bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunization of Cats against Feline Immunodeficiency Virus (FIV) Infection by Using Minimalistic Immunogenic Defined Gene Expression Vector Vaccines Expressing FIV gp140 Alone or with Feline Interleukin-12 (IL-12), IL-16, or a CpG Motif
Authors: Leutenegger Christian M. , Boretti Felicitas S. , Mislin Caroline N. , Flynn J. Norman , Schroff Matthias , Habel Andre , Junghans Claas , Koenig-Merediz Sven A. , Sigrist Brigitte , Aubert Andre , Pedersen Niels C. , Wittig Burghardt , Lutz Hans
Published: July 2002

Four groups of cats, each containing four animals, were immunized at 0, 3, and 6 weeks with minimalistic immunogenic defined gene expression vector (MIDGE) vaccines containing the gene(s) for feline immunodeficiency virus (FIV) gp140, FIV gp140 and feline interleukin-12 (IL-12), FIV gp140 and feline IL-16, or FIV gp140 and a CpG motif. MIDGEs were coated onto gold beads and injected intradermally with a gene gun. A fifth group of four cats were immunized in an identical manner but with blank gold beads. All cats were challenge exposed to virulent FIV 4 weeks following the final immunization, and the course of infection was monitored. The two groups of cats immunized with the FIV gp140 gene alone or with blank gold particles became highly viremic and seroconverted as early as 4 weeks after infection. In contrast, three of four cats immunized with FIV gp140 in combination with feline IL-12 failed to become viremic or seropositive, as has been shown elsewhere (F. S. Boretti, C. M. Leutenegger, C. Mislin, et al., AIDS 14:1749–1757, 2000). Here we show the effect of IL-12 when used as an adjuvant on the viral RNA and DNA load and on the cytokine profile. In addition, the two groups of cats immunized either with gp140 and IL-16 or with gp140 and the CpG had greatly reduced viremia. Protection correlated weakly with cytotoxic T-lymphocyte activity and increased cytokine transcription of IL-12, gamma interferon, and IL-10 by peripheral blood mononuclear cells in the postchallenge period. This study extends the data on IL-12 and provides new results on CpG motifs and IL-16 used as adjuvants in the FIV cat model.
Expression of the Two Major Envelope Proteins of Equine Arteritis Virus as a Heterodimer Is Necessary for Induction of Neutralizing Antibodies in Mice Immunized with Recombinant Venezuelan Equine Encephalitis Virus Replicon Particles
Authors: Balasuriya Udeni B. R. , Heidner Hans W. , Hedges Jodi F. , Williams Jacqueline C. , Davis Nancy L. , Johnston Robert E. , MacLachlan N. James
Published: July 2002

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G
L
and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G
L
/M). Open reading frame 5 (ORF5) encodes the G
L
protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114–128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in
trans
in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G
L
, pVR21-M, and pVR100-G
L
/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G
L
/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G
L
protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.
Separable Mechanisms of Attachment and Cell Uptake during Retrovirus Infection
Authors: Sharma Sanjai , Miyanohara Atsushi , Friedmann Theodore
Published: July 2002

In the absence of viral envelope gene expression, cells expressing human immunodeficiency virus type 1 (HIV-1)
gag
and
pol
, accessory HIV functions, and a vector genome RNA produce and secrete large amount of noninfectious virus-like particles (VLPs) into the conditioned medium. After partial purification, such HIV-1 VLPs can be made infectious in cell-free conditions in vitro by complex formation with lipofection reagents or with the G protein of vesicular stomatitis virus (VSV-G). The resulting in vitro-modified HIV-1 particles are able to infect nondividing cells. Infectivity of envelope-free HIV VLPs can also be induced by prior modification of target cells through exposure to partially purified VSV-G vesicles. Similarly, infection can be carried out by attachment of envelope-free noninfectious VLPs to unmodified cells followed by subsequent treatment of cells with VSV-G. We interpret these findings to indicate that interaction between a viral envelope and a cell surface receptor is not necessary for the initial virus binding to the cells but is required for subsequent cell entry and infection.
Role of Vif in Stability of the Human Immunodeficiency Virus Type 1 Core
Authors: Öhagen Åsa , Gabuzda Dana
Published: July 2002

