bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection and Characterization of Genetic Recombination in Cytopathic Type 2 Bovine Viral Diarrhea Viruses
Authors: Ridpath Julia F. , Neill John D.
Published: July 2002

In cytopathic bovine viral diarrhea virus genotype 1 (BVDV1) isolates, insertions are reported at position A (amino acid [aa] 1535) and position B (aa 1589). Insertions at position B predominate. In this survey it was found that in BVDV2, insertions at position A predominate. Possible reasons for this difference in relative frequency are discussed.
Effects of Genital Tract Inflammation on Human Immunodeficiency Virus Type 1 V3 Populations in Blood and Semen
Authors: Ping Li-Hua , Cohen Myron S. , Hoffman Irving , Vernazza Pietro , Seillier-Moiseiwitsch Françoise , Chakraborty Hrishikesh , Kazembe Peter , Zimba Dick , Maida Martin , Fiscus Susan A. , Eron Joseph J. , Swanstrom Ronald , Nelson Julie A. E.
Published: July 2002

We have examined cell-free viral populations in the blood plasma and seminal plasma compartments of men infected with subtype C human immunodeficiency virus type 1 (HIV-1) using the V3-specific heteroduplex tracking assay (V3-HTA). We studied two cohorts of subjects who had visited either a sexually transmitted disease (STD) clinic for genital tract inflammation in the form of urethritis (
n
= 43) or a dermatology clinic (controls,
n
= 14) in Malawi. We have previously shown that the presence of urethritis is associated with an eightfold increase in virus load in the seminal plasma compartment (M. S. Cohen et al., Lancet 349:1868–1873, 1997). The purpose of this study was to determine whether genital tract inflammation and its treatment caused genetic instability in cell-free HIV-1 populations. In a cross-sectional analysis at study entry, three-fourths of the STD and control subjects had multiple V3 populations in their blood while 60% of the STD subjects and 79% of the control subjects had multiple V3 populations in their semen. Overall, one-fourth of all of the subjects showed discordance between results with blood and semen specimens when samples were compared for the presence and absence of subpopulations. When differences in the relative levels of abundance of bands were also taken into account, two-fifths of all of the subjects showed discordance between the compartments. Among the subset of subjects in whom multiple virus populations could be detected, half showed discordance between the compartments. There were no differences between STD and control cohorts for these comparisons of the compartments in this cross-sectional analysis at study entry. Longitudinal analysis of the viral populations from two separate clinic visits over 1 to 4 weeks showed that the complexity of each V3 population as measured by Shannon entropy was different in blood and semen at the two time points, indicating that the blood and semen constitute different compartments for HIV-1. The seminal plasma compartment was more dynamic than the blood plasma compartment for the STD subjects who were treated for urethritis, with changes being noted in the presence or absence of V3-HTA bands in the semen of 29% of these subjects but in the blood of only 9% of these subjects. However, the changes were generally small. Overall, our results suggest that 40% of male subjects show discordance between seminal and blood viral populations and that the complexity of each V3 population was different between the two compartments. Both of these results point to the partial independence of the seminal compartment as a viral niche within the body.
Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation
Authors: Sauder Christian , Hallensleben Wiebke , Pagenstecher Axel , Schneckenburger Stefanie , Biro Laszlo , Pertlik Doris , Hausmann Jürgen , Suter Mark , Staeheli Peter
Published: July 2002

Borna disease virus (BDV) causes CD8+T-cell-mediated meningoencephalitis in immunocompetent mice and rats, thus providing a valuable animal model for studying the mechanisms of virus-induced central nervous system (CNS) immunopathology. Chemokine-mediated leukocyte recruitment to the CNS is a crucial step in the development of neurological disease. We found increased mRNA levels of IP-10 and other chemokines in brains of adult rats following infection with BDV. The marked increase in chemokine gene expression at about day 8 postinfection seemed to immediately precede the inflammatory process. In brains of rats infected as newborns, in which inflammation was only mild and transient, sustained expression of IP-10 and RANTES genes was observed. In situ hybridization studies revealed that astrocytes were the major source of IP-10 mRNAs in brains of rats infected as newborns and as adults. In brains of infected mice lacking CD8+T cells (β2m0/0), transcripts encoding IP-10 and RANTES were also observed. IP-10 transcripts were also present in a small number of scattered astrocytes of infected knockout mice lacking mature B and T cells as well as functional alpha/beta and gamma interferon receptors, indicating that BDV can induce chemokine synthesis in the absence of interferons and other B- or T-cell-derived cytokines. These data provide strong evidence that CNS-resident cells are involved in the early localized host immune response to infection with BDV and support the concept that chemokines are pivotal for the initiation of virus-induced CNS inflammation.
Epstein-Barr Virus Recombinants from BC-1 and BC-2 Can Immortalize Human Primary B Lymphocytes with Different Levels of Efficiency and in the Absence of Coinfection by Kaposi's Sarcoma-Associated Herpesvirus
Authors: Aguirre Andrew J. , Robertson Erle S.
Published: July 2002

