bluuebunny/crossref_metadata_2025 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

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

The 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

Retrovirus 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

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

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

Cyclin-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

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

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

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

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

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

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.
Complex Formation by Human Cytomegalovirus Glycoproteins M (gpUL100) and N (gpUL73)
Authors: Mach M. , Kropff B. , Dal Monte P. , Britt W.
Published: July 2002

The envelope glycoproteins of human cytomegalovirus (HCMV) virions are incompletely characterized. We have analyzed complex formation between glycoprotein M (gM or gpUL100) and a second glycoprotein. gM-homologous proteins are conserved throughout the herpesvirus family and represent type III membrane proteins containing multiple hydrophobic sequences. In extracellular HCMV particles, gM was found to be complexed through disulfide bonds to a second protein with an apparent molecular mass of 50 to 60 kDa. The 50- to 60-kDa protein was found to be derived from reading frame UL73 of HCMV strain AD169. UL73-homologous genes are also conserved within herpesviruses. When transiently expressed by itself, the UL73 gene product consisted of a protein of 18 kDa. However, in the presence of gM, the UL73 gene product was posttranslationally modified to the 50- to 60-kDa species. Thus, gM and the UL73 gene product, which represents the gN homolog of herpesviruses, form a disulfide-linked complex in HCMV virions. Transient expression of gM and gN followed by fluorescence imaging with monoclonal antibodies against either protein demonstrated that complex formation was required for transport of the proteins from the endoplasmic reticulum to the Golgi and
trans
-Golgi compartments. Finally, we tested the gM-gN complex for reactivity with sera from HCMV-seropositive donors. Whereas most sera failed to react with either gM or gN when expressed alone, 62% of sera were positive for the gM-gN complex. Because a murine monoclonal antibody reactive with gN in the gM-gN complex efficiently neutralizes infectious virus, the gM-gN complex may represent a major antigenic target of antiviral antibody responses.
Multiple Integrations of Human Foamy Virus in Persistently Infected Human Erythroleukemia Cells
Authors: Meiering Christopher D. , Comstock Kenine E. , Linial Maxine L.
Published: July 2002

Foamy viruses are complex retroviruses whose replication strategy resembles that of conventional retroviruses. However, foamy virus replication also resembles that of hepadnaviruses in many respects. Because hepadnaviruses replicate in an integrase-independent manner, we were interested in investigating the characteristics of human foamy virus (HFV) integration. We have shown that HFV requires a functional integrase protein for infectivity. Our analyses have revealed that in single-cell clones derived from HFV-infected erythroleukemia-derived cells (H92), there were up to 20 proviral copies per host cell genome as determined by Southern blot and fluorescent in situ hybridization analysis. Use of specific probes has also shown that a majority of the proviruses contain the complete
tas
gene, which encodes the viral transactivator, and are not derived from Δ
tas
cDNAs, which have been shown to arise rapidly in infected cells. To demonstrate that the multiple proviral sequences are due to integration instead of recombination, we have sequenced the junctions between the proviral sequences and the host genome and found that the proviruses have authentic long terminal repeat ends and that each integration is at a different chromosomal site. A virus lacking the Gag nuclear localization signal accumulates fewer proviruses, suggesting that nuclear translocation is important for high proviral load. Since persistently infected H92 clones are not resistant to superinfection, the relative importance of an intracellular versus extracellular mechanism in proviral acquisition has yet to be determined.
Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein
Authors: Schaley Joel , O'Connor Robert J. , Taylor Laura J. , Bar-Sagi Dafna , Hearing Patrick
Published: July 2002

The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.
Hypervariable Region 1 Sequence Stability during Hepatitis C Virus Replication in Chimpanzees
Authors: Ray Stuart C. , Mao Qing , Lanford Robert E. , Bassett Suzanne , Laeyendecker Oliver , Wang Yu-Ming , Thomas David L.
Published: July 2002

