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At least 1,045 records · Page 58Linked to original sources
PubMed1998

Coding sequences enhance internal initiation of translation by hepatitis A virus RNA in vitro.

Hepatitis A virus (HAV), unlike other picornaviruses, has a slow-growth phenotype in permissive cell lines and in general does not induce host cell cytopathology. Although there are no published reports of productive infection of HeLa cells by HAV, HAV RNA appears to be readily translated in HeLa cells when transcribed by T7 RNA polymerase provided by a recombinant vaccinia virus. The 5' noncoding region of HAV was fused to poliovirus (PV) coding sequences to determine the effect on translation efficiency in HeLa cell extracts in vitro. Conditions were optimized for utilization of the HAV internal ribosome entry segment (IRES). Transcripts from chimeric constructs fused precisely at the initiation codon were translated very poorly. However, chimeric RNAs which included 114 or more nucleotides from the HAV capsid coding sequences downstream of the initiation codon were translated much more efficiently than those lacking these sequences, making HAV-directed translation efficiency similar to that directed by the PV IRES. Sixty-six nucleotides were insufficient to confer increased translation efficiency. The most 5'-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an inhibitory effect on translation efficiency. Constructs lacking these terminal sequences, or those in which the PV 5'-terminal sequences replaced those from HAV, translated three- to fourfold better than those with the intact HAV 5'-terminal end.

Cell Extracts↗
PubMed1992

Cleavage of concatemeric DNA at the internal junction of "translocation" mutants of pseudorabies virus and inversion of their L component appear to be linked.

When pseudorabies virus (PrV) strains are grown in chicken embryo fibroblasts (CEF), variants ("translocation" mutants) arise in which there is a duplication of the leftmost sequences of the genome and their translocation in inverted orientation next to the internal inverted repeat bracketing the S component. In these variants, the UL becomes bracketed by inverted repeats and is found in two orientations relative to the Us. To study the cis-functions involved in cleavage of concatemeric DNA as well as those involved in inversion of the L component and to ascertain whether the two events are linked in the "translocation" mutants, a viral mutant (vLD68) was constructed in which the terminal 64 bp of the L component (that include sequences with homology to the pac 2 site of HSV) and the 4 terminal bp of the S component were deleted from the internal junction. Although revertants that have acquired the 68 bp at the internal junction emerge rapidly in populations of vLD68, analysis of the characteristics of this mutant revealed that: (1) the termini derived from both orientations of the L component include the 64 bp that have been deleted from the internal junction of vLD68; (2) in contrast to other "translocation" mutants, the internal junction of the vLD68 genome is not a good substrate for cleavage; (3) inversion of the L component of true vLD68 DNA does not occur or is rare; a good correlation exists in the populations of vLD68 between the proportion of revertants that have acquired an intact internal junction and the proportion of genomes with an L component that inverts. These results show that an intact internal junction in "translocation" mutants is necessary for both inversion of their L components and cleavage at their alternative internal junction. Since cleavage at the alternative junction will result in inversion of the L component, we conclude that inversion of the L component of "translocation" mutants of PrV can be attributed to cleavage of concatemeric DNA at the internal alternative junction.

Animals↗
PubMed2004

Use of an internal positive control in a multiplex reverse transcription-PCR to detect West Nile virus RNA in mosquito pools.

We report on the use of West Nile virus Armored RNA as an internal positive control (IPC) for the extraction and reverse transcription-PCR (RT-PCR) of RNA extracted from field-collected mosquitoes and on a multiplex real-time Taqman RT-PCR to simultaneously detect the 3' noncoding region of West Nile virus and the West Nile virus NS5-2 region comprising the IPC. Mosquito pools from the province of British Columbia, Canada (n = 635), were tested in duplicate and found to be negative for West Nile virus and positive for the IPC. Known West Nile virus-positive supernatants from mosquito pools from the provinces of Alberta and Manitoba were tested in duplicate and found to be positive for both regions of the West Nile virus genome. The mean cycle threshold (Ct) value for the IPC in batch extraction controls +/- 2 standard deviations was found to be 36.43 +/- 1.78 cycles. IPCs of 98.4% (624) of West Nile virus-negative pools fell within this range, indicating the reproducibility of RNA extraction and RT-PCR for pools varying in mosquito genus and number. A comparison of mosquito pool genera revealed no significant genus effect on the Ct value of the IPC. The incorporation of West Nile virus Armored RNA as an IPC allows monitoring of RNA extraction and RT-PCR and detection of false-negative results due to failures in these processes or to PCR inhibition, respectively.