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown that
vif
-defective virions exhibit structural abnormalities in the virus core and are defective in the ability to complete proviral DNA synthesis in acutely infected cells. We developed novel assays to assess the relative stability of the core in HIV-1 virions. Using these assays, we examined the role of Vif in the stability of the HIV-1 core. The integrity of the core was examined following virion permeabilization or removal of the lipid envelope and treatment with various triggers, including S100 cytosol, deoxynucleoside triphosphates, detergents, NaCl, and buffers of different pH to mimic aspects of the uncoating and disassembly process which occurs after virus entry but preceding or during reverse transcription.
vif
mutant cores were more sensitive to disruption by all triggers tested than wild-type cores, as determined by endogenous reverse transcriptase (RT) assays, biochemical analyses, and electron microscopy. RT and the p7 nucleocapsid protein were released more readily from
vif
mutant virions than from wild-type virions, suggesting that the internal nucleocapsid is less stably packaged in the absence of Vif. Purified cores could be isolated from wild-type but not
vif
mutant virions by sedimentation through detergent-treated gradients. These results demonstrate that Vif increases the stability of virion cores. This may permit efficient viral DNA synthesis by preventing premature degradation or disassembly of viral nucleoprotein complexes during early events after virus entry.
Lentivirus Nef Specifically Activates Pak2
Authors: Arora Vivek K. , Molina Rene P. , Foster John L. , Blakemore John L. , Chernoff Jonathan , Fredericksen Brenda L. , Garcia J. Victor
Published: July 2002

Nef proteins from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have been found to associate with an active cellular serine/threonine kinase designated Nef-associated kinase (Nak). The exact identity of Nak remains controversial, with two recent studies indicating that Nak may be either Pak1 or Pak2. In this study, we investigated the hypothesis that such discrepancies arise from the use of different Nef alleles or different cell types by individual investigators. We first confirm that Pak2 but not Pak1 is cleaved by caspase 3 in vitro and then demonstrate that Nak is caspase 3 sensitive, regardless of Nef allele or cell type used. We tested
nef
alleles from three lentiviruses (HIV-1 SF2, HIV-1 NL4-3, and SIVmac239) and used multiple cell lines of myeloid, lymphoid, and nonhematopoietic origin to evaluate the identity of Nak. We demonstrate that ectopically expressed Pak2 can substitute for Nak, while ectopically expressed Pak1 cannot. We then show that Nef specifically mediates the robust activation of ectopically expressed Pak2, directly demonstrating that Nef regulates Pak2 activity and does not merely associate with activated Pak2. We report that most of the active Pak2 is found bound to Nef, although a fraction is not. In contrast, only a small amount of Nef is found associated with Pak2. We conclude that Nak is Pak2 and that Nef specifically mediates Pak2 activation in a low-abundance complex. These results will facilitate both the elucidation of the role of Nef in pathogenesis and the development of specific inhibitors of this highly conserved function of Nef.
Ongoing Viral Replication Is Required for Gammaherpesvirus 68-Induced Vascular Damage
Authors: Dal Canto Albert J. , Virgin Herbert W. , Speck Samuel H.
Published: July 2002

The role of autoimmunity in large-vessel vasculitis in humans remains unclear. We have previously shown that infection of gamma interferon receptor knockout (IFN-γR
−/−
) mice with gammaherpesvirus 68 (γHV68) results in severe inflammation of the large elastic arteries that is pathologically similar to the lesions observed in Takayasu's arteritis, the nongranulomatous variant of temporal arteritis, and Kawasaki's disease (K. E. Weck et al., Nat. Med. 3:1346–1353, 1997). Here we define the mechanism of damage to the elastic arteries. We show that there is a persistent productive infection of the media of the large elastic vessels. In addition, we demonstrate that persistent virus replication is necessary for chronic arteritis, since antiviral therapy of mice with established disease resulted in increased survival, clearance of viral antigen from the media of the affected vessel, and dramatic amelioration of arteritic lesions. These data argue that ongoing virus replication, rather than autoimmunity, is the cause of γHV68-induced elastic arteritis.
Ternary Complex Factors and Cofactors Are Essential for Human T-Cell Leukemia Virus Type 1 Tax Transactivation of the Serum Response Element
Authors: Shuh Maureen , Derse David
Published: July 2002

The human T-cell leukemia virus type 1 Tax protein activates the expression of cellular immediate early genes controlled by the serum response element (SRE), which contains both the serum response factor (SRF) binding element (CArG box) and the ternary complex factor (TCF) binding element (Ets box). We show that TCF binding is necessary for Tax activation of the SRE and that Tax directly interacts with TCFs in vitro. In addition, Tax interactions with CREB binding protein (CBP) and p300- and CBP-associated factor were found to be essential for Tax activation of SRF-mediated transcription.
Profound Protection against Respiratory Challenge with a Lethal H7N7 Influenza A Virus by Increasing the Magnitude of CD8+T-Cell Memory
Authors: Christensen Jan P. , Doherty Peter C. , Branum Kristen C. , Riberdy Janice M.
Published: July 2002

