bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exploitation of the Low Fidelity of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase and the Nucleotide Composition Bias in the HIV-1 Genome To Alter the Drug Resistance Development of HIV
Authors: Balzarini Jan , Camarasa Maria-José , Pérez-Pérez Maria-Jesus , San-Félix Ana , Velázquez Sonsoles , Perno Carlo-Federico , De Clercq Erik , Anderson John N. , Karlsson Anna 
Published: July 2002

ABSTRACTThe RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G→A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G→A) to an adenine-to-guanine (A→G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development.
Genetic Retargeting of Adenovirus: Novel Strategy Employing “Deknobbing” of the Fiber
Authors: Magnusson Maria K. , Hong Saw See , Boulanger Pierre , Lindholm Leif 
Published: July 2002

ABSTRACT
For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.
Human Parainfluenza Virus Type 3 HN-Receptor Interaction: Effect of 4-Guanidino-Neu5Ac2en on a Neuraminidase-Deficient Variant
Authors: Porotto Matteo , Greengard Olga , Poltoratskaia Natalia , Horga Maria-Arantxa , Moscona Anne 
Published: July 2002

ABSTRACT
The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4°C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37°C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.
Phylogenetic Analysis of Enterovirus 71 Strains Isolated during Linked Epidemics in Malaysia, Singapore, and Western Australia
Authors: McMinn Peter , Lindsay Katie , Perera David , Chan Hung Ming , Chan Kwai Peng , Cardosa Mary Jane 
Published: July 2002

ABSTRACT
Enterovirus 71 (EV71) is a frequent cause of hand, foot, and mouth disease (HFMD) epidemics associated with severe neurological sequelae in a small proportion of cases. There has been a significant increase in EV71 epidemic activity throughout the Asia-Pacific region since 1997. Recent HFMD epidemics in this region have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case fatality rates. In this study, we show that four genetic lineages of EV71 have been prevalent in the Asia-Pacific region since 1997, including two previously undescribed genogroups (B3 and B4). Furthermore, we show that viruses belonging to genogroups B3 and B4 have circulated endemically in Southeast Asia during this period and have been the primary cause of several large HFMD or encephalitis epidemics in Malaysia, Singapore, and Western Australia.
Autonomous Role of 3′-Terminal CCCA in Directing Transcription of RNAs by Qβ Replicase
Authors: Tretheway David M. , Yoshinari Shigeo , Dreher Theo W. 
Published: July 2002

ABSTRACT

We have studied transcription in vitro by Qβ replicase to deduce the minimal features needed for efficient end-to-end copying of an RNA template. Our studies have used templates ca. 30 nucleotides long that are expected to be free of secondary structure, permitting unambiguous analysis of the role of template sequence in directing transcription. A 3′-terminal CC
C
A (3′-CC
C
A) directs transcriptional initiation to opposite the underlined C; the amount of transcription is comparable between RNAs possessing upstream (CCA)

n

tracts, A-rich sequences, or a highly folded domain and is also comparable in single-round transcription assays to transcription of two amplifiable RNAs. Predominant initiation occurs within the 3′-CCCA initiation box when a wide variety of sequences is present immediately upstream, but CCA or a closely similar sequence in that position results in significant internal initiation. Removal of the 3′-A from the 3′-CCCA results in 5- to 10-fold-lower transcription, emphasizing the importance of the nontemplated addition of 3′-A by Qβ replicase during termination. In considering whether 3′-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that tRNA
His
is the only stable
Escherichia coli
RNA with 3′-CCCA. In vitro-generated transcripts corresponding to tRNA
His
served as poor templates for Qβ replicase; this was shown to be due to the inaccessibility of the partially base-paired CCCA. These studies demonstrate that 3′-CCCA plays a major role in the control of transcription by Qβ replicase and that the abundant RNAs present in the host cell should not be efficient templates.
Altered Cellular mRNA Levels in Human Cytomegalovirus-Infected Fibroblasts: Viral Block to the Accumulation of Antiviral mRNAs
Authors: Browne Edward P. , Wing Bret , Coleman David , Shenk Thomas 
Published: July 2002