Animals↗
PubMed2004

Lack of clinical significance of variability in the internal ribosome entry site of hepatitis C virus.

The extreme 5'-proximal sequence of the hepatitis C virus (HCV) genome including the 5' non-coding region (5'NCR) of 341 nucleotide long and the first 30 nucleotides of the core region is highly conserved among different HCV genotypes. It contains a segment termed Internal Ribosome Entry Site (IRES) that regulates the cap-independent translation of HCV-RNA to polyprotein. Sequence variability in this region has important implications for structural organisation and function of the IRES element and could correlate with HCV RNA concentration or response to antiviral therapy. Fourteen patients (seven women, seven men) with chronic hepatitis C were separated into two groups according to their response to antiviral therapy. Seven of these were sustained responders to treatment by Interferon alpha 2b and Ribavirin and seven were non-responders. After cloning-sequencing, the IRES (nt 21 to 374) appears to be structurally highly conserved. However some variability was found between the different isolates obtained: 209 substitutions with a median of four variants/patients. Comparison of the number of variants present in the viral population of the sustained responders and non-responders patients do not show any difference. Positioning of the mutations on the predicted IRES secondary structure showed that the distribution of the mutations and their apparition frequency were different between the two groups. The translation initiator AUG-4 codon, located in the stem-loop IV, is never modified. Variations observed in the IRES are not a parameter of response to antiviral therapy, but the integrity of this region is a necessary condition to maintain its activity.

5' Untranslated Regions↗
PubMed1987

The atomic structure of Mengo virus at 3.0 A resolution.

The structure of Mengo virus, a representative member of the cardio picornaviruses, is substantially different from the structures of rhino- and polioviruses. The structure of Mengo virus was solved with the use of human rhinovirus 14 as an 8 A resolution structural approximation. Phase information was then extended to 3 A resolution by use of the icosahedral symmetry. This procedure gives promise that many other virus structures also can be determined without the use of the isomorphous replacement technique. Although the organization of the major capsid proteins VP1, VP2, and VP3 of Mengo virus is essentially the same as in rhino- and polioviruses, large insertions and deletions, mostly in VP1, radically alter the surface features. In particular, the putative receptor binding "canyon" of human rhinovirus 14 becomes a deep "pit" in Mengo virus because of polypeptide insertions in VP1 that fill part of the canyon. The minor capsid peptide, VP4, is completely internal in Mengo virus, but its association with the other capsid proteins is substantially different from that in rhino- or poliovirus. However, its carboxyl terminus is located at a position similar to that in human rhinovirus 14 and poliovirus, suggesting the same autocatalytic cleavage of VP0 to VP4 and VP2 takes place during assembly in all these picornaviruses.

Antigens, Viral↗
PubMed1995

Internal controls as performance monitors and quantitative standards in the detection by polymerase chain reaction of herpes simplex virus and cytomegalovirus in clinical specimens.

Internal controls (IC) were produced and characterized for an HSV and a CMV PCR assay which serve both as test performance monitors and as quantitative standards. In each of the PCR assays the IC and native targets were amplified with equal efficiency and were detected with the same sensitivity, i.e. < 10 target copies. An algorithm was developed for the use of IC as a quantitative standard which entailed coamplifying a test specimen with four two-fold dilutions of the respective IC target (63-500 copies), followed by regression analysis of the relative yield of amplification products. This approach allowed the determination of both the initial virus genome copy number and the variability of the results, which provided a confidence index for the PCR assay. The relative yields of PCR products were determined by Southern blot and probe hybridization and by densitometry of digitized ethidium bromide-stained gels. Both methods produced estimations of the initial target copy numbers within +/- 40% of the expected value. Such a comprehensive analysis of an internal control for a PCR assay provides a rigorous control of test performance and permits reliable quantitative interpretation of a PCR assay result.

AIDS-Related Opportunistic Infections↗
PubMed1992

Sequence determination and genetic content of an 8.9-kb restriction fragment in the short unique region and the internal inverted repeat of Marek's disease virus type 1 DNA.