The recall of CD8+T-cell memory established by infectingH-2bmice with an H1N1 influenza A virus provided a measure of protection against an extremely virulent H7N7 virus. The numbers of CD8+effector and memory T cells specific for the shared, immunodominant DbNP366epitope were greatly increased subsequent to the H7N7 challenge, and though lung titers remained as high as those in naive controls for 5 days or more, the virus was cleared more rapidly. Expanding the CD8+memory T-cell pool (<0.5 to >10%) by sequential priming with two different influenza A viruses (H3N2→H1N1) gave much better protection. Though the H7N7 virus initially grew to equivalent titers in the lungs of naive and double-primed mice, the replicative phase was substantially controlled within 3 days. This tertiary H7N7 challenge caused little increase in the magnitude of the CD8+DbNP366+T-cell pool, and only a portion of the memory population in the lymphoid tissue could be shown to proliferate. The great majority of the CD8+DbNP366+set that localized to the infected respiratory tract had, however, cycled at least once, though recent cell division was shown not to be a prerequisite for T-cell extravasation. The selective induction of CD8+T-cell memory can thus greatly limit the damage caused by a virulent influenza A virus, with the extent of protection being directly related to the number of available responders. Furthermore, a large pool of CD8+memory T cells may be only partially utilized to deal with a potentially lethal influenza infection.
Development of a Primary Tamarin Hepatocyte Culture System for GB Virus-B: a Surrogate Model for Hepatitis C Virus
Authors: Beames Burton , Chavez Deborah , Guerra Bernadette , Notvall Lena , Brasky Kathleen M. , Lanford Robert E.
Published: July 2002

GB virus-B (GBV-B) causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Phylogenetically, GBV-B is the closest relative to hepatitis C virus (HCV), and thus GBV-B infection of tamarins represents a powerful surrogate model system for the study of HCV. In this study, the course of infection of GBV-B in tamarins was followed using a real-time 5′ exonuclease (TaqMan) reverse transcription-PCR assay to determine the level of GBV-B in the serum. Peak viremia levels exceeded 10
9
genome equivalents/ml, followed by viral clearance within 14 to 16 weeks. Rechallenge of animals that had cleared infection resulted in viremia that was limited to 1 week, suggestive of a strong protective immune response. A robust tissue culture system for GBV-B was developed using primary cultures of tamarin hepatocytes. Hepatocytes obtained from a GBV-B-infected animal maintained high levels of cell-associated viral RNA and virion secretion for 42 days of culture. In vitro infection of normal hepatocytes resulted in rapid amplification of cell-associated viral RNA and secretion of up to 10
7
genome equivalents/ml of culture supernatant. In addition, infection could be monitored by immunofluorescence staining for GBV-B nonstructural NS3 protein. This model system overcomes many of the current obstacles to HCV research, including low levels of viral replication, lack of a small primate animal model, and lack of a reproducible tissue culture system.
Lytic but Not Latent Replication of Epstein-Barr Virus Is Associated with PML and Induces Sequential Release of Nuclear Domain 10 Proteins
Authors: Bell Peter , Lieberman Paul M. , Maul Gerd G.
Published: July 2002

Nuclear domains called ND10 (nuclear domain 10) are discrete nuclear protein aggregations characterized by a set of interferon-upregulated proteins including Sp100 and PML, where papova-, adeno-, and herpesviruses begin their transcription and DNA replication. Both the alpha- and betaherpesvirus subfamilies disrupt ND10 upon infection by dispersing and/or destroying ND10-associated proteins. We studied the effect of the gammaherpesvirus Epstein-Barr virus (EBV) on ND10 and its spatial distribution in the nucleus of cells during latency and lytic reactivation. In latently infected Burkitt's lymphoma, lymphoblastoid, and D98/HR1 cells, ND10 were intact, as judged by immunofluorescence localization of PML, Sp100, NDP55, and Daxx. Fluorescent in situ hybridization revealed no association between viral episomes and ND10 during latency, implying that the maintenance replication of EBV, which depends on host cell proliferation, occurs independent of ND10. As in mitosis, the EBV genomes were attached to interphase chromosomes, suggesting that they are unable to move freely within the interchromosomal space and thus unable to associate with the interchromosomally located ND10 or other nuclear domains. Upon lytic activation, ND10 became dispersed in cells expressing lytic proteins. Redistribution of ND10 proteins occurred sequentially at different stages of the lytic cycle, with Sp100, Daxx, and NDP55 dispersed before and PML dispersed after the onset of lytic replication. ND10 remnants were retained until the early stages of lytic replication, and replicating EBV genomes were frequently found beside this nuclear domain; the number of replication domains was usually lower than the average latent virus frequency. Thus, latency does not require or induce interaction of EBV with ND10 for transcription and replication, whereas lytic replication triggers dispersion of ND10 proteins and occurs in close association with PML aggregates. The required movement of chromosome-attached latent EBV episomes to ND10 after reactivation from latency might include physical release of the chromosome-bound episomes. Only episomes contacting ND10 after such a release might be able to begin lytic replication.
Evolution of the Human Immunodeficiency Virus Type 1 Envelope during Infection Reveals Molecular Corollaries of Specificity for Coreceptor Utilization and AIDS Pathogenesis
Authors: Hu Qin-xue , Barry Ashley Perkins , Wang Zi-xuan , Connolly Shanon M. , Peiper Stephen C. , Greenberg Michael L.
Published: July 2002