ABSTRACTThe effect of human cytomegalovirus (HCMV) infection on cellular mRNA accumulation was analyzed by gene chip technology. During a 48-h time course after infection of human diploid fibroblasts, 1,425 cellular mRNAs were found to be up-regulated or down-regulated by threefold or greater in at least two consecutive time points. Several classes of genes were prominently affected, including interferon response genes, cell cycle regulators, apoptosis regulators, inflammatory pathway genes, and immune regulators. The number of mRNAs that were up-regulated or down-regulated were roughly equal over the complete time course. However, for the first 8 h after infection, the number of up-regulated mRNAs was significantly less than the number of down-regulated mRNAs. By analyzing the mRNA expression profile of cells infected in the presence of cycloheximide, it was found that a minimum of 25 mRNAs were modulated by HCMV in the absence of protein synthesis. These included mRNAs encoded by a small number of interferon-responsive genes, as well as beta interferon itself. Cellular mRNA levels in cytomegalovirus-infected cells were compared to the levels in cells infected with UV-inactivated virus. The inactivated virus caused the up-regulation of a much greater number of mRNAs, many of which encoded proteins with antiviral roles, such as interferon-responsive genes and proinflammatory cytokines. These data argue that one or more newly synthesized viral gene products block the induction of antiviral pathways that are triggered by HCMV binding and entry.
Polypeptide p41 of a Norwalk-Like Virus Is a Nucleic Acid-Independent Nucleoside Triphosphatase
Authors: Pfister Thomas , Wimmer Eckard 
Published: July 2002

ABSTRACT

Southampton virus (SHV) is a member of the Norwalk-like viruses (NLVs), one of four genera of the family
Caliciviridae
. The genome of SHV contains three open reading frames (ORFs). ORF 1 encodes a polyprotein that is autocatalytically processed into six proteins, one of which is p41. p41 shares sequence motifs with protein 2C of picornaviruses and superfamily 3 helicases. We have expressed p41 of SHV in bacteria. Purified p41 exhibited nucleoside triphosphate (NTP)-binding and NTP hydrolysis activities. The NTPase activity was not stimulated by single-stranded nucleic acids. SHV p41 had no detectable helicase activity. Protein sequence comparison between the consensus sequences of NLV p41 and enterovirus protein 2C revealed regions of high similarity. According to secondary structure prediction, the conserved regions were located within a putative central domain of alpha helices and beta strands. This study reveals for the first time an NTPase activity associated with a calicivirus-encoded protein. Based on enzymatic properties and sequence information, a functional relationship between NLV p41 and enterovirus 2C is discussed in regard to the role of 2C-like proteins in virus replication.
Woodchuck Gamma Interferon Upregulates Major Histocompatibility Complex Class I Transcription but Is Unable To Deplete Woodchuck Hepatitis Virus Replication Intermediates and RNAs in Persistently Infected Woodchuck Primary Hepatocytes
Authors: Lu Mengji , Lohrengel Beate , Hilken Gero , Kemper Thekla , Roggendorf Michael 
Published: July 2002

ABSTRACTGamma interferon (IFN-γ) is an important mediator with multiple functions in the host defense against viral infection. IFN-γ, in concert with tumor necrosis factor alpha (TNF-α), leads to a remarkable reduction of intrahepatic replication intermediates and specific mRNAs of hepatitis B virus (HBV) by a noncytolytic mechanism in the transgenic mouse model. Thus, it is rational to evaluate the potential value of IFN-γ for the treatment of chronic HBV infection. In the present study, we expressed recombinant woodchuck IFN-γ (wIFN-γ) in Escherichia coli and mammalian cells. wIFN-γ protected woodchuck cells against infection of murine encephalomyocarditis virus in a species-specific manner. It upregulated the mRNA level of the woodchuck major histocompatibility complex class I (MHC-I) heavy chain in permanent woodchuck WH12/6 cells and regulated differentially the gene expression. However, the level of the replication intermediates and specific RNAs of woodchuck hepatitis virus (WHV) in persistently WHV-infected primary woodchuck hepatocytes did not change despite a treatment with 1,000 U of wIFN-γ per ml or with a combination of wIFN-γ and woodchuck TNF-α. Rather, hepatocytes derived from chronic carriers had an elevated level of the MHC-I heavy-chain mRNAs, most probably due to the exposure to inflammatory cytokines in vivo. Treatment with high doses of wIFN-γ led to an abnormal cell morphology and loss of hepatocytes. Thus, wIFN-γ regulates the gene expression in woodchuck hepatocytes but could not deplete WHV replication intermediates and mRNAs in persistently infected hepatocytes. The cellular response to wIFN-γ may be changed in hepatocytes from chronically WHV-infected woodchucks. It should be clarified in the future whether the continuous exposure of hepatocytes to inflammatory cytokines or the presence of viral proteins leads to changes of the cellular response to wIFN-γ.
Genetic Analysis of Sequences in the 3′ Nontranslated Region of Hepatitis C Virus That Are Important for RNA Replication
Authors: Friebe Peter , Bartenschlager Ralf 
Published: July 2002

