bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of the COBAS Amplicor HBV Monitor Test for Quantitation of Serum Hepatitis B Virus DNA Levels
Authors: Lopez Vincent A. , Bourne Eric J. , Lutz Michael W. , Condreay Lynn D. 
Published: July 2002

ABSTRACT

Treatment of patients with chronic hepatitis B virus (HBV) infections with potent antiviral therapy often results in dramatic reductions in the levels of viremia to very low levels. Monitoring of serum HBV DNA levels is a consistent method for the assessment of antiviral potency; however, widely used hybridization assays for the monitoring of HBV DNA levels have limited sensitivities and are not effective for the monitoring of patients whose serum HBV DNA levels have decreased to below approximately 700,000 HBV genomes/ml. The objective of the present study was to assess a PCR-based assay (the COBAS-AM assay) for quantitation of serum HBV DNA levels and to compare the results of the COBAS-AM assay with those of a solution hybridization assay with a radiolabeled probe. The precision and accuracy of the assay were determined with low-positive and high-positive controls consisting of a plasmid DNA molecule containing HBV-specific primer binding regions, and the sensitivity of the assay was determined by using serial dilutions of sera from subjects with chronic HBV infection. HBV DNA levels were quantitated in 1,695 serum samples from subjects with chronic HBV infection who were enrolled in clinical trials of lamivudine in North America or Asia. The COBAS-AM assay demonstrated high levels of inter- and intra-assay precision and accuracy, and the linear range of the COBAS-AM assay was greater than that of the solution hybridization assay. The assay is linear over a 3-log
10
range and is able to quantitate serum HBV DNA at levels 3 log
10
lower than those that can be detected by the solution hybridization assay. We found that the COBAS-AM assay is an accurate PCR-based assay for quantitation of serum HBV DNA levels in subjects with chronic HBV infection.
Macrophages Escape Inhibition of Major Histocompatibility Complex Class I-Dependent Antigen Presentation by Cytomegalovirus
Authors: Hengel Hartmut , Reusch Uwe , Geginat Gernot , Holtappels Rafaela , Ruppert Thomas , Hellebrand Eva , Koszinowski Ulrich H. 
Published: July 2002

ABSTRACTThe mouse cytomegalovirus (MCMV)m152- andm06-encoded glycoproteins gp40 and gp48, respectively, independently downregulate major histocompatibility complex (MHC) class I surface expression during the course of productive MCMV infection in fibroblasts. As a result, presentation of an immediate-early protein pp89-derived nonapeptide toH-2Ld-restricted CD8+cytotoxic T cells is completely prevented in fibroblasts. Here we demonstrate that MCMV-infected primary bone marrow macrophages and the macrophage cell line J774 constitutively present pp89 peptides during permissive MCMV infection to cytotoxic T lymphocytes (CTL). In contrast to fibroblasts, expression of them152andm06genes in macrophages does not affect surface expression of MHC class I. Assessment of pp89 synthesis and quantification of extracted peptide revealed a significantly higher efficiency of macrophages than of fibroblasts to process pp89 into finally trimmed peptide. The yield of pp89 peptide determined in MCMV-infected tissues of bone marrow chimeras confirmed that bone marrow-derived cells represent a prime source of pp89 processing in parenchymal organs. The finding that macrophages resist the viral control of MHC I-dependent antigen presentation reconciles the paradox of efficient induction of CMV-specific CD8+CTL in vivo despite extensive potential of CMVs to subvert MHC class I.
Efficient Cell Infection by Moloney Murine Leukemia Virus-Derived Particles Requires Minimal Amounts of Envelope Glycoprotein
Authors: Bachrach Estanislao , Marin Mariana , Pelegrin Mireia , Karavanas Georgios , Piechaczyk Marc 
Published: July 2002

ABSTRACTRetrovirus entry into cells is mediated by specific interactions between the retrovirally encoded Env envelope glycoprotein and a host cell surface receptor. Though a number of peptide motifs responsible for the structure as well as for the binding and fusion activities of Env have been identified, only a few quantitative data concerning the infection process are available. Using an inducible expression system, we have expressed various amounts of ecotropic and amphotropic Env at the surfaces of Moloney murine leukemia virus-derived vectors and assayed for the infectivity of viral particles. Contrary to the current view that numerous noncooperative Env-viral receptor interactions are required for cell infection, we report here that very small amounts of Env are sufficient for optimal infection. However, increasing Env density clearly accelerates the rate at which infectious attachment to cells occurs. Moreover, our data also show that a surprisingly small number of Env molecules are sufficient to drive infection, albeit at a reduced efficiency, and that, under conditions of low expression, Env molecules act cooperatively. These observations have important consequences for our understanding of natural retroviral infection as well as for the design of cell-targeted infection techniques involving retroviral vectors.
Low-Level Secretion of Human Hepatitis B Virus Virions Caused by Two Independent, Naturally Occurring Mutations (P5T and L60V) in the Capsid Protein
Authors: Le Pogam Sophie , Yuan Thomas Ta-Tung , Sahu Gautam Kumar , Chatterjee Soma , Shih Chiaho 
Published: July 2002