The evolution of human immunodeficiency virus type 1 infection is associated with a shift in the target cell population, driven by variability in coreceptor utilization resulting from diversity in
env
. To elucidate the potential consequences of these changes for Env-mediated fusion over the course of AIDS, we examined the biological properties of serial viral isolates and determined coreceptor utilization by the products of
env
cloned from two individuals, followed from the detection of seroconversion throughout the course of their infection. One had a typical course, and the other had an accelerated progression. Early isolates were non-syncytium inducing, and the corresponding Env exclusively utilized CCR5, whereas Env from late phases of infection showed restricted utilization of CXCR4 in both patients. Env from subject SC24, who had a standard progression, demonstrated multitropism, manifested by utilization of CCR3, CXCR4, and CCR5 in the intervening period. In contrast, Env from patient SC51, who experienced early conversion to the syncytium-inducing phenotype, developed dualtropic coreceptor utilization of CCR5 and CXCR4. Genetic analysis of
env
from each isolate revealed that those with an X4 phenotype formed a distinct subcluster within each subject. Analysis of chimeras constructed from R5 and multispecific
env
from patient SC24 demonstrated that while the V3 domain played a dominant role in determining coreceptor utilization, sequences in the V4–V5 region also contributed to the latter phenotype. Immunoprecipitation experiments confirmed that the hybrid Env proteins were expressed at similar levels. These experiments demonstrate that progression from the R5 to X4 phenotype may occur through a multi- or dual-tropic intermediate and that multiple domains contribute to this process.
Expanded Host Cell Tropism and Cytopathic Properties of Feline Immunodeficiency Virus Strain PPR Subsequent to Passage through Interleukin-2-Independent T Cells
Authors: Lerner Danica L. , Elder John H.
Published: July 2002

A cytopathic variant of feline immunodeficiency virus (FIV) strain PPR emerged after passage of wild-type virus on an interleukin-2-independent cell line. The virus, termed FIV-PPRglial, displayed a phenotype markedly different from the parental virus, including the ability to productively infect previously refractory cell lines, induction of large syncytia, and accelerated kinetic properties. A chimeric molecular clone, FIV-PPRchim42, containing the FIV-PPRglial envelope within the backbone of FIV-PPR, exhibited all the characteristics of the FIV-PPRglial phenotype, demonstrating that the viral envelope was responsible for the acquired traits. Subsequent molecular characterization revealed that the FIV-PPRglial envelope contained five amino acid substitutions relative to wild-type FIV-PPR. Mutagenic analyses further demonstrated that the acquired phenotype was minimally attributable to a combination of three mutations, specifically, a glutamine-to-proline change within the second constant domain of the surface protein (SU); a threonine-to-proline change within the V4 loop, also in the SU; and a premature stop codon in the cytoplasmic tail of the transmembrane protein. All three changes were required to produce the FIV-PPRglial phenotype. Cotransfection studies with mutant viruses in combination with each other and with FIV-PPR indicated that the truncated cytoplasmic tail was responsible for the induction of syncytium formation. Receptor usage analyses were pursued, and distinctions were observed between FIV-PPR and FIV-PPRglial. In vitro infections with FIV-PPR, FIV-PPRglial, and FIV-34TF10 on two adherent cell lines were ablated in the presence of SDF1α, the natural ligand for CXCR4. In contrast, viral infection of T cells was not limited to CXCR4 usage, and inhibition studies indicate the potential involvement of a CC chemokine receptor.
Mutation of Predicted Virion Pit Residues Alters Binding of Theiler's Murine Encephalomyelitis Virus to BHK-21 Cells
Authors: Hertzler Shannon , Luo Ming , Lipton Howard L.
Published: July 2002