ABSTRACT
The genome of the hepatitis C virus (HCV) is a plus-strand RNA molecule that carries a single long open reading frame. It is flanked at either end by highly conserved nontranslated regions (NTRs) that mediate crucial steps in the viral life cycle. The 3′ NTR of HCV has a tripartite structure composed of an about 40-nucleotide variable region, a poly(U/UC) tract that has a heterogeneous length, and a highly conserved 98-nucleotide 3′-terminal sequence designated the X tail or 3′X. Conflicting data as to the role the sequences in the 3′ NTR play in RNA replication have been reported. By using the HCV replicon system, which is based on the self-replication of subgenomic HCV RNAs in human hepatoma cell line Huh-7, we mapped in this study the sequences in the 3′ NTR required for RNA replication. We found that a mutant with a complete deletion of the variable region is viable but that replication is reduced significantly. Only replicons in which the poly(U/UC) tract was replaced by a homouridine stretch of at least 26 nucleotides were able to replicate, whereas RNAs with homopolymeric guanine, adenine, or cytosine sequences were inactive. Deletions of individual or all stem-loop structures in 3′X were not tolerated, demonstrating that this region is most crucial for efficient RNA replication. Finally, we found that none of these deletions or substitutions within the 3′ NTR affected RNA stability or translation, demonstrating that the primary effect of the mutations was on RNA replication. These data represent the first detailed mapping of sequences in the 3′ NTR assumed to act as a promoter for initiation of minus-strand RNA synthesis.
Efficient Integration of Recombinant Adeno-Associated Virus DNA Vectors Requires a p5-
rep
Sequence in
cis
Authors: Philpott Nicola J. , Giraud-Wali Catherine , Dupuis Carolyn , Gomos Janette , Hamilton Henry , Berns Kenneth I. , Falck-Pedersen Erik 
Published: July 2002

ABSTRACT

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in
cis
to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-
rep
promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.
Restriction of Measles Virus RNA Synthesis by a Mouse Host Cell Line:
trans
-Complementation by Polymerase Components or a Human Cellular Factor(s)
Authors: Vincent Séverine , Tigaud Isabelle , Schneider Henriette , Buchholz Christian J. , Yanagi Yusuke , Gerlier Denis 
Published: July 2002

ABSTRACT

The mouse epithelial MODE-K cell line expressing human CD46 or CD150 cellular receptors was found to be nonpermissive for measles virus (MV) replication. The virus binding and membrane fusion steps were unimpaired, but only very limited amounts of virus protein and RNA synthesized were detected after the infection. In a minigenome chloramphenicol acetyltransferase assay, MODE-K cells were as able as the permissive HeLa cells in supporting MV polymerase activity. The restriction phenotype of MODE-K cells could be alleviated by providing, in
trans
, either N-P-L or N-P functional protein complexes but not by P-L complexes or individual N, P, and L proteins. Several human × mouse (HeLa × MODE-K) somatic hybrid clones expressing human CD46 were isolated and found to be either nonpermissive or permissive according to their human chromosomal contents. The MV-restricted phenotype exhibited by the MODE-K cell line suggests that a cellular factor(s) can control MV transcription, possibly by stabilizing the incoming virus polymerase templates.
Feline Calicivirus: Recovery of Wild-Type and Recombinant Viruses after Transfection of cRNA or cDNA Constructs
Authors: Thumfart Jörg Oliver , Meyers Gregor 
Published: July 2002