Epstein-Barr virus
(EBV) and
Kaposi's sarcoma-associated herpesvirus
(KSHV) are human gammaherpesviruses associated with numerous malignancies. Primary effusion lymphoma or body cavity-based lymphoma is a distinct clinicopathological entity that, in the majority of cases, manifests coinfection with KSHV and EBV. In previous analyses, we have characterized the EBV in the BC-1 and BC-2 cell lines as potential intertypic recombinants of the EBV types 1 and 2. In order to examine the infectious and transforming capacities of KSHV and the intertypic EBV recombinants from the BC-1 and BC-2 cell lines, viral replication was induced in these cell lines and fresh human primary B lymphocytes were infected with progeny virus. The transformed clones were analyzed by PCR and Western blotting. All analyzed clones were infected with the intertypic progeny EBV but had no detectable signal for progeny KSHV. Additionally, primary B lymphocytes incubated with viral supernatant containing KSHV alone showed an unsustained initial proliferation, but prolonged growth or immortalization of these cells in vitro was not observed. We also show that the EBV recombinants from BC-1 were less efficient than the EBV recombinants from BC-2 in the ability to maintain the transformed phenotype of the infected human B lymphocytes. From these findings, we conclude that the BC-1 and BC-2 intertypic EBV recombinants can immortalize human primary B lymphocytes, albeit at different levels of efficiency. However, the KSHV induced from BC-1 and BC-2 alone cannot transform primary B cells, nor can it coinfect EBV-positive B lymphocytes under our experimental conditions with B lymphocytes from EBV-seropositive individuals. These results are distinct from those in one previous report and suggest a possible requirement for other factors to establish coinfection with both viral agents.
Nucleotide and Amino Acid Complexity of Hepatitis C Virus Quasispecies in Serum and Liver
Authors: Cabot Beatriz , Martell María , Esteban Juan I. , Sauleda Sílvia , Otero Teresa , Esteban Rafael , Guàrdia Jaime , Gómez Jordi
Published: July 2002

The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral replication. Several studies have tried to unravel, through the parameters that characterize HCV circulating quasispecies, prognostic markers of the disease. In a previous work we demonstrated that the parameters of circulating viral quasispecies do not always reflect those of the intrahepatic virus. Here, we have analyzed paired serum and liver quasispecies from 39 genotype 1b-infected patients with different degrees of liver damage, ranging from minimal changes to cirrhosis. Viral level was quantified by real-time reverse transcription-PCR, and viral heterogeneity was characterized through the cloning and sequencing of 540 HCV variants of a genomic fragment encompassing the E2-NS2 junction. Although in 95% of patients, serum and liver consensus HCV amino acid sequences were identical, quasispecies complexity varied considerably between the viruses isolated from each compartment. Patients with HCV quasispecies in serum more complex (26%) than, less complex (28%) than, or similarly complex (41%) to those in liver were found. Among the last, a significant correlation between fibrosis and all the parameters that measure the viral amino acid complexity was found. Correlation between fibrosis and serum viral load was found as well (
R
= 0.7). With regard to the origin of the differences in quasispecies complexity between serum and liver populations, sequence analysis argued against extrahepatic replication as a quantitatively important contributing factor and supported the idea of a differential effect or different selective forces on the virus depending on whether it is circulating in serum or replicating in the liver.
A New Primary Effusion Lymphoma-Derived Cell Line Yields a Highly Infectious Kaposi's Sarcoma Herpesvirus-Containing Supernatant
Authors: Cannon Jennifer S. , Ciufo Dolores , Hawkins Anita L. , Griffin Constance A. , Borowitz Michael J. , Hayward Gary S. , Ambinder Richard F.
Published: July 2002

A primary effusion lymphoma (PEL) cell line, JSC-1, that yields highly infectious Kaposi's sarcoma herpesvirus (KSHV) supernatants was established from the ascitic fluid of a human immunodeficiency virus-positive patient. Flow cytometry showed strong expression of CD45 and lambda light-chain restriction. Southern blot hybridization showed immunoglobulin heavy-chain gene rearrangements in the tumor and the resultant cell line consistent with B-cell lineage. Expression of viral genes was assessed by reverse transcription-PCR and immunohistochemistry. Only latent Epstein-Barr virus (EBV) gene expression was detected, and this was at a low level. In contrast, lytic and latent KSHV gene expression were detected. Tetradecanoyl phorbol acetate and butyrate upregulated KSHV lytic expression, but not EBV lytic expression. Viral supernatant from JSC-1 was much more efficient at infecting primary human dermal microvascular endothelial cells (DMVECs) with KSHV than supernatants from BC-3 or BCP-1 PEL cell lines. Quantitation of viral yields produced by the PEL lines showed at least 2 orders of magnitude more DNase I-resistant KSHV DNA in the JSC-1 supernatant compared to BC-3 or BCP-1 supernatants. KSHV infection in DMVECs was associated with a change from a cobblestone to a spindle shape, LANA expression, and an increased number of mitoses.
A Significant Number of Human Immunodeficiency Virus Epitope-Specific Cytotoxic T Lymphocytes Detected by Tetramer Binding Do Not Produce Gamma Interferon
Authors: Goepfert Paul A. , Bansal Anju , Edwards Bradley H. , Ritter G. Douglas , Tellez Ildefonso , McPherson Sylvia A. , Sabbaj Steffanie , Mulligan Mark J.
Published: July 2002

Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8+T lymphocytes are discussed.
In Vivo Selection of Protease Cleavage Sites by Using Chimeric Sindbis Virus Libraries
Authors: Pacini Laura , Vitelli Alessandra , Filocamo Gessica , Bartholomew Linda , Brunetti Mirko , Tramontano Anna , Steinkühler Christian , Migliaccio Giovanni
Published: July 2002

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.
Tegument-Specific, Virus-Reactive CD4 T Cells Localize to the Cornea in Herpes Simplex Virus Interstitial Keratitis in Humans
Authors: Koelle David M. , Reymond Sigrid N. , Chen Hongbo , Kwok William W. , McClurkan Christopher , Gyaltsong Tashi , Petersdorf Effie W. , Rotkis Walter , Talley Audrey R. , Harrison Devin A.
Published: July 2002

Herpes stromal keratitis (HSK) is a prevalent and frequently vision-threatening disease associated with herpes simplex virus type 1 (HSV-1) infection. In mice, HSK progression occurs after viral clearance and requires T cells and neutrophils. One model implicates Th1-like CD4 T cells with cross-reactivity between the HSV-1 protein UL6 and a corneal autoantigen. HSK can be prevented by establishing specific immunological tolerance. However, HSK can also occur in T-cell receptor-transgenic X SCID mice lacking HSV-specific T cells. To study the pathogenesis of HSK in the natural host species, we measured local HSV-specific T-cell responses in HSK corneas removed at transplant surgery (n= 5) or control corneas (n= 2). HSV-1 DNA was detected by PCR in two specimens. HSV-specific CD4 T cells were enriched in three of the five HSK specimens and were not detectable in the control specimens. Reactivity with peptide epitopes within the tegument proteins UL21 and UL49 was documented. Responses to HSV-1 UL6 were not detected. Diverse HLA DR and DP alleles restricted these local responses. Most clones secreted gamma interferon, but not interleukin-5, in response to antigen. HSV-specific CD8 cells were also recovered. Some clones had cytotoxic-T-lymphocyte activity. The diverse specificities and HLA-restricting alleles of local virus-specific T cells in HSK are consistent with their contribution to HSK by a proinflammatory effect.
Rous Sarcoma Virus Translation Revisited: Characterization of an Internal Ribosome Entry Segment in the 5′ Leader of the Genomic RNA
Authors: Deffaud Clarence , Darlix Jean-Luc
Published: July 2002

The 5′ leader of Rous sarcoma virus (RSV) genomic RNA and of retroviruses in general is long and contains stable secondary structures that are critical in the early and late steps of virus replication such as RNA dimerization and packaging and in the process of reverse transcription. The initiation of RSV Gag translation has been reported to be 5′ cap dependent and controlled by three short open reading frames located in the 380-nucleotide leader upstream of the Gag start codon. Translation of RSV Gag would thus differ from that prevailing in other retroviruses such as murine leukemia virus, reticuloendotheliosis virus type A, and simian immunodeficiency virus, in which an internal ribosome entry segment (IRES) in the 5′ end of the genomic RNA directs efficient Gag expression despite stable 5′ secondary structures. This prompted us to investigate whether RSV Gag translation might be controlled by an IRES-dependent mechanism. The results show that the 5′ leaders of RSV and v-Src RNA exhibit IRES properties, since these viral elements can promote efficient translation of monocistronic RNAs in conditions inhibiting 5′ cap-dependent translation. When inserted between two cistrons in a canonical bicistronic construct, both the RSV and v-Src leaders promote expression of the 3′ cistron. A genetic analysis of the RSV leader allowed the identification of two nonoverlapping 5′ and 3′ leader domains with IRES activity. In addition, the v-Src leader was found to contain unique 3′ sequences promoting an efficient reinitiation of translation. Taken together, these data lead us to propose a new model for RSV translation.
Genetic Studies with the Fission Yeast
Schizosaccharomyces pombe
Suggest Involvement of Wee1, Ppa2, and Rad24 in Induction of Cell Cycle Arrest by Human Immunodeficiency Virus Type 1 Vpr
Authors: Masuda Michiaki , Nagai Yukiko , Oshima Norihito , Tanaka Koichi , Murakami Hiroshi , Igarashi Hiroko , Okayama Hiroto
Published: July 2002

Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G
2
/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast
Schizosaccharomyces pombe
, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (
cdc
phenotype) typical of G
2
/M cell cycle arrest in wild-type and various mutant strains of
S. pombe
was examined. Our results indicated that Vpr caused the
cdc
phenotype in wild-type
S. pombe
as well as in strains carrying mutations, such as the
cdc2-3w
, Δ
cdc25
,
rad1-1
, Δ
chk1
, Δ
mik1
, and Δ
ppa1
strains. However, other mutants, such as the
cdc2-1w
, Δ
wee1
, Δ
ppa2
, and Δ
rad24
strains, failed to show a distinct
cdc
phenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the
cdc
phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.
Identification of Amino Acid Residues in CD81 Critical for Interaction with Hepatitis C Virus Envelope Glycoprotein E2
Authors: Higginbottom Adrian , Quinn Elizabeth R. , Kuo Chiung-Chi , Flint Mike , Wilson Louise H. , Bianchi Elisabetta , Nicosia Alfredo , Monk Peter N. , McKeating Jane A. , Levy Shoshana
Published: July 2002

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).
Mutation Analysis of the GDD Sequence Motif of a Calicivirus RNA-Dependent RNA Polymerase
Authors: Vázquez Ana López , Alonso José M. Martín , Parra Francisco
Published: July 2002

The RNA-dependent RNA polymerase from
rabbit hemorrhagic disease virus
, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in
Escherichia coli
, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate.
Reciprocal Interactions between Human T-Lymphotropic Virus Type 1 and Prostaglandins: Implications for Viral Transmission
Authors: Moriuchi Masako , Inoue Hiroyasu , Moriuchi Hiroyuki
Published: July 2002

Human T-lymphotropic virus type 1 (HTLV-1), the etiologic agent of adult T-cell leukemia/lymphoma, is transmitted through breast milk and seminal fluid, which are rich in prostaglandins (PGs). We demonstrate that PGE2upregulates the HTLV-1 long terminal repeat promoter through the protein kinase A pathway, induces replication of HTLV-1 in peripheral blood mononuclear cells (PBMC) derived from asymptomatic carriers, and enhances transmission of HTLV-1 to cord blood mononuclear cells (CBMC). Furthermore, HTLV-1 Tax transactivates a promoter for cyclooxygenase 2, a PG synthetase, and induces PGE2expression in PBMC or CBMC. Thus, HTLV-1 interacts with and benefits from PGs, constituents of its own vehicle for transmission.
Characterization of Adenovirus-Induced Inverted Terminal Repeat-Independent Amplification of Integrated Adeno-Associated Virus
rep-cap
Sequences
Authors: Tessier Jacques , Chadeuf Gilliane , Nony Pascale , Avet-Loiseau Hervé , Moullier Philippe , Salvetti Anna
Published: July 2002

Stable packaging cell lines expressing the
rep
and
cap
genes for recombinant adeno-associated virus type 2 (rAAV-2) assembly constitute an attractive alternative to transient transfection protocols. We recently characterized a stable HeLa
rep-cap
cell clone (HeRC32) and demonstrated that upon vector transfection and adenovirus infection, efficient rAAV assembly correlated with a 100-fold amplification of the integrated
rep-cap
sequence with the inverted terminal repeats (ITRs) deleted. We now report a more detailed analysis of this phenomenon and highlight the key cellular and viral factors involved. Determination of the
rep-cap
copy number of HeRC32 cells indicated that maximum
rep-cap
amplification occurred between 24 and 48 h following adenovirus infection. Analysis by pulsed-field gel electrophoresis of adenovirus-infected HeRC32 cells indicated that amplified
rep-cap
sequences were found in an extrachromosomal form. Amplification of the
rep-cap
sequence with the ITRs deleted was not dependent on adenovirus replication and still occurred when the highly specific adenovirus polymerase was inactivated. In contrast, amplification was inhibited in the presence of aphidicolin, indicating that cellular polymerases were needed. Our study also documented that among the adenovirus gene products, the DNA-binding protein (DBP) was essential, since
rep-cap
amplification was severely abrogated when HeRC32 cells were infected at a nonpermissive temperature with an adenovirus mutant encoding a thermosensitive DBP. Furthermore, expression of DBP alone in HeRC32 cells was sufficient to induce a sustained level of
rep-cap
amplification. Finally, immunofluorescence analysis showed that HeRC32 cells expressing the DBP also simultaneously expressed the Rep proteins, suggesting a possible involvement of the latter in
rep-cap
amplification. Indeed, the lack of detectable amplification in an adenovirus-infected stable
rep-cap
HeLa cell clone unable to produce Rep proteins further supported that, among the viral gene products, both the DBP and Rep proteins are necessary to induce the targeted amplification of the integrated
rep-cap
sequences in the absence of the AAV ITRs.
Induction of Polyomavirus-Specific CD8+T Lymphocytes by Distinct Dendritic Cell Subpopulations
Authors: Drake Donald R. , Shawver Mandy L. , Hadley Annette , Butz Eric , Maliszewski Charles , Lukacher Aron E.
Published: July 2002