Theiler's murine encephalomyelitis virus
(TMEV), a natural pathogen of mice, is a member of the genus
Cardiovirus
in the family
Picornaviridae
. Structural studies indicate that the cardiovirus pit, a deep depression on the surface of the virion, is involved in receptor attachment; however, this notion has never been systematically tested. Therefore, we used BeAn virus, a less virulent TMEV, to study the effect of site-specific mutation of selected pit amino acids on viral binding as well as other replicative functions of the virus. Four amino acids within the pit, V1091, P1153, A1225 and P3179, were selected for mutagenesis to evaluate their role in receptor attachment. Three amino acid replacements were made at each site, the first a conservative replacement, followed by progressively more radical amino acid changes in order to detect variable effects at each site. A total of seven viable mutant viruses were recovered and characterized for their binding properties to BHK-21 cells, capsid stability at 40°C, viral RNA replication, single- and multistep growth kinetics, and virus translation. Our data implicate three of these residues in TMEV-cell receptor attachment.
Resistance to Replication of Human Immunodeficiency Virus Challenge in SCID-Hu Mice Engrafted with Peripheral Blood Mononuclear Cells of Nonprogressors Is Mediated by CD8+T Cells and Associated with a Proliferative Response to p24 Antigen
Authors: de Quiros Juan Carlos Lopez Bernaldo , Shupert W. Lesley , McNeil Andrew C. , Gea-Banacloche Juan C. , Flanigan Mark , Savage Ann , Martino Lisa , Weiskopf Emma E. , Imamichi Hiromi , Zhang Yi-Ming , Adelsburger Joseph , Stevens Randy , Murphy Philip M. , Zimmerman Peter A. , Hallahan Claire W. , Davey Richard T. , Connors Mark
Published: July 2002

High levels of resistance to challenge with human immunodeficiency virus type 1 SF162 were observed in animals engrafted with peripheral blood mononuclear cells of four long-term nonprogressors (LTNPs). Resistance was abrogated by depletion of CD8+T cells in vivo and was observed only in LTNPs with proliferative responses to p24. In a subgroup of nonprogressors, CD8+T cells mediated restriction of challenge viruses, and this response was associated with strong proliferative responses to p24 antigen.
Hepatitis C Virus-Encoded Enzymatic Activities and Conserved RNA Elements in the 3′ Nontranslated Region Are Essential for Virus Replication In Vivo
Authors: Kolykhalov Alexander A. , Mihalik Kathy , Feinstone Stephen M. , Rice Charles M.
Published: July 2002

Hepatitis C virus (HCV) infection is a widespread major human health concern. Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection in two chimpanzees by intrahepatic injection of in vitro-transcribed RNA (A. A. Kolykhalov et al., Science 277:570–574, 1997). In order to validate potential targets for development of anti-HCV therapeutics, we constructed six mutant derivatives of this prototype infectious clone. Four clones contained point mutations ablating the activity of the NS2-3 protease, the NS3-4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highly conserved 98-base sequence at the 3′ terminus of the HCV genome RNA. The RNA transcript from each of the six clones was injected intrahepatically into a chimpanzee. No signs of HCV infection were detected in the 8 months following the injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion. These data suggest that these four HCV-encoded enzymatic activities and the conserved 3′ terminal RNA element are essential for productive replication in vivo.
Sensitivity to a Nonpeptidic Compound (RPR103611) Blocking Human Immunodeficiency Virus Type 1 Env-Mediated Fusion Depends on Sequence and Accessibility of the gp41 Loop Region
Authors: Labrosse Béatrice , Treboute Carole , Alizon Marc
Published: July 2002

The triterpene RPR103611 is an efficient inhibitor of membrane fusion mediated by the envelope proteins (Env, gp120-gp41) of CXCR4-dependent (X4) human immunodeficiency virus type 1 (HIV-1) strains, such as HIV-1
LAI
(LAI). Other X4 strains, such as HIV-1
NDK
(NDK), and CCR5-dependent (R5) HIV-1 strains, such as HIV-1
ADA
(ADA), were totally resistant to RPR103611. Analysis of chimeric LAI-NDK Env proteins identified a fragment of the NDK gp41 ectodomain determining drug resistance. A single difference at position 91, leucine in LAI and histidine in NDK, apparently accounted for their sensitivity or resistance to RPR103611. We had previously identified a mutation of isoleucine 84 to serine in a drug escape LAI variant. Both I84 and L91 are located in the “loop region” of gp41 separating the proximal and distal helix domains. Nonpolar residues in this region therefore appear to be important for the antiviral activity of RPR103611 and are possibly part of its target. However, another mechanism had to be envisaged to explain the drug resistance of ADA, since its gp41 loop region was almost identical to that of LAI. Fusion mediated by chimeric Env consisting of LAI gp120 and ADA gp41, or the reciprocal construct, was fully blocked by RPR103611. The gp120-gp41 complex of R5 strains is stable, relative to that of X4 strains, and this stability could play a role in their drug resistance. Indeed, when the postbinding steps of ADA infection were performed under mildly acidic conditions (pH 6.5 or 6.0), a treatment expected to favor dissociation of gp120, we achieved almost complete neutralization by RPR103611. The drug resistance of NDK was partially overcome by preincubating virus with soluble CD4, a gp120 ligand inducing conformational changes in the Env complex. The antiviral efficacy of RPR103611 therefore depends on the sequence of the gp41 loop and the stability of the gp120-gp41 complex, which could limit the accessibility of this target.
Erythroid Cells Rendered Erythropoietin Independent by Infection with Friend Spleen Focus-Forming Virus Show Constitutive Activation of Phosphatidylinositol 3-Kinase and Akt Kinase: Involvement of Insulin Receptor Substrate-Related Adapter Proteins
Authors: Nishigaki Kazuo , Hanson Charlotte , Ohashi Takashi , Thompson Delores , Muszynski Karen , Ruscetti Sandra
Published: July 2002