ABSTRACT
The functional significance of naturally occurring variants of human hepatitis B virus (HBV) remains largely unknown. Previously, we reported an immature secretion phenotype caused by a highly frequent mutation at amino acid 97 of the HBV core (capsid) protein (HBcAg). This phenotype is characterized by a nonselective and excessive secretion of virions containing an immature genome of single-stranded viral DNA. To extend our study of virion secretion to other naturally occurring variants, we have characterized mutations at HBcAg codons 5, 38, and 60 via site-directed mutagenesis. Although the phenotype of the mutation at codon 38 is nearly identical to that for the wild-type virus, our study reveals that a single mutation at codon 5 or 60 exhibits a new extracellular phenotype with significantly reduced virion secretion yet maintains normal intracellular viral DNA replication. A complementation study indicates that the mutant core protein alone is sufficient for the “low-secretion” phenotype. Furthermore, the low-secretion phenotype of the codon 5 mutant appears to be induced by the loss of a parental proline residue, rather than by the gain of a new amino acid. Our study underscores the core protein as another crucial determinant in virion secretion, in addition to the known envelope proteins. Our present results suggest that a very precise structure of both α-helical and nonhelical loop regions of the entire HBcAg molecule is important for virion secretion. The low-secretion variants may contribute to the phenomenon of gradually decreasing viremia in chronic carriers during the late phase of persistent infection.
Mutations of the Kissing-Loop Dimerization Sequence Influence the Site Specificity of Murine Leukemia Virus Recombination In Vivo
Authors: Mikkelsen Jacob Giehm , Lund Anders H. , Duch Mogens , Pedersen Finn Skou 
Published: July 2002

ABSTRACT

The genetic information of retroviruses is retained within a dimeric RNA genome held together by intermolecular RNA-RNA interactions near the 5′ ends. Coencapsidation of retrovirus-derived RNA molecules allows frequent template switching of the virus-encoded reverse transcriptase during DNA synthesis in newly infected cells. We have previously shown that template shifts within the 5′ leader of murine leukemia viruses occur preferentially within the kissing stem-loop motif, a
cis
element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that modifications of the kissing-loop structure, ranging from a deletion of the entire sequence to introduction of a single point mutation in the loop motif, significantly disturb site specificity of recombination within the highly structured 5′ leader region. In addition, we find that an intact kissing-loop sequence favors optimal RNA encapsidation and vector transduction. Our data are consistent with the kissing-loop dimerization model and suggest that a direct intermolecular RNA-RNA interaction, here mediated by palindromic loop sequences within the mature genomic RNA dimer, facilitates hotspot template switching during retroviral cDNA synthesis in vivo.
Cell Cycle-Regulated Transcription by the Human Immunodeficiency Virus Type 1 Tat Transactivator
Authors: Kashanchi Fatah , Agbottah Emmanuel T. , Pise-Masison Cynthia A. , Mahieux Renaud , Duvall Janet , Kumar Ajit , Brady John N. 
Published: July 2002

ABSTRACTCyclin-dependent kinases are required for the Tat-dependent transition from abortive to productive elongation. Further, the human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G2phase of the cell cycle. These findings suggest that the life cycle of the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle. Tat-dependent long terminal repeat activation is observed in G1. This activation is TAR dependent and requires a functional Sp1 binding site. A second phase of transactivation by Tat is observed in G2and is TAR independent. This later phase of transcription is enhanced by a natural cell cycle blocker of HIV-1,vpr, which arrests infected cells at the G2/M boundary. These studies link the HIV-1 Tat protein to cell cycle-specific biological functions.
Long-Term Transgene Expression in Mice Infected with a Herpes Simplex Virus Type 1 Mutant Severely Impaired for Immediate-Early Gene Expression
Authors: Marshall Ker R. , Lachmann Robin H. , Efstathiou Stacey , Rinaldi Angela , Preston Chris M. 
Published: July 2002