Dendritic cells are pivotal antigen-presenting cells for generating adaptive T-cell responses. Here, we show that dendritic cells belonging to either the myeloid-related or lymphoid-related subset are permissive for infection by mouse polyomavirus and, when loaded with a peptide corresponding to the immunodominant anti-polyomavirus CD8+T-cell epitope or infected by polyomavirus, are each capable of driving expansion of primary polyomavirus-specific CD8+T-cell responses in vivo.
Conservation of the Conformation and Positive Charges of Hepatitis C Virus E2 Envelope Glycoprotein Hypervariable Region 1 Points to a Role in Cell Attachment
Authors: Penin François , Combet Christophe , Germanidis Georgios , Frainais Pierre-Olivier , Deléage Gilbert , Pawlotsky Jean-Michel
Published: July 2002

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.
Hantavirus Infection Induces the Expression of RANTES and IP-10 without Causing Increased Permeability in Human Lung Microvascular Endothelial Cells
Authors: Sundstrom J. Bruce , McMullan Laura K. , Spiropoulou Christina F. , Hooper W. Craig , Ansari Aftab A. , Peters Clarence J. , Rollin Pierre E.
Published: July 2002

Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1β [IL-1β], IL-6, IL-8, MCP-1, MIP-1α, and MIP-1β) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-γ)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-γ for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-κB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.
Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50 Represses p53-Induced Transcriptional Activity and Apoptosis
Authors: Gwack Yousang , Hwang Seungmin , Byun Hyewon , Lim Chunghun , Kim Jin Woo , Choi Eui-Ju , Choe Joonho
Published: July 2002

Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) encodes a viral transcriptional activator which stimulates the transcription of viral early and late genes of KSHV. Here we show that ORF50 represses transcriptional activity of p53 and p53-induced apoptosis through interaction with CREB binding protein (CBP). This inhibitory effect of ORF50 on the transcriptional activity of p53 was relieved by the addition of CBP. ORF50 mutants, which are defective in interaction with CBP, lost the inhibitory effects on p53. Our data provide a framework for delineating the regulatory mechanisms used by KSHV to modulate cellular transcription and the cell cycle.
Minute Virus of Mice Initiator Protein NS1 and a Host KDWK Family Transcription Factor Must Form a Precise Ternary Complex with Origin DNA for Nicking To Occur
Authors: Christensen Jesper , Cotmore Susan F. , Tattersall Peter
Published: July 2002

Parvoviral rolling hairpin replication generates palindromic genomic concatemers whose junctions are resolved to give unit-length genomes by a process involving DNA replication initiated at origins derived from each viral telomere. The left-end origin of minute virus of mice (MVM), oriL, contains binding sites for the viral initiator nickase, NS1, and parvovirus initiation factor (PIF), a member of the emerging KDWK family of transcription factors. oriL is generated as an active form, oriL
TC
, and as an inactive form, oriL
GAA
, which contains a single additional nucleotide inserted between the NS1 and PIF sites. Here we examined the interactions on oriL
TC
which lead to activation of NS1 by PIF. The two subunits of PIF, p79 and p96, cooperatively bind two ACGT half-sites, which can be flexibly spaced. When coexpressed from recombinant baculoviruses, the PIF subunits preferentially form heterodimers which, in the presence of ATP, show cooperative binding with NS1 on oriL, but this interaction is preferentially enhanced on oriL
TC
compared to oriL
GAA
. Without ATP, NS1 is unable to bind stably to its cognate site, but PIF facilitates this interaction, rendering the NS1 binding site, but not the nick site, resistant to DNase I. Varying the spacing of the PIF half-sites shows that the distance between the NS1 binding site and the NS1-proximal half-site is critical for nickase activation, whereas the position of the distal half-site is unimportant. When expressed separately, both PIF subunits form homodimers that bind site specifically to oriL, but only complexes containing p79 activate the NS1 nickase function.
Sindbis Virus-Induced Neuronal Death Is both Necrotic and Apoptotic and Is Ameliorated by
N
-Methyl-
d
-Aspartate Receptor Antagonists
Authors: Nargi-Aizenman Jennifer L. , Griffin Diane E.
Published: July 2002

