regard to the outcome of virus evolution. The results underline the observation that greater insight into evolutionary processes of viruses may be gained from detailed clonal analyses of the mutant swarms at the sequence level.

Adaptation, Physiological↗

PubMed2006

Viral interactions with the cytoskeleton: a hitchhiker's guide to the cell.

The actin and microtubule cytoskeleton play important roles in the life cycle of every virus. During attachment, internalization, endocytosis, nuclear targeting, transcription, replication, transport of progeny subviral particles, assembly, exocytosis, or cell-to-cell spread, viruses make use of different cellular cues and signals to enlist the cytoskeleton for their mission. Viruses induce rearrangements of cytoskeletal filaments so that they can utilize them as tracks or shove them aside when they represent barriers. Viral particles recruit molecular motors in order to hitchhike rides to different subcellular sites which provide the proper molecular environment for uncoating, replicating and packaging viral genomes. Interactions between subviral components and cytoskeletal tracks also help to orchestrate virus assembly, release and efficient cell-to-cell spread. There is probably not a single virus that does not use cytoskeletal and motor functions in its life cycle. Being well informed intracellular passengers, viruses provide us with unique tools to decipher how a particular cargo recruits one or several motors, how these are activated or tuned down depending on transport needs, and how cargoes switch from actin tracks to microtubules to nuclear pores and back.

Animals↗

PubMed2003

Generation and characterization of an H9N2 cold-adapted reassortant as a vaccine candidate.

H9N2 subtype avian influenza viruses have been identified in avian species worldwide, and infections in pigs were confirmed in Hong Kong in 1998. Subsequently, H9N2 viruses were isolated from two children in Hong Kong in 1999, and five human infections were reported from China, raising the possibility that H9N2 viruses pose a potential pandemic threat for humans. These events prompted us to develop a vaccine candidate to protect humans against this subtype of influenza A viruses. Reassortant H1N1 and H3N2 human influenza A viruses with the six internal gene segments of A/Ann Arbor/6/60 (H2N2)(AA) cold-adapted (ca) virus have been tested extensively in humans and have proved to be attenuated and safe as live virus vaccines. Using classical genetic reassortment, we generated a reassortant that contains the hemagglutinin and neuraminidase genes from A/chicken/Hong Kong/G9/97 (H9N2) and six internal gene segments from the AAca virus. The G9/AAca reassortant virus exhibits the ca phenotype and the temperature-sensitive phenotypes of the AAca virus and was attenuated in mice. The reassortant virus was immunogenic and protected mice from wild-type H9N2 virus challenge. The G9/AAca virus bears the in vitro and in vivo phenotypes specified by the AAca virus and will be evaluated as a potential vaccine candidate in humans.

Acclimatization↗

PubMed1991

Interaction of a cellular 57-kilodalton protein with the internal translation initiation site of foot-and-mouth disease virus.

A cellular 57-kDa protein (p57) that binds specifically to the internal translation initiation site in the 5' untranslated region of foot-and-mouth disease virus RNA was detected in cell extracts of different mammalian species by UV cross-linking. The protein binds to two distinct sites of the translation control region which have as the only common sequence a UUUC motif. The first binding site consists of a conserved hairpin structure, whereas the second binding site contains an essential pyrimidine-rich region without obvious secondary structure. Competition experiments indicate that the complexes with the two binding sites were formed by a single p57 species. The protein binds also to the 5' untranslated region of other picornaviruses. Results from footprint analyses with foot-and-mouth disease RNA suggest the participation of additional cellular factors in the translation initiation complex.

Animals↗