The putative envelope 2 (E2) gene of hepatitis C virus (HCV) contains a highly variable region referred to as hypervariable region 1 (HVR1). We hypothesized that this genetic variability is driven by immune selection pressure, rather than representing the accumulation of random mutations in a region with relatively little functional constraint. To test this hypothesis, we examined the E2 sequence of a human inoculum that was passaged through eight chimpanzees, which appear to have a replicative rate (opportunity for chance mutation) similar to that of humans. Acute-phase plasma samples from a human (the inoculum) and six of eight serially infected chimpanzees were studied. For each, 33 cloned cDNAs were examined by a combined heteroduplex–single-stranded conformational polymorphism assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns (clonotypes) for sequencing. The sequence diversity of HCV was significantly lower in the chimpanzees than in the humans, and during eight serial passages there was no change in the sequence of the majority clonotype from each animal examined. Similarly, the rates of protein sequence altering (nonsynonymous) substitution were lower in the chimpanzees than in the humans. These findings demonstrate that nonsynonymous mutations indicate selection pressure rather than being an incidental result of HCV replication.
Identification of Antigenic Proteins Encoded by Human Herpesvirus 8 and Seroprevalence in the General Population and among Patients with and without Kaposi's Sarcoma
Authors: Katano Harutaka , Iwasaki Takuya , Baba Nobuyoshi , Terai Masanori , Mori Shigeo , Iwamoto Aikichi , Kurata Takeshi , Sata Tetsutaro
Published: July 2002

To establish a sensitive and specific antibody assay, potent antigenic proteins encoded by human herpesvirus 8 (HHV8) were studied. Fifteen recombinant HHV8-encoded proteins were produced as glutathione
S
-transferase fusion proteins. The sera from AIDS-associated Kaposi's sarcoma (KS) patients reacted with four proteins encoded by open reading frames (ORFs) K8.1, 59, 65, and 73 in a Western blot assay. An enzyme-linked immunosorbent assay (ELISA) using these four proteins as antigens (mixed-antigen ELISA) revealed that all 26 sera derived from KS patients (24 with and 2 without human immunodeficiency virus infection) became positive for anti-HHV8 antibodies. The presence of HHV8 was demonstrated in 14 (1.4%) of 1,004 sera from the Japanese general population and 10 (1.9%) of 527 sera from patients without HHV8-associated diseases. The presence of immunoglobulin G (IgG) and IgM antibodies against HHV8 examined further by the mixed-antigen ELISA and Western blotting revealed IgG antibody in all ELISA-positive sera, while IgM antibody against ORF K8.1 was absent. These data suggest that the ORF 73 and 65 proteins are potent antigens for a sensitive serological assay.
Sustained Activation of Mitogen-Activated Protein Kinases and Activator Protein 1 by the Hepatitis B Virus X Protein in Mouse Hepatocytes In Vivo
Authors: Nijhara Ruchika , Jana Siddhartha S. , Goswami Shyamal K. , Rana Ajay , Majumdar Subeer S. , Kumar Vijay , Sarkar Debi P.
Published: July 2002

Transcriptional activation of diverse cellular genes by the X protein (HBx) of hepatitis B virus (HBV) has been suggested as one of the mechanisms for HBV-associated hepatocellular carcinoma. However, such functions of HBx have been studied using transformed cells in culture and have not been examined in the normal adult hepatocytes, a natural host of HBV. Using an efficient hepatocyte-specific virus-based gene delivery system developed in our laboratory earlier, we studied the HBx action in vivo. We demonstrate that following virosome-mediated delivery of HBx DNA, a large population (>50%) of hepatocytes express the HBx protein in a dose-dependent manner, which induces a significant increase in the activity of extracellular signal-regulated kinases (ERKs) in the livers of HBx-transfected mice. Inhibition of HBx-induced ERK activation following intravenous administration of PD98059, a mitogen-activated protein kinase kinase kinase (MEK) inhibitor, confirmed the requirement for MEK in the activation of ERKs by HBx. Induction of ERK activity by HBx was sustained for up to 30 days. Interestingly, sustained activation of c-Jun N-terminal kinases (JNKs) for up to 30 days was also noted. Such constitutive ERK and JNK activation as a consequence of continued HBx expression also led to sustained stimulation of further downstream events, such as increased levels of c-Jun and c-Fos proteins along with the persistent induction of activator protein 1 binding activity. Taken together, our data suggest a critical role of these molecules in HBx-mediated cell transformation.
Cloning and Sequencing of Defective Particles Derived from the Autonomous Parvovirus Minute Virus of Mice for the Construction of Vectors with Minimal
cis
-Acting Sequences
Authors: Clément Nathalie , Avalosse Bernard , El Bakkouri Karim , Velu Thierry , Brandenburger Annick
Published: July 2002

The production of wild-type-free stocks of recombinant parvovirus minute virus of mice [MVM(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. We have therefore cloned and sequenced spontaneously occurring defective particles of MVM(p) with very small genomes to identify the minimal
cis
-acting sequences required for DNA amplification and virus production. One of them has lost all capsid-coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in
trans
by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with new, matched, helper plasmids that present no homology downstream from the transgene.
Role of Vaccinia Virus A20R Protein in DNA Replication: Construction and Characterization of Temperature-Sensitive Mutants
Authors: Ishii Koji , Moss Bernard
Published: July 2002

