bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determinants of Syncytium Formation in Microglia by Human Immunodeficiency Virus Type 1: Role of the V1/V2 Domains
Authors: Shieh Joseph T. C. , Martín Julio , Baltuch Gordon , Malim Michael H. , González-Scarano Francisco 
Published: July 2002

ABSTRACT

Microglia are the main reservoir for human immunodeficiency virus type 1 (HIV-1) in the central nervous system (CNS), and multinucleated giant cells, the result of fusion of HIV-1-infected microglia and brain macrophages, are the neuropathologic hallmark of HIV dementia. One potential explanation for the formation of syncytia is viral adaptation for these CD4
+
CNS cells. HIV-1
BORI-15
, a virus adapted to growth in microglia by sequential passage in vitro, mediates high levels of fusion and replicates more efficiently in microglia and monocyte-derived-macrophages than its unpassaged parent (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. Gonzalez-Scarano, J. Virol. 70:7654–7662, 1996). Since the interaction between the viral envelope glycoprotein and CD4 and the chemokine receptor mediates fusion and plays a key role in tropism, we have analyzed the HIV-1
BORI-15
env
as a fusogen and in recombinant and pseudotyped viruses. Its syncytium-forming phenotype is not the result of a switch in coreceptor use but rather of the HIV-1
BORI-15
envelope-mediated fusion of CD4
+
CCR5
+
cells with greater efficiency than that of its parental strain, either by itself or in the context of a recombinant virus. Genetic analysis indicated that the syncytium-forming phenotype was due to four discrete amino acid differences in V1/V2, with a single-amino-acid change between the parent and the adapted virus (E153G) responsible for the majority of the effect. Additionally, HIV-1
BORI-15
env
-pseudotyped viruses were less sensitive to decreases in the levels of CD4 on transfected 293T cells, leading to the hypothesis that the differences in V1/V2 alter the interaction between this envelope and CD4 or CCR5, or both. In sum, the characterization of the envelope of HIV-1
BORI-15
, a highly fusogenic glycoprotein with genetic determinants in V1/V2, may lead to a better understanding of the relationship between HIV replication and syncytium formation in the CNS and of the importance of this region of gp120 in the interaction with CD4 and CCR5.
A Role for Single-Stranded Templates in Cell-Free Adeno-Associated Virus DNA Replication
Authors: Ward Peter , Linden R. Michael 
Published: July 2002

ABSTRACT
Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.
Impaired Intracellular Trafficking of Adeno-Associated Virus Type 2 Vectors Limits Efficient Transduction of Murine Fibroblasts
Authors: Hansen Jonathan , Qing Keyun , Kwon Hyung-Joo , Mah Cathryn , Srivastava Arun 
Published: July 2002

ABSTRACT
Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the more commonly used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer and transgene expression by AAV vectors require that the following two obstacles be overcome. First, the target cell must express the receptor and the coreceptor for AAV infection, and second, the cell must allow for viral second-strand DNA synthesis. We now describe a third obstacle, impaired intracellular trafficking of AAV to the nucleus, which results in the lack of transgene expression in murine fibroblasts which do express the AAV receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis. We document that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently transduced by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-mediated gene transfer has implications for the optimal use of these vectors in human gene therapy.
Rous Sarcoma Virus DR Posttranscriptional Elements Use a Novel RNA Export Pathway
Authors: Paca Robert E. , Ogert Robert A. , Hibbert Catherine S. , Izaurralde Elisa , Beemon Karen L. 
Published: July 2002

ABSTRACT

Rous sarcoma virus (RSV), a simple retrovirus, needs to export unspliced viral RNA from the nucleus to the cytoplasm, circumventing the host cell restriction on cytoplasmic expression of intron-containing RNA. The cytoplasmic accumulation of full-length viral RNA is promoted by two
cis
-acting direct repeat (DR) elements that flank the
src
gene; at least one copy of the DR sequence is necessary for viral replication. We show here that the DR mediates export of a reporter construct from the nucleus, suggesting it is a constitutive transport element (CTE). In contrast, human immunodeficiency virus type 1 (HIV-1) and other complex retroviruses encode accessory proteins, Rev or Rex, which promote export of incompletely spliced viral transcripts. This RNA export pathway is CRM1 dependent and can be blocked by the cytotoxic agent leptomycin B. We show here that DR-mediated export is CRM1 independent, suggesting that RSV uses a different export pathway from that of HIV-1 and other complex retroviruses. The simian retroviruses have a CTE which interacts with the cellular Tap export protein. However, we were unable to detect binding of the RSV DR RNA to Tap, suggesting it may use a different export pathway from that of the simian retroviruses. These data suggest that the RSV DR element uses a novel nucleocytoplasmic export pathway.
Impairment of Human Immunodeficiency Virus Type 1 (HIV-1) Entry into Jurkat T Cells by Constitutive Expression of the HIV-1 Vpr Protein: Role of CD4 Down-Modulation
Authors: Conti Lucia , Varano Barbara , Gauzzi M. Cristina , Matarrese Paola , Federico Maurizio , Malorni Walter , Belardelli Filippo , Gessani Sandra 
Published: July 2002

ABSTRACT
Jurkat T-cell clones, stably expressing the human immunodeficiency virus type 1 (HIV-1) Vpr protein, exhibited an impaired susceptibility to HIV-1 infection. A marked down-modulation of surface CD4 receptors was detected in Vpr-expressing clones with respect to control cells. Likewise, a reduced CD4 expression was also observed in parental Jurkat cells infected with wild-type but not with Vpr-mutant HIV-1. Notably, Vpr-expressing clones were fully susceptible to infection with a vesicular stomatitis virus G protein-pseudotyped HIV-1 virus, indicating that a block at the level of viral entry was responsible for the inhibition of viral