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). In an effort to understand how SFFV causes Epo independence, we have been examining erythroid cells rendered factor independent by SFFV infection for constitutive activation of signal-transducing molecules. Previous studies from our laboratory showed that various signal-transducing molecules known to be activated by Epo, including Stat proteins and components of the Raf-1/MAP kinase pathway, are constitutively activated in SFFV-infected erythroid cells in the absence of Epo. Since another signal transduction pathway involving activation of phosphatidylinositol 3-kinase (PI 3-kinase) after Epo stimulation plays an important role in erythroid cell proliferation and differentiation, we carried out studies to determine if this pathway was also activated in SFFV-infected cells in the absence of Epo. Our studies show that PI 3-kinase is constitutively activated in erythroid cells rendered factor independent by infection with SFFV and that PI 3-kinase activity, but not Epo receptor tyrosine phosphorylation, is required for the proliferation of these cells in the absence of Epo. We further show that in SFFV-infected erythroid cells grown in the absence of Epo, PI 3-kinase associates with the insulin receptor substrate (IRS)-related adapter molecules IRS-2, Gab1, and Gab2, which are constitutively tyrosine phosphorylated in SFFV-infected cells. Finally, Akt, a protein kinase that is one of the downstream effectors of PI 3-kinase, and SHIP, a lipid phosphatase that is important for Akt activation through PI 3-kinase, are both tyrosine phosphorylated in SFFV-infected cells grown in the absence of Epo. Our results indicate that induction of Epo independence by SFFV requires the activation of PI 3-kinase and suggest that constitutive activation of this kinase in SFFV-infected cells may occur primarily through interaction of PI 3-kinase with constitutively phosphorylated IRS-related adapter molecules.
Structure and Coding Content of CST (BART) Family RNAs of Epstein-Barr Virus
Authors: Smith Paul R. , de Jesus Orlando , Turner David , Hollyoake Martine , Karstegl Claudio Elgueta , Griffin Beverly E. , Karran Lorraine , Wang Yilong , Hayward S. Diane , Farrell Paul J.
Published: July 2002

CST (BART BARF0) family viral RNAs are expressed in several types of Epstein-Barr virus (EBV) infection, including EBV-associated cancers. Many different spliced forms of these RNAs have been described; here we have clarified the structures of some of the more abundant splicing patterns. We report the first cDNAs representing a full-length CST mRNA from a clone library and further characterize the transcription start. The relative abundance of splicing patterns and genomic analysis of the open reading frames (ORFs) suggest that, in addition to the much studied BARF0 ORF, there may be important products made from some of the upstream ORFs in the CST RNAs. Potential biological functions are identified for two of these. The product of the RPMS1 ORF is shown to be a nuclear protein that can bind to the CBF1 component of Notch signal transduction. RPMS1 can inhibit the transcription activation induced through CBF1 by NotchIC or EBNA-2. The protein product of another CST ORF, A73, is shown to be a cytoplasmic protein which can interact with the cell RACK1 protein. Since RACK1 modulates signaling from protein kinase C and Src tyrosine kinases, the results suggest a possible role for CST products in growth control, perhaps consistent with the abundant transcription of CST RNAs in cancers such as nasopharyngeal carcinoma.
Bystander Sensitization to Activation-Induced Cell Death as a Mechanism of Virus-Induced Immune Suppression
Authors: Zarozinski Christopher C. , McNally James M. , Lohman Barbara L. , Daniels Keith A. , Welsh Raymond M.
Published: July 2002