The DNA sequence (8.9 kb) covering about 70% of the short unique region (Us) and part of the short inverted repeat of the Marek's disease virus type 1 GA strain was determined. Computer analysis of the sequence showed the presence of nine potential open reading frames (ORFs), consisting of more than 300 nucleotides in the Us region. Of these ORFs, four were found to be homologous to US10 (minor virion protein), US3 (protein kinase), US2, and US6 (gD) in the Us region of alpha-herpesvirus herpes simplex virus type 1. The protein kinase homologue is especially well conserved in alpha-herpesviruses. No counterpart of the nine MDV1 ORFs was found in the beta-herpes virus human cytomegalovirus and gamma-herpesvirus Epstein-Barr virus, suggesting that MDV1 is more similar to the alpha-herpesviruses. The junction of the Us region and the short inverted repeat was also determined by comparison between the sequences of the DNA fragments, including the terminal and internal repeats. Northern blot analysis showed that the Us region within the 8.9 kb sequence was transcriptionally active in MDV1-infected cells.

Amino Acid Sequence↗
PubMed1998

A functional antigenomic promoter for the paramyxovirus simian virus 5 requires proper spacing between an essential internal segment and the 3' terminus.

A previous analysis of naturally occurring defective interfering (DI) RNA genomes of the prototypic paramyxovirus simian virus 5 (SV5) indicated that 113 bases at the 3' terminus of the antigenome were sufficient to direct RNA encapsidation and replication. A nucleotide sequence alignment of the antigenomic 3'-terminal 113 bases of members of the Rubulavirus genus of the Paramyxoviridae family identified two regions of sequence identity: bases 1 to 19 at the 3' terminus (conserved region I [CRI]) and a more distal region consisting of antigenome bases 73 to 90 (CRII) that was contained within the 3' coding region of the L protein gene. To determine whether these regions of the antigenome were essential for SV5 RNA replication, a reverse genetics system was used to analyze the replication of copyback DI RNA analogs that contained a foreign gene (GL, encoding green fluorescence protein) flanked by 113 5'-terminal bases and various amounts of SV5 3'-terminal antigenomic sequences. Results from a deletion analysis showed that efficient encapsidation and replication of SV5-GL DI RNA analogs occurred when the 90 3'-terminal bases of the SV5 antigenomic RNA were retained, but replication was reduced approximately 5- to 14-fold in the case of truncated antigenomes that lacked the 3'-end CRII sequences. A chimeric copyback DI RNA containing the 3'-terminal 98 bases including the CRI and CRII sequences from the human parainfluenza virus type 2 (HPIV2) antigenome in place of the corresponding SV5 sequences was efficiently replicated by SV5 cDNA-derived components. However, replication was reduced approximately 20-fold for a truncated SV5-HPIV2 chimeric RNA that lacked the HPIV2 CRII sequences between antigenome bases 72 and 90. Progressive deletions of 6 to 18 bases in the region located between the SV5 antigenomic CRI and CRII segments (3'-end nucleotides 21 to 38) resulted in a approximately 25-fold decrease in SV5-GL RNA synthesis. Surprisingly, replication was restored to wild-type levels when these length alterations between CRI and CRII were corrected by replacing the deleted bases with nonviral sequences. Together, these data suggest that a functional SV5 antigenomic promoter requires proper spacing between an essential internal region and the 3' terminus. A model is presented for the structure of the 3' end of the SV5 antigenome which proposes that positioning of CRI and CRII along the same face of the helical nucleocapsid is an essential feature of a functional antigenomic promoter.

Animals↗
PubMed1994

Cell tropism of influenza virus mediated by hemagglutinin activation at the stage of virus entry.

When influenza virus A/WSN/33 (H1N1) was grown in MDBK or CV-1 cells in serum-free medium, progeny virus released from these cells contained only uncleaved hemagglutinin (HA). This virus was unable to infect CV-1 cells unless subjected to cleavage activation by trypsin, but it was infectious for MDBK cells without such treatment. Temperature shift experiments demonstrated that HA of radiolabeled input virus was cleaved within 30 min after inoculation of MDBK cells. As indicated by its resistance to trypsin cleavage, the virus was already internalized at this stage. Cleavage was not observed after inoculation of CV-1 cells. The serine-protease inhibitor leupeptin blocked virus growth when added to MDBK cells during the initial phase of the replication cycle. The inhibitor did not show this effect, when the input virus contained already cleaved HA. These results demonstrate that activation of HA of the WSN strain can occur in MDBK cells, but not in CV-1 cells, at the stage of virus entry, presumably by an endosomal protease.