Virus infection of neurons leads to different outcomes ranging from latent and noncytolytic infection to cell death. Viruses kill neurons directly by inducing either apoptosis or necrosis or indirectly as a result of the host immune response. Sindbis virus (SV) is an alphavirus that induces apoptotic cell death both in vitro and in vivo. However, apoptotic changes are not always evident in neurons induced to die by alphavirus infection. Time lapse imaging revealed that SV-infected primary cortical neurons exhibited both apoptotic and necrotic morphological features and that uninfected neurons in the cultures also died. Antagonists of the
N
-methyl-
d
-aspartate (NMDA) subtype of glutamate receptors protected neurons from SV-induced death without affecting virus replication or SV-induced apoptotic cell death. These results provide evidence that SV infection activates neurotoxic pathways that result in aberrant NMDA receptor stimulation and damage to infected and uninfected neurons.
Human Herpesvirus 8 Envelope Glycoprotein K8.1A Interaction with the Target Cells Involves Heparan Sulfate
Authors: Wang Fu-Zhang , Akula Shaw M. , Pramod Naranatt P. , Zeng Ling , Chandran Bala
Published: July 2002

Human herpesvirus-8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus K8.1 gene encodes for two immunogenic glycoproteins, gpK8.1A and gpK8.1B, originating from spliced messages. The 228-amino-acid (aa) gpK8.1A is the predominant form associated with the virion envelope, consisting of a 167-aa region identical to gpK8.1B and a 61-aa unique region (L. Zhu, V. Puri, and B. Chandran, Virology 262:237–249, 1999). HHV-8 has a broad in vivo and in vitro cellular tropism, and our studies showed that this may be in part due to HHV-8's interaction with the ubiquitous host cell surface molecule, heparan sulfate (HS). Since HHV-8 K8.1 gene is positionally colinear to the Epstein-Barr virus (EBV) gene encoding the gp350/gp220 protein involved in EBV binding to the target cells, gpK8.1A's ability to interact with the target cells was examined. The gpK8.1A without the transmembrane and carboxyl domains (ΔTMgpK8.1A) was expressed in a baculovirus system and purified. Radiolabeled purified ΔTMgpK8.1A protein bound to the target cells, which was blocked by unlabeled ΔTMgpK8.1A. Unlabeled ΔTMgpK8.1A blocked the binding of [
3
H]thymidine-labeled purified HHV-8 to the target cells. Binding of radiolabeled ΔTMgpK8.1A to the target cells was inhibited in a dose-dependent manner by soluble heparin, a glycosaminoglycan (GAG) closely related to HS, but not by other GAGs such as chondroitin sulfate A and C,
N
-acetyl heparin and de-
N
-sulfated heparin. Cell surface absorbed ΔTMgpK8.1A was displaced by soluble heparin. Radiolabeled ΔTMgpK8.1A also bound to HS expressing Chinese hamster ovary (CHO-K1) cells, and binding to mutant CHO cell lines deficient in HS was significantly reduced. The ΔTMgpK8.1A specifically bound to heparin-agarose beads, which was inhibited by HS and heparin, but not by other GAGs. Virion envelope-associated gpK8.1A was specifically precipitated by heparin-agarose beads. These findings suggest that gpK8.1A interaction with target cells involves cell surface HS-like moieties, and HHV-8 interaction with HS could be in part mediated by virion envelope-associated gpK8.1A.
Jaagsiekte Sheep Retrovirus Env Protein Stabilizes Retrovirus Vectors against Inactivation by Lung Surfactant, Centrifugation, and Freeze-Thaw Cycling
Authors: Coil David A. , Strickler John H. , Rai Sharath K. , Miller A. Dusty
Published: July 2002

Jaagsiekte sheep retrovirus (JSRV) replicates in the lungs of sheep and causes the secretion of copious lung fluid containing the virus. Adaptation of JSRV to infection and replication in the lung and its apparent resistance to the denaturing activity of lung fluid suggest that vectors based on JSRV would be useful for gene therapy targeted to the lung. We show here that a retrovirus vector bearing the JSRV Env is stable during treatment with lung surfactant while an otherwise identical vector bearing an amphotropic Env is inactivated. Furthermore, the JSRV vector was stable during centrifugation, allowing facile vector concentration, and showed no loss of activity after six freeze-thaw cycles. However, the JSRV vector was inactivated by standard disinfectants, indicating that JSRV vectors pose no unusual safety risk related to their improved stability under other conditions.
A Novel Nonnucleoside Inhibitor Specifically Targets Cytomegalovirus DNA Maturation via the UL89 and UL56 Gene Products
Authors: Buerger Iris , Reefschlaeger Juergen , Bender Wolfgang , Eckenberg Peter , Popp Andreas , Weber Olaf , Graeper Sascha , Klenk Hans-Dieter , Ruebsamen-Waigmann Helga , Hallenberger Sabine
Published: July 2002