Viral infections which induce strong T-cell responses are often characterized by a period of transient immunodeficiency associated with the failure of host T cells to proliferate in response to mitogens or to mount memory recall responses to other antigens. During acute infections, most of the activated, proliferating virus-specific T cells are sensitized to undergo apoptosis on strong T-cell receptor (TCR) stimulation, but it has not been known why memory T cells not specific for the virus fail to proliferate on exposure to their cognate antigen. Using a lymphocytic choriomeningitis virus (LCMV) infection model in which LCMV-immune Thy 1.1+splenocytes are adoptively transferred into Thy 1.2+LCMV carrier mice, we demonstrate here that T cells clearly defined as not specific for the virus are sensitized to undergo activation-induced cell death on TCR stimulation in vitro. This bystander sensitization was in part dependent on the expression of Fas ligand (FasL) on the activated virus-specific cells and gamma interferon (IFN-γ) receptor expression on the bystander T cells. We propose that FasL from highly activated antiviral T cells may sensitize IFN-γ-conditioned T cells not specific for the virus to undergo apoptosis rather than to proliferate on encountering antigen. This may in part explain the failure of memory T cells to respond to recall antigens during acute and persistent viral infections.
Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+T-Lymphocyte Reactivity during Combination Antiretroviral Therapy in HIV-1-Infected Patients with Advanced Immunodeficiency
Authors: Rinaldo Charles R. , Huang Xiao-Li , Fan Zheng , Margolick Joseph B. , Borowski Luann , Hoji Aki , Kalinyak Christine , McMahon Deborah K. , Riddler Sharon A. , Hildebrand William H. , Day Richard B. , Mellors John W.
Published: July 2002

The long-term efficacy of combination antiretroviral therapy may relate to augmentation of anti-human immunodeficiency virus type 1 (HIV-1) CD8+T-cell responses. We found that prolonged treatment of late-stage HIV-1-infected patients with a protease inhibitor and two nucleoside reverse transcriptase inhibitors failed to restore sustained, high levels of HIV-1-specific, HLA class I-restricted, cytotoxic-T-lymphocyte precursors and gamma interferon (IFN-γ) production by CD8+T cells. In some patients, particularly those initiating three-drug combination therapy simultaneously rather than sequentially, there were early, transient increases in the frequency of anti-HIV-1 CD8+T cells that correlated with decreases in HIV-1 RNA and increases in T-cell counts. In the other patients, HIV-1-specific T-cell functions either failed to increase or declined from baseline during triple-drug therapy, even though some of these patients showed suppression of plasma HIV-1 RNA. These effects of combination therapy were not unique to HIV-1 specific T-cell responses, since similar effects were noted for CD8+T cells specific for the cytomegalovirus pp65 matrix protein. The level and breadth of CD8+cell reactivity to HLA A*02 HIV-1 epitopes, as determined by IFN-γ production and HLA tetramer staining after combination therapy, were related to the corresponding responses prior to treatment. There was, however, a stable, residual population of potentially immunocompetent HIV-1-specific T cells remaining after therapy, as shown by tetramer staining of CD8+CD45RO+cells. These results indicate that new strategies will be needed to target residual, immunocompetent HIV-1-specific CD8+T cells to enhance the effectiveness of antiretroviral therapy in patients with advanced immunodeficiency.
Duck Hepatitis B Virus Expresses a Regulatory HBx-Like Protein from a Hidden Open Reading Frame
Authors: Chang Shau-Feng , Netter Hans Jürgen , Hildt Eberhard , Schuster Ralph , Schaefer Stephan , Hsu Yin-Chen , Rang Andreas , Will Hans
Published: July 2002

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf–mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.
Use of Intron-Disrupted Polyadenylation Sites To Enhance Expression and Safety of Retroviral Vectors
Authors: Ismail Said I. , Rohll Jonathan B. , Kingsman Susan M. , Kingsman Alan J. , Uden Mark
Published: July 2002

Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256–5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy.
Kinetics of Hepadnavirus Loss from the Liver during Inhibition of Viral DNA Synthesis
Authors: Zhu Yuao , Yamamoto Toshiki , Cullen John , Saputelli Jeffry , Aldrich Carol E. , Miller Darren S. , Litwin Samuel , Furman Phillip A. , Jilbert Allison R. , Mason William S.
Published: July 2002

Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl-β-
l
-arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of ∼33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis.
Tissue Distribution and Timing of Appearance of Polytropic Envelope Recombinants during Infection with SL3-3 Murine Leukemia Virus or Its Weakly Pathogenic SL3ΔMyb5 Mutant
Authors: Rulli Karen , Lobelle-Rich Patricia A. , Trubetskoy Alla , Lenz Jack , Levy Laura S.
Published: July 2002