Cells, Cultured↗
PubMed1993

An improved method for the detection of hepatitis C virus RNA in plasma utilizing heminested primers and internal control RNA.

The majority of transfusion-associated, non-A, non-B hepatitis cases are caused by hepatitis C virus (HCV), a positive-stranded RNA virus. Although high titers of HCV in clinical specimens have been reported, in some cases extremely low titers of virus are not uncommon. Therefore, an extremely sensitive and reliable assay is required to determine viremia and replication of HCV accurately. We report here the systematic investigation of factors influencing the detection of HCV RNA by a reverse transcription-polymerase chain reaction (RT-PCR) assay utilizing "drop in-drop out" heminested primers derived from the conserved 5' non-coding region of the viral genome. A genetically engineered 5' noncoding region has been constructed and used as an internal control. Addition of the control RNA to each test not only allowed semiquantitation of positive reactions but also validated the performance of reverse transcription and PCR for every specimen. The optimized heminested PCR (HN-PCR) protocol is capable of amplifying one molecule of cloned HCV DNA or 10 molecules of in vitro-transcribed HCV RNA to levels detectable in ethidium bromide-stained agarose gels. We evaluated the improved method for the detection of HCV RNA on a human plasma sample containing the pedigreed strain H of HCV with a chimpanzee infectious dose of 10(6)/ml. Utilizing the internal control RNA, we calculated 2 x 10(7) virions in 1 ml of the original human plasma. The HN-PCR achieves the sensitivity and specificity of the double-nested PCR (DN-PCR) in a simplified format that avoids the false-positive results associated with DN-PCR.

Base Sequence↗
PubMed2005

Functional display of an alpha2 integrin-specific motif (RKK) on the surface of baculovirus particles.

The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an alpha2 integrin, the alpha2I-domain. However, the interaction was not strong enough to overcome binding of wild type gp64 to the unknown cellular receptor(s) on the surface of alpha2 integrin-expressing cells (CHO-alpha2beta1) or enhance the viral uptake. After treatment of these cells with phospholipase C, internalization of all viruses was blocked or decreased significantly. However, one of the RKK displaying viruses, AcGFP(K)gp64, was still able to internalize into CHO-alpha2beta1 cells, although at a lower level as compared to non-treated cells. This may indicate the possible utilization of a PLC independent alternative route via, in this case, the alpha2beta1 integrin.

Amino Acid Motifs↗
PubMed1979

Structural elements in adenovirus cores. Evidence for a "core shell" and linear structures in "relaxed" cores.

"Freeze-fracture negative staining" of adenovirus type 5 revealed the virus cores as internal bodies with a fine granular surface which at high magnification shows reticular and ring-like patterns. This indicates that the virus protein V forms a thin surface layer for which a name "core shell" is proposed. Virus cores prepared by heating virus particles in sodium deoxycholate (DOC) relaxed into curved filaments or several rods by means of EGTA and high salt, respectively. High pH treatment had similar effects as high salt. Rod-like elements were also observed in ultrathin sections of the "DOC-cores". Fresh cores exhibited circular dichroism (C.D.) with similar features as described for nucleosomes (COWMAN and FASMAN, ref. 5). This indicates that the DNA has an orderly arrangement in the cores. EGTA had no effect on C.D. spectra but high salt treatment abolished the positive peak in 10 minutes. It is concluded that the adenovirus nucleocapsid is a linear structure, presumably a 120-150 Angstrom thick filament folded 5 to 6 times into "rods" as previously observed by freeze-fracturing.

Adenoviruses, Human↗
PubMed1984

Structure and origin of a novel class of defective interfering particle of vesicular stomatitis virus.

The genome structure and terminal sequences of a 'copyback' defective interfering (DI) particle ST1, and a novel complexly rearranged 'snapback' DI particle ST2 of vesicular stomatitis virus have been determined. The ST1 DI genome RNA possesses 54 base long inverted complementary termini, the 5' end of which is homologous to the standard virus genome 5' end. Following this region of inverted complementarity the DI RNA 5' end continues to be homologous to standard virus RNA 5' sequences, whereas the 3' end diverges into sequences within the virus L gene internal sequences. ST2 DI genome RNA does not contain colinear covalently linked plus and minus sense RNA copies of the standard infectious virus RNA 5' terminus as predicted from the prototype snapback DI structure, but instead appears to be a hairpin copy of the ST1 DI RNA genome. This is the first evidence suggesting that DI particles may be generated from RNA templates other than the standard virus RNA. Generation models and the implications of these findings for RNA virus evolution are discussed.