ABSTRACT

The role of viral immediate-early (IE) gene expression in herpes simplex virus type 1 (HSV-1) latency was investigated. The HSV-1 multiple mutant
in
1312, defective for the expression of the virion transactivator VP16 and the IE proteins ICP0 and ICP4, was used as the parent for these studies. The coding sequences of the
Escherichia coli lacZ
gene, preceded by the encephalomyocarditis virus internal ribosome entry site, were inserted into the region of
in
1312 that encodes the latency-associated transcripts (LATs) such that transcription of the transgene was controlled by the LAT promoter. This insert has previously been shown to direct long-term latent-phase expression of β-galactosidase in a wild-type HSV-1 genome (R. H. Lachmann and S. Efstathiou, J. Virol. 71, 3197–3207, 1997). The resulting recombinant,
in
1388, was apathogenic after inoculation into mice via the footpad and did not detectably replicate in dorsal root ganglia (DRG) or footpads. Mutant
in
1388 established latency in DRG, and β-galactosidase was expressed in increasing numbers of neurons over the first 25 days of infection. During latency, more than 1% of neurons in ganglia that innervate the footpad expressed β-galactosidase, with the number of positive cells remaining constant for at least 5 months. Rescue of the VP16, ICP0, or ICP4 mutations of
in
1388 did not affect the number of β-galactosidase-expressing neurons detected during latency. The results demonstrate that HSV-1 mutants severely impaired for IE gene expression are capable of establishing latency and efficiently expressing a foreign gene product under control of the LAT promoter.
Evidence of a Role for the Q151L Mutation and the Viral Background in Development of Multiple Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1
Authors: García-Lerma J. Gerardo , Gerrish Philip J. , Wright Anthony C. , Qari Shoukat H. , Heneine Walid 
Published: July 2002

ABSTRACT

The majority of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with zidovudine (AZT) plus zalcitabine (ddC) and didanosine (ddI) develop AZT resistance mediated by mutations such as T215Y and M41L. Only a small proportion of patients develop multiple dideoxynucleoside resistance (MDNR) mediated by the Q151M mutation. To gain insight into the factors responsible for the low frequency of selection of Q151M, we evaluated the replication capabilities of recombinant viruses carrying two possible intermediates (151L or 151K) of the Q151M mutation generated in different reverse transcriptase (RT) genetic backgrounds. The 151L and 151K mutations were introduced by site-directed mutagenesis in RTs from two patient-derived HIV-1 isolates that had either wild type (WT) Q or the Q151M (posttreatment isolate) mutation. For comparison, both mutations were also introduced in a laboratory-adapted HIV-1 strain (HIV-1
HXB2
). Analysis of replication capabilities showed that both 151L and 151K were lethal in RT genetic backgrounds of the WT isolate and in HIV-1
HXB2
. In contrast, 151L but not 151K allowed virus replication in RT backgrounds of the posttreatment isolate. Three mutations (V35I, S68G, and I178M) were present in the RT background of the posttreatment isolate but not in the WT isolate. Introduction of S68G in the RT of both the WT isolate and HIV-1
HXB2
partially restored replication capacity of recombinants carrying the 151L mutation. The S68G mutation alone did not confer a significant replicative disadvantage in WT viruses. Like HIV-1
151M
, HIV-1
151L
RT was found to have six- to eightfold resistance to AZT-triphosphate (TP), ddA-TP, and ddC-TP, indicating an MDNR phenotype. However, HIV-1
151L
was found to be less fit than HIV-1
151M
, which may explain the preferential selection of HIV-1
151M
observed in vivo. The demonstrated ability of HIV-1
151L/68G
to replicate and the associated MDNR suggest that 151L is a potential intermediate of Q151M. The dependence of HIV-1
151L
on other mutations, such as S68G, for replication may explain the low frequency of the Q151M-mediated pathway of resistance.
Vaccination with Inactivated Virus but Not Viral DNA Reduces Virus Load following Challenge with a Heterologous and Virulent Isolate of Feline Immunodeficiency Virus
Authors: Hosie Margaret J. , Dunsford Thomas , Klein Dieter , Willett Brian J. , Cannon Celia , Osborne Robert , MacDonald Julie , Spibey Norman , Mackay Nancy , Jarrett Oswald , Neil James C. 
Published: July 2002