ABSTRACT

The RNA genome of the vaccine strain 2024 of feline calicivirus was cloned as cDNA and analyzed by nucleotide sequencing. A full-length DNA copy of the viral genome was established and proved to be a source of infectious cRNA after in vitro transcription and RNA transfection. Virus could also be recovered when the DNA construct was introduced into cells containing phage T7 RNA polymerase that was provided by vaccinia virus MVA-T7. After insertion of the sequence encoding the green fluorescent protein into the structural protein-encoding region of the infectious cDNA clone, a defective replicon was recovered that was able to replicate autonomously and was packaged into virus particles when the structural proteins were provided in
trans
.
The S4 Genome Segment of Baboon Reovirus Is Bicistronic and Encodes a Novel Fusion-Associated Small Transmembrane Protein
Authors: Dawe Sandra , Duncan Roy 
Published: July 2002

ABSTRACT
We demonstrate that the S4 genome segment of baboon reovirus (BRV) contains two sequential partially overlapping open reading frames (ORFs), both of which are functional in vitro and in virus-infected cells. The 15-kDa gene product (p15) of the 5"-proximal ORF induces efficient cell-cell fusion when expressed by itself in transfected cells, suggesting that p15 is the only viral protein required for induction of syncytium formation by BRV. The p15 protein is a small, hydrophobic, basic, integral membrane protein, properties shared with the p10 fusion-associated small transmembrane (FAST) proteins encoded by avian reovirus and Nelson Bay reovirus. As with p10, the BRV p15 protein is also a nonstructural protein and, therefore, is not involved in virus entry. Sequence analysis indicates that p15 shares no significant sequence similarity with the p10 FAST proteins and contains a unique repertoire and arrangement of sequence-predicted structural and functional motifs. These motifs include a functional N-terminal myristylation consensus sequence, an N-proximal proline-rich motif, two potential transmembrane domains, and an intervening polybasic region. The unique structural properties of p15 suggest that this protein is a novel member of the new family of FAST proteins.
PrP
c
Expression Influences the Establishment of Herpes Simplex Virus Type 1 Latency
Authors: Thackray Alana M. , Bujdoso Raymond 
Published: July 2002

ABSTRACT

PrP
c
is a glycophosphatidylinositol-linked cell-surface protein expressed principally by neural tissue. The normal function of this protein is unestablished, although a role in either transmembrane signaling, cell-cell adhesion, or copper metabolism has been proposed. In this study we have investigated the effect of the neurotropic virus herpes simplex virus type 1 (HSV-1) in strains of mice which express different levels of PrP
c
. Viral gene expression under the control of the HSV-1 early promoter IE110, detected either by in situ hybridization for RNA transcripts or by β-galactosidase (β-Gal) activity from an inserted
lacZ
gene, showed that the magnitude of HSV replication was retarded in PrP
−/−
mice. This was reflected in the lower level of acute viral titers in tissues from these virus-inoculated mice. However, HSV-inoculated PrP
−/−
mice contained higher levels of latent virus in both peripheral and central nervous tissue than those seen in mice which express PrP
c
. Our observations show that lack of PrP
c
expression favors the establishment of HSV latency whereas HSV replication proceeds more efficiently in neuronal tissue that expresses this protein. The data further suggest that PrP
c
may be involved in a metabolic pathway that culminates in apoptosis of neurons that have been infected by neurotropic viruses.
Inhibition of In Vitro Leukocyte Proliferation by Morbilliviruses
Authors: Heaney J. , Barrett T. , Cosby S. L. 
Published: July 2002

ABSTRACT

Immune suppression associated with morbillivirus infections may influence the mortality rate by allowing secondary bacterial infections that are lethal to the host to flourish. Using an in vitro proliferation assay, we have shown that all members of the genus
Morbillivirus
inhibit the proliferation of a human B-lymphoblast cell line (BJAB). Proliferation of freshly isolated, stimulated bovine and caprine peripheral blood lymphocytes is also inhibited by UV-inactivated rinderpest (RPV) and peste-des-petits ruminants viruses. As for measles virus, coexpression of both the fusion and the hemagglutinin proteins of RPV is necessary and sufficient to induce immune suppression in vitro.