Base Composition↗
PubMed1972

Group-specific antigens of RNA tumor viruses as markers for subinfectious expression of the RNA virus genome.

Antigenic markers associated with the major internal protein of RNA tumor viruses of the C-type have proven extremely useful in natural history studies of these viruses. This protein possesses species-specific antigenic determinants, and, in the case of mammalian C-type viruses, the protein possesses crossreactive determinants as well. These determinants are, thus, useful for species identification and classification of mammalian viruses. A unique distribution of antigens in embryonic tissues of several species (where tests are available) was detected, and in addition, antigen expression in tissues appears to be controlled by a dominant gene. These data have contributed greatly to the theory that RNA tumor-virus information is inherited as part of the cellular genome.

Animals↗
PubMed2026

Programmatic access to ICTV virus taxonomy through a public ontology API.

The International Committee on Taxonomy of Viruses (ICTV) is responsible for developing and maintaining a universal virus taxonomy. As the reference framework for organising the viral world, it is essential for virology and related fields. Despite its widespread use in research and public health, programmatic access to ICTV taxonomy has remained limited, posing challenges for integration, versioning, and interoperability across databases and bioinformatics resources requiring up-to-date virus taxonomy. To address this, we developed a public and sustainable solution leveraging ontology-based APIs. Successive ICTV Master Species List (MSL) releases were transformed into a structured ontology and deployed as a unified representation through the Ontology Lookup Service (OLS). The framework also provides ICTV-NCBI mappings and helper libraries for integration into downstream systems. This enables, for the first time, public programmatic retrieval of current and historical virological taxon names, taxonomic relationships, metadata, and persistent identifiers through stable endpoints. More broadly, this work illustrates a general strategy for transforming structured biological datasets into semantically enriched graph resources exposed through scalable public APIs. These developments enhance interoperability, reduce manual curation, and support FAIR-aligned taxonomic data management in virology and pandemic preparedness.

API↗
PubMed2003

Herpes simplex virion entry into and intracellular transport within mammalian cells.

Alphaherpesviruses, membrane-enveloped DNA viruses that are responsible for a host of human ailments, bind to, enter and are directly targeted to specific intracellular domains within their mammalian host cells. This review emphasizes recent work on the best studied of the alphaherpesviruses, Herpes simplex virus type 1 (HSV1). One area of focus is on recent work that has identified viral glycoproteins that are important in binding and internalization of the virus to the host cell. Complementary work on the receptors for those viral glycoproteins that reside on the host cell surface is also presented, with some discussion of how receptor variety might lead to the tissue tropism demonstrated by alphaherpes viruses. An additional area of focus in this review is how HSV uses the host cell transport systems to achieve intracellular targeting of the incoming virion toward the cell nucleus, and, after production of newly synthesized and assembled viral progeny, targeting them toward the plasma membrane for release.

Animals↗
PubMed2005

Novel compound enables high-level adenovirus transduction in the absence of an adenovirus-specific receptor.

Viral vectors are extensively used to deliver foreign DNA to cells for applications ranging from basic research to potential clinical therapies. A limiting step in this process is virus uptake and internalization into the target cells, which is mediated by membrane receptors. Although it is possible to modify viral capsid proteins to target the viruses, such procedures are complex and often unsuccessful. Here we present a rapid, inexpensive system for improving transduction of cells, including those that lack receptors for adenovirus fiber proteins. Addition of GeneJammer (Stratagene, La Jolla, CA) during the adenovirus transduction led to a significant increase in both the total number of transduced cells and the level of transgene expression per cell. Studies using cell lines deficient in adenovirus receptors demonstrated that addition of GeneJammer provided a novel cellular entry mechanism for the virus. These findings were tested in a cell-based gene therapy system for the induction of bone, which is contingent on high-level expression of the transgene. Inclusion of GeneJammer in either Ad5BMP2 or Ad5F35BMP2 transduction of a variety of cells demonstrated a correlating increase in bone formation. The results suggest a novel and versatile method for achieving high-level transduction using adenovirus.

Adenoviridae↗