Previous analyses of randomly generated, temperature-sensitive vaccinia virus mutants led to the mapping of DNA synthesis negative complementation groups to the B1R, D4R, D5R, and E9L genes. Evidence from the yeast two-hybrid system that the D4R and D5R proteins can interact with the A20R protein suggested that A20R was also involved in DNA replication. We found that the A20R gene was transcribed early after infection, consistent with such a role. To investigate the function of the A20R protein, targeted mutations were made by substituting alanines for charged amino acids occurring in 11 different clusters. Four mutants were not isolated, suggesting that they were lethal, two mutants exhibited no temperature sensitivity, two mutants exhibited partial temperature sensitivity, and two mutants formed no plaques or infectious virus at 39°C. The two mutants with stringent phenotypes were further characterized. Temperature shift-up experiments indicated that the crucial period was between 6 and 12 h after infection, making it unlikely that the defect was in virus entry, early gene expression, or a late stage of virus assembly. Similar patterns of metabolically labeled viral early proteins were detected at permissive and nonpermissive temperatures, but one mutant showed an absence of late proteins under the latter conditions. Moreover, no viral DNA synthesis was detected when cells were infected with either stringent mutant at 39°C. The previous yeast two-hybrid analysis together with the present characterization of A20R temperature-sensitive mutants suggested that the A20R, D4R, and D5R proteins are components of a multiprotein DNA replication complex.
Evolution of Replication Efficiency following Infection with a Molecularly Cloned Feline Immunodeficiency Virus of Low Virulence
Authors: Hosie Margaret J. , Willett Brian J. , Klein Dieter , Dunsford Thomas H. , Cannon Celia , Shimojima Masayuki , Neil James C. , Jarrett Oswald
Published: July 2002

The development of an effective vaccine against human immunodeficiency virus is considered to be the most practicable means of controlling the advancing global AIDS epidemic. Studies with the domestic cat have demonstrated that vaccinal immunity to infection can be induced against feline immunodeficiency virus (FIV); however, protection is largely restricted to laboratory strains of FIV and does not extend to primary strains of the virus. We compared the pathogenicity of two prototypic vaccine challenge strains of FIV derived from molecular clones; the laboratory strain PET
F14
and the primary strain GL8
414
. PET
F14
established a low viral load and had no effect on CD4
+
- or CD8
+
-lymphocyte subsets. In contrast, GL8
414
established a high viral load and induced a significant reduction in the ratio of CD4
+
to CD8
+
lymphocytes by 15 weeks postinfection, suggesting that PET
F14
may be a low-virulence-challenge virus. However, during long-term monitoring of the PET
F14
-infected cats, we observed the emergence of variant viruses in two of three cats. Concomitant with the appearance of the variant viruses, designated 627
W135
and 628
W135,
we observed an expansion of CD8
+
-lymphocyte subpopulations expressing reduced CD8 β-chain, a phenotype consistent with activation. The variant viruses both carried mutations that reduced the net charge of the V3 loop (K409Q and K409E), giving rise to a reduced ability of the Env proteins to both induce fusion and to establish productive infection in CXCR4-expressing cells. Further, following subsequent challenge of naïve cats with the mutant viruses, the viruses established higher viral loads and induced more marked alterations in CD8
+
-lymphocyte subpopulations than did the parent F14 strain of virus, suggesting that the E409K mutation in the PET
F14
strain contributes to the attenuation of the virus.
Mutant Tax Protein from Bovine Leukemia Virus with Enhanced Ability To Activate the Expression of c
-fos
Authors: Tajima Shigeru , Aida Yoko
Published: July 2002

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. We previously identified several mutants of the BLV Tax protein with an ability to transactivate transcription via the BLV enhancer that is significantly greater than that of the wild-type Tax protein. Moreover, the mutant proteins also activated other viral enhancers, such as the enhancer of human T-cell leukemia virus type 1, which cannot be activated by wild-type BLV Tax. In this study, we demonstrated that the mutant proteins but not wild-type protein activate the upstream sequence of the human c
-fos
gene, which contains two major
cis
-acting elements, the CArG box and cyclic AMP-responsive element (CRE) motif. The mutant protein also strongly increased levels of endogenous c-
fos
mRNA in both human and bovine cell lines. On the other hand, the wild-type Tax protein has no activity to activate the expression of human c-
fos
, indicating that wild-type BLV Tax might discriminate between human and bovine c-
fos
promoter sequences. Deletion and point-mutational analysis of the
cis
-acting elements revealed that both the CArG box and the CRE motif were indispensable for the activation of c-
fos
by the mutant BLV Tax protein. Our results suggest that the mutant BLV Tax proteins might not only have the ability to enhance the production of virus particles but might also have increased ability to induce leukemia.
High Frequency of Virus-Specific B Lymphocytes in Germinal Centers of Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys
Authors: Margolin David H. , Helmuth Saunders Erika F. , Bronfin Benjamin , de Rosa Nicole , Axthelm Michael K. , Alvarez Xavier , Letvin Norman L.
Published: July 2002