3-Hydroxy-2,2-dimethyl-
N
-[4({[5-(dimethylamino)-1-naphthyl]sulfonyl}amino)-phenyl]propanamide (BAY 38-4766) is a novel selective nonnucleoside inhibitor of cytomegalovirus (CMV) replication with an excellent safety profile. This compound and structural analogues inhibit neither viral DNA synthesis nor viral transcription and translation. Accumulation of dense bodies and noninfectious enveloped particles coincides with inhibition of both concatemer processing and functional cleavage at intergenomic transitions, pointing to interference with viral DNA maturation and packaging of monomeric genome lengths. Resistant virus populations, including a murine CMV (MCMV) isolate with 566-fold-decreased drug sensitivity, were selected in vitro. Sequencing of the six open reading frames (ORFs) known to be essentially involved in viral DNA cleavage and packaging identified mutations in ORFs UL56, UL89, and UL104. Construction of MCMV recombinants expressing different combinations of murine homologues of mutant UL56, UL89, and UL104 and analysis of drug susceptibilities clearly demonstrated that mutant ORFs UL89 exon II (M360I) and M56 (P202A I208N) individually confer resistance to BAY 38-4766. A combination of both mutant proteins exhibited a strong synergistic effect on resistance, reconstituting the high-resistance phenotype of the in vitro mutant. These findings are consistent with genetic mapping of resistance to TCRB (2,5,6-trichloro-1-β-
d
-ribofuranosyl benzimidazole) (P. M. Krosky et al., J. Virol. 72:4721–4728, 1998) and provide further indirect evidence that proteins encoded by UL89 and UL56 function as two subunits of the CMV terminase. While these studies also suggest that the molecular mechanism of BAY 38-4766 is distinct from that of benzimidazole ribonucleosides, they also offer an explanation for the excellent specificity and tolerability of BAY 38-4766, since mammalian DNA does not undergo comparable maturation steps.
The N-Terminal Region of the VP1 Protein of Swine Vesicular Disease Virus Contains a Neutralization Site That Arises upon Cell Attachment and Is Involved in Viral Entry
Authors: Jiménez-Clavero M. A. , Escribano-Romero E. , Douglas A. J. , Ley V.
Published: July 2002

The N-terminal region of VP1 of swine vesicular disease virus (SVDV) is highly antigenic in swine, despite its internal location in the capsid. Here we show that antibodies to this region can block infection and that allowing the virus to attach to cells increases this blockage significantly. The results indicate that upon binding to the cell, SVDV capsid undergoes a conformational change that is temperature independent and that exposes the N terminus of VP1. This process makes this region accessible to antibodies which block virus entry.
Preexisting Immunity to Poliovirus Does Not Impair the Efficacy of Recombinant Poliovirus Vaccine Vectors
Authors: Mandl Stefanie , Hix Laura , Andino Raul
Published: July 2002

Recombinant viruses are attractive candidates for the development of novel vaccines. A number of viruses have been engineered as vaccine vectors to express antigens from other pathogens or tumors. Inoculation of susceptible animals with this type of recombinant virus results in the induction of both humoral and cellular immune responses directed against the foreign antigens. A general problem to this approach is that existing immunity to the vector can diminish or completely abolish the efficacy of the viral vector. In this study, we investigated whether poliovirus recombinants are capable of inducing effective immunity to the foreign antigen in previously vaccinated animals. Antipoliovirus immunity was induced in susceptible mice by intraperitoneal immunization with live poliovirus. Immunized mice developed antibodies directed against capsid proteins that effectively neutralized poliovirus in vitro and protected animals from a lethal challenge with a high dose of pathogenic poliovirus. To test whether preexisting immunity reduces the efficacy of vaccination with recombinant poliovirus, immunized mice were inoculated with a recombinant poliovirus expressing the C-terminal half of chicken ovalbumin (Polio-Ova). Animals developed ovalbumin-specific antibodies and cytotoxic T lymphocytes (CTL). While the antibody titers observed in preimmune and naive mice were similar, the overall CTL response appeared to be reduced in preimmune mice. Importantly, vaccination with Polio-Ova was able to effectively protect preimmune mice against lethal challenge with a tumor expressing the antigen. Thus, preexisting immunity to poliovirus does not compromise seriously the efficacy of replication-competent poliovirus vaccine vectors. These results contrast with those observed for other viral vaccine vectors and suggest that preexisting immunity does not equally affect the vaccine potential of individual viral vectors.
Cocirculation of Avian H9N2 and Contemporary “Human” H3N2 Influenza A Viruses in Pigs in Southeastern China: Potential for Genetic Reassortment?
Authors: Peiris J. S. M. , Guan Y. , Markwell D. , Ghose P. , Webster R. G. , Shortridge K. F.
Published: July 2002