A time course analysis was performed to identify the sites of formation and timing of appearance of polytropic recombinant viruses following infection of NIH/Swiss mice with the murine retrovirus SL3-3 murine leukemia virus (SL3) or with a weakly pathogenic mutant termed SL3ΔMyb5. The results indicated that (i) polytropic recombinant viruses occur initially in the thymus of SL3-infected animals, (ii) the timing of appearance of polytropic recombinants in bone marrow is not consistent with their participation in the previously reported formation of transplantable tumor-forming cells at 3 to 4 week postinoculation, and (iii) the efficient generation of recombinant virus is correlated with efficient tumor induction.
Amino Acids of Epstein-Barr Virus Nuclear Antigen 3A Essential for Repression of Jκ-Mediated Transcription and Their Evolutionary Conservation
Authors: Dalbiès-Tran Rozenn , Stigger-Rosser Evelyn , Dotson Travis , Sample Clare E.
Published: July 2002

Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor Jκ. This interaction downregulates transcription mediated by EBNA-2 and Jκ. To identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with Jκ. Using a reporter gene assay based on the recruitment of Jκ by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of Jκ to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with Jκ. To determine the biological significance of the interaction of EBNA-3A with Jκ, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with Jκ through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and Jκ. The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes.
Cell Cycle Analysis of Epstein-Barr Virus-Infected Cells following Treatment with Lytic Cycle-Inducing Agents
Authors: Rodriguez Antonio , Jung Eun Joo , Flemington Erik K.
Published: July 2002

While Epstein-Barr virus (EBV) latency-associated gene expression is associated with cell cycle progression, the relationship between the EBV lytic program and the cell cycle is less clear. Using four different EBV lytic induction systems, we address the relationship between lytic cycle activation and the cell cycle. In three of these systems, G
0
or G
1
cell growth arrest signaling is observed prior to detection of the EBV immediate-early gene product Zta. In tetradecanoyl phorbol acetate-treated P3HR1 cultures and in 5-iodo-2′-deoxyuridine-treated NPC-KT cultures, cell cycle analysis of Zta-expressing cell populations showed a significant G
1
bias during the early stages of lytic cycle progression. In contrast, treatment of the cell line Akata with anti-immunoglobulin (Ig) results in rapid induction of immediate-early gene expression, and accordingly, activation of the immediate-early gene product Zta precedes significant anti-Ig-induced cell cycle effects. Nevertheless, cell cycle analysis of the Zta-expressing population following anti-Ig treatment shows a bias for cells in G
1
, indicating that anti-Ig-mediated induction of Zta occurs more efficiently in cells traversing G
1
. Last, although 5-azacytidine treatment of Rael cells results in a G
1
arrest in the total cell population which precedes the induction of Zta, cell cycle analysis of the Zta-expressing population shows a significant bias for cells with an apparent G
2
/M DNA content. This bias may result, in part, from activation of Zta expression following demethylation of the Zta promoter during S-phase. Together, these studies indicate that induction of Zta occurs through several distinct mechanisms, some of which may involve checkpoint signaling.
Efficient Conversion of Normal Prion Protein (PrP) by Abnormal Hamster PrP Is Determined by Homology at Amino Acid Residue 155
Authors: Priola Suzette A. , Chabry Joëlle , Chan Kaman
Published: July 2002

In the transmissible spongiform encephalopathies, disease is closely associated with the conversion of the normal proteinase K-sensitive host prion protein (PrP-sen) to the abnormal proteinase K-resistant form (PrP-res). Amino acid sequence homology between PrP-res and PrP-sen is important in the formation of new PrP-res and thus in the efficient transmission of infectivity across species barriers. It was previously shown that the generation of mouse PrP-res was strongly influenced by homology between PrP-sen and PrP-res at amino acid residue 138, a residue located in a region of loop structure common to PrP molecules from many different species. In order to determine if homology at residue 138 also affected the formation of PrP-res in a different animal species, we assayed the ability of hamster PrP-res to convert a panel of recombinant PrP-sen molecules to protease-resistant PrP in a cell-free conversion system. Homology at amino acid residue 138 was not critical for the formation of protease-resistant hamster PrP. Rather, homology between PrP-sen and hamster PrP-res at amino acid residue 155 determined the efficiency of formation of a protease-resistant product induced by hamster PrP-res. Structurally, residue 155 resides in a turn at the end of the first alpha helix in hamster PrP-sen; this feature is not present in mouse PrP-sen. Thus, our data suggest that PrP-res molecules isolated from scrapie-infected brains of different animal species have different PrP-sen structural requirements for the efficient formation of protease-resistant PrP.