The etiology of the lymphadenopathy and follicular hyperplasia associated with human immunodeficiency virus type 1 (HIV-1) infection has remained unclear. To determine whether the B-lymphocyte expansions characteristic of this syndrome represent polyclonal and virus-specific processes, the antigen specificity of B cells in lymphoid tissues of monkeys infected with simian-human immunodeficiency virus (SHIV) chimeras was assessed using an inverse immunohistochemical assay with biotinylated HIV-1 envelope gp120 (Env) as an antigen probe. Env-binding B cells were found aggregated in lymph node and splenic germinal centers (GCs). Most Env-binding GCs also contained an unstained population of B cells, suggesting the GCs were formed by a polyclonal (oligoclonal) process. By day 42 following infection, Env-binding B cells were present in 19% of all lymph node GCs. Env-binding cells were present in 25% of GCs even during chronic infection. This extraordinarily high frequency of Env-specific B lymphocytes suggests that the expansion of virus-specific B cells may largely account for the follicular hyperplasia in AIDS virus-infected individuals.
Preparation of s-Triazolo[4,3-c]quinazolines
Authors: Špirková Katarína , Stankovský Štefan , Horňaček Jakub
Published: July 2002

Several biological activities have been attributed to the quinazoline and triazole derivatives. Combining two known active heterocyclic moieties, we prepared a series of 3-aryl-5-morpholino-9-chloro-s-triazolo[4,3-c]quinazolines by an oxidative cyclization of the corresponding arylhydrazones, with the hope, that the known activities might result in a novel type of activity.
New Pyrazoline Derivatives
Authors: Latif F. M. Abd El , Maghraby A. S. , Barsy M. A. , Badr M. Z. A. , Dopp D.
Published: July 2002

The biological activity of azoles, pyrimidines as well as pyrazolines are of interest. Formation of pyrazolines and related compounds from hydrazine derivatives and α,β-unsaturated carbonyl compounds is very common. Pyrazoline fused with different heterocyclic nuclei at position 4 and 5 have been reported. Here we report the synthesis of some new monocyclic or fused azoles and pyrimidinones linked to C-4 or 5-chloro-3-methyl-1-phenylpyrazoline.
Derivatives of Furo[3,2-b]pyrrole
Authors: Krutošíková Alžbeta , Dandárová Miloslava , Bobošík Vladimír
Published: July 2002

A number of methyl 2-substituted, 2,3-disubstituted-4H-furo[3,2-b]pyrrole-5-carboxylates (V - VII) and methyl 1H-benzo[b]furo[3,2-b]pyrrole-2-carboxylate (VIII) was prepared by the thermolysis of corresponding methyl 2-azido-3-(R1,R2-substituted-2-furyl)propenoates (I - IV). N-Methyl and N-benzyl derivatives IX - XVI were prepared at the phase-transfer catalysis conditions. Hydrolysis of some N-methyl and N-benzyl substituted esters furnished corresponding acids XVII - XXII. The hydrazides XXIII - XXX were obtained from corresponding esters and hydrazine hydrate. The preparation of 1-benzylbenzo[b]furo[3,2-b]pyrrole (XXXI) is described as well.
Regioselectivity of Complexations of Substituted Phenyl Benzoates and Phenyl Phenylacetates with Cr(CO)6
Authors: Hrnčiar Pavol , Černák Peter , Gajda Vladimír , Toma Štefan
Published: July 2002

Selectivity of complexation of substituted phenyl benzoates is very low. In most cases, comparable yields of both regioisomeric complexes are isolated. Exception is 4-chlorophenyl ester, where benzoic acid moiety is complexed nearly exclusively. Very high regioselectivity of complexation was observed with substituted phenyl phenylacetates. The substituent of substituted phenols has not any effect on the complexation, and only phenylacetic acid moiety is complexed. This observation supports the recently proposed mechanism of the catalytic activity of the esters at arene complexation.
Synthesis of 2-Deoxy-β-D-ribonucleosides and 2,3-Dideoxy-β-D-pentofuranosides on Immobilized Bacterial Cells
Authors: Votruba Ivan , Holý Antonín , Dvořáková Hana , Günter Jaroslav , Hocková Dana , Hřebabecký Hubert , Cihlar Tomas , Masojídková Milena
Published: July 2002