ABSTRACT

It has been shown that cats can be protected against infection with the prototypic Petaluma strain of feline immunodeficiency virus (FIV
PET
) using vaccines based on either inactivated virus particles or replication-defective proviral DNA. However, the utility of such vaccines in the field is uncertain, given the absence of consistent protection against antigenically distinct strains and the concern that the Petaluma strain may be an unrepresentative, attenuated isolate. Since reduction of viral pathogenicity and dissemination may be useful outcomes of vaccination, even in the absence of complete protection, we tested whether either of these vaccine strategies ameliorates the early course of infection following challenge with heterologous and more virulent isolates. We now report that an inactivated virus vaccine, which generates high levels of virus neutralizing antibodies, confers reduced virus loads following challenge with two heterologous isolates, FIV
AM6
and FIV
GL8
. This vaccine also prevented the marked early decline in CD4/CD8 ratio seen in FIV
GL8
-infected cats. In contrast, DNA vaccines based on either FIV
PET
or FIV
GL8
, which induce cell-mediated responses but no detectable antiviral antibodies, protected a fraction of cats against infection with FIV
PET
but had no measurable effect on virus load when the infecting virus was FIV
GL8
. These results indicate that the more virulent FIV
GL8
is intrinsically more resistant to vaccinal immunity than the FIV
PET
strain and that a broad spectrum of responses which includes virus neutralizing antibodies is a desirable goal for lentivirus vaccine development.
Epstein-Barr Virus Small RNAs Potentiate Tumorigenicity of Burkitt Lymphoma Cells Independently of an Effect on Apoptosis
Authors: Ruf Ingrid K. , Rhyne Paul W. , Yang Chunying , Cleveland John L. , Sample Jeffery T. 
Published: July 2002

ABSTRACT

The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-
MYC
in BL.
Conserved Regions in the Epstein-Barr Virus Leader Protein Define Distinct Domains Required for Nuclear Localization and Transcriptional Cooperation with EBNA2
Authors: Peng RongSheng , Tan Jie , Ling Paul D. 
Published: July 2002

ABSTRACT
Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood. Recent studies have shown that EBNA-LP may be an important EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introduced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the latency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are located in the repeat domains that are essential for the EBNA2 cooperativity function. In addition, three serine residues are also well conserved in the repeat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA-LP with two repeats, which is the minimal protein able to cooperate with EBNA2. Mutations introduced into CR1a had no effect on EBNA-LP function, while mutations introduced into CR1b resulted in EBNA-LP with slightly decreased activity. Mutations in CR1c and CR2 resulted in proteins that no longer localized exclusively to the nucleus and also had no EBNA2 cooperation activity. Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved serines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP proteins with substantially reduced activity. The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleotides encoding these positively charged amino acid groupings into a cytoplasmic test protein, herpes simplex virus ΔIE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (109 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function independently as a nuclear localization signal. However, a combination of amino acids 29 to 50 resulted in more efficient nuclear localization than with amino acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is essential for EBNA2 cooperativity function. Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explanation for why this isoform has no activity. In addition, two conserved serine residues which are distinct from nuclear import functions are important for EBNA2 cooperativity function.
Integrase-LexA Fusion Proteins Incorporated into Human Immunodeficiency Virus Type 1 That Contains a Catalytically Inactive Integrase Gene Are Functional To Mediate Integration
Authors: Holmes-Son Michelle L. , Chow Samson A. 
Published: July 2002

ABSTRACT

Purified fusion proteins made up of a retroviral integrase and a sequence-specific DNA-binding protein have been tested in in vitro assays for their ability to direct integration into specific target sites. To determine whether these fusion proteins can be incorporated into human immunodeficiency virus type 1 (HIV-1) and are functional to mediate integration, we used an in
trans
approach to deliver various integrase-LexA proteins to an integrase-defective virus containing an integrase mutation at aspartate residue 64. Integrase-LexA, integrase-LexA DNA-binding domain, or N- or C-terminally truncated integrase-LexA proteins were fused to the HIV-1 accessory protein, Vpr. Coexpression of the Vpr fusion proteins and an integrase-defective HIV-1 molecular clone by a producer cell line resulted in efficient incorporation of the fusion protein into the integrase-mutated virus. In addition, each of these viruses was infectious and capable of performing integration, as determined by two independent cellular assays that measure reporter gene expression. With the exception of the N-terminally truncated integrase fused to LexA, which was at about 1%, all of the fusion proteins restored integration to a similar level, at 17 to 24% of that of the wild-type virus. The low level observed with the N-terminally truncated integrase fused to LexA is consistent with previous results implying that the N terminus of integrase is involved in multiple steps of the retroviral life cycle. These data indicate that the integrase-fusion proteins retain catalytic function in the integrase-mutated viruses and demonstrate the feasibility of incorporating integrase fusion proteins into HIV-1 for the development of site-directed retroviral vectors.