Pigs are permissive to both human and avian influenza viruses and have been proposed to be an intermediate host for the genesis of pandemic influenza viruses through reassortment or adaptation of avian viruses. Prospective virological surveillance carried out between March 1998 and June 2000 in Hong Kong, Special Administrative Region, People's Republic of China, on pigs imported from southeastern China, provides the first evidence of interspecies transmission of avian H9N2 viruses to pigs and documents their cocirculation with contemporary human H3N2 (A/Sydney/5/97-like, Sydney97-like) viruses. All gene segments of the porcine H9N2 viruses were closely related to viruses similar to chicken/Beijing/1/94 (H9N2), duck/Hong Kong/Y280/97 (H9N2), and the descendants of the latter virus lineage. Phylogenetic analysis suggested that repeated interspecies transmission events had occurred from the avian host to pigs. The Sydney97-like (H3N2) viruses isolated from pigs were related closely to contemporary human H3N2 viruses in all gene segments and had not undergone genetic reassortment. Cocirculation of avian H9N2 and human H3N2 viruses in pigs provides an opportunity for genetic reassortment leading to the emergence of viruses with pandemic potential.
Endothelial Cells Enhance Human Immunodeficiency Virus Type 1 Replication in Macrophages through a C/EBP-Dependent Mechanism
Authors: Lee Eileen S. , Zhou Huiyu , Henderson Andrew J.
Published: July 2002

Macrophages are early targets of human immunodeficiency virus type 1 (HIV-1) infection and serve as potential reservoirs for long-term infection. Through inflammatory mediators and direct cell contact, infected macrophages interact with neighboring cell populations, such as the endothelium, which create a microenvironment favorable for HIV-1 replication. We hypothesize that the transcriptional activator C/EBPβ is critical for macrophages to respond to endothelial cell-derived signals. We show that endothelial cells significantly enhance C/EBPβ binding activity and HIV-1 replication in macrophages. This increase in HIV-1 transcription is due to cell-cell contact as well as the production of soluble factors, mediated in part by ICAM-1 and interleukin 6, respectively. Furthermore, C/EBP factors are necessary for endothelial cell-dependent activation of HIV-1 transcription in macrophages, and HIV-1 induction can be inhibited by a C/EBP dominant-negative protein. In addition, C/EBP binding sites are necessary for efficient LTR activity and HIV-1 replication in the presence of endothelial cells. Taken together, these results indicate that endothelial cells, through the activation of C/EBPβ, provide a microenvironment that supports HIV-1 replication in monocytes/macrophages.
Gene Array Analysis Reveals Changes in Peripheral Nervous System Gene Expression following Stimuli That Result in Reactivation of Latent Herpes Simplex Virus Type 1: Induction of Transcription Factor Bcl-3
Authors: Tsavachidou Dimitra , Podrzucki Wawrzyniec , Seykora John , Berger Shelley L.
Published: July 2002

The earliest events within the peripheral mammalian nervous system that cause herpes simplex virus type 1 (HSV-1) to reactivate from latency are unknown but are highly likely to include altered regulation of cellular transcription factors. Using gene array analysis, we have examined the changes that occur in cellular mRNA levels in mouse trigeminal ganglia following explantation, a stimulus that results in HSV-1 reactivation from latency. We have detected both increased and decreased expression levels of particular cellular transcripts, which include RNAs encoding neuronal factors, transcription factors, and factors involved in the cell cycle. Among the transcription factors that are upregulated is Bcl-3, a coactivator for NFκB. We have confirmed these increases in Bcl-3 transcription levels using reverse transcription-PCR and S1 nuclease protection assays. In addition, we have shown Bcl-3 upregulation at the protein level. Importantly, Bcl-3 RNA levels were found to increase specifically in neuronal cells within the trigeminal ganglia. We discuss a potential role for this factor in upregulating ICP0 transcription, which is an important viral event for initiation of HSV-1 reactivation.
Requirements for Minus-Strand Transfer Catalyzed by Rous Sarcoma Virus Reverse Transcriptase
Authors: Werner Susanne , Vogel-Bachmayr Karin , Hollinderbäumer Britta , Wöhrl Birgitta M.
Published: July 2002

We have examined the specific minus-strand transfer reactions that occur after the synthesis of minus strong-stop DNA and nonspecific strand switching on homopolymeric poly(rA) templates with different types of Rous sarcoma virus (RSV) reverse transcriptases. Three different types of reverse transcriptases can be isolated from virions of RSV: heterodimeric αβ and homodimeric α and β. The mechanism of minus-strand transfer was examined using a model primer-template substrate corresponding to the 5′- and 3′-terminal RNA regions of the RSV genome. The results reveal that the RNase H activity of RSV reverse transcriptases is required for minus-strand transfer. Less than 2% of strand transfer of the extended product is detectable with RNase H-deficient enzymes. We could show that the α homodimer lacking the integrase domain can perform strand transfer almost as efficiently as the αβ and αPol heterodimers. In contrast, the activities of β and Pol for minus-strand transfer are reduced. Furthermore, a two- to fivefold increase in minus-strand transfer activities was observed in the presence of human immunodeficiency virus type 1 nucleocapsid protein.