Alginate gel-entrapped cells of auxotrophic thymine-dependent strain of E. coli catalyze the transfer of 2-deoxy-D-ribofuranosyl moiety of 2'-deoxyuridine to purine and pyrimidine bases as well as their aza and deaza analogs. All experiments invariably gave β-anomers; in most cases, the reaction was regiospecific, affording N9-isomers in the purine and N1-isomers in the pyrimidine series. Also a 2,3-dideoxynucleoside can serve as donor of the glycosyl moiety. The acceptor activity of purine bases depends only little on substitution, the only condition being the presence of N7-nitrogen atom. On the other hand, in the pyrimidine series the activity is limited to only a narrow choice of mostly short 5-alkyl and 5-halogeno uracil derivatives. Heterocyclic bases containing amino groups are deaminated; this can be avoided by conversion of the base to the corresponding N-dimethylaminomethylene derivative which is then ammonolyzed. The method was verified by isolation of 9-(2-deoxy-β-D-ribofuranosyl) derivatives of adenine, guanine, 2-chloroadenine, 6-methylpurine, 8-azaadenine, 8-azaguanine, 1-deazaadenine, 3-deazaadenine, 1-(2-deoxy-β-D-ribofuranosyl) derivatives of 5-ethyluracil, 5-fluorouracil, and 9-(2,3-dideoxy-β-D-pentofuranosyl)hypoxanthine, 9-(2,3-dideoxy-β-D-pentofuranosyl)-6-methylpurine, and other nucleosides.
Electrosynthesis of Organic Amalgam from Tetraethylammonium Tetrafluoroborate in Aqueous Medium
Authors: Krásenský Simon , Studničková Marie
Published: July 2002

The electroreduction of neutral aqueous solutions of 0.1 - 0.5 M Et4NBF4 on a mercury cathode at room temperature at -2.8 - -2.4 V produces a black precipitate of Et4N . Hgx with simultaneous evolution of hydrogen (the pH value increases to 12). No anodic oxidation of the amalgam has been observed in the interval of 0 - 2.8 V. The composition of the precipitate, collected by filtration under nitrogen, has been determined from the decrease of Et4N+ in the solution after electrolysis and from analysis of mercury in the binary compound Et4N . Hgx. The value x = 2.9 ± 0.8 has been found.
Adsorption and Thermal Decomposition of Tetrakis(dimethylamido)titanium on Si and SiO2/Si Surfaces: an XPS Study
Authors: Janovská Michaela , Bastl Zdeněk
Published: July 2002

X-Ray photoelectron spectroscopy (XPS) has been used to study adsorption of tetrakis(dimethylamido)titanium (TDMT) on a clean Si surface and surface on which an oxide layer was grown prior to TDMT adsorption. Measurements were carried out at temperatures 80 and 295 K. In addition, decomposition of TDMT on the above substrates yielding titanium nitride coatings was investigated. The change in surface composition as a function of TiNx layer thickness was monitored by XPS. Adsorption at 80 K proceeds without dissociation on both substrates. Photoelectron spectra measured at 295 K indicate presence of surface species containing C-N multiple bond on both substrates and on SiO2/Si substrate formation of species that contain Ti-N-C rings. At 580 K an overlayer consisting predominantly of TiNx was produced by pyrolysis of TDMT. The photoelectron spectra revealed also presence of species with C-N-C, Si-CHx and Ti-C bonds the concentration of which depended on an overlayer thickness. On SiO2/Si substrate decomposition of SiO2 accompanied by titanium oxide formation occurs during initial stages of TDMT pyrolysis.
Structure and Properties of Quaternized 2- and 4-Aminonicotinamides
Authors: Smrčková-Voltrová Svatava , Říha Jaroslav , Prutianov Viktor
Published: July 2002

Compounds 2- and 4-aminonicotinamide were quaternized with eight 4-substituted 1-bromomethylbenzenes to form 1-(4-substituted benzyl)-3-carbamoyl-1,2(1,4)-dihydropyridin-2(4)-iminium bromides. The optimal reaction conditions were found and the resulting iminium salts were characterized by 1H and 13C NMR spectra. No transmission of electronic effect of the substituent at the benzene ring on the spectral properties of dihydropyridine moiety was observed. The possible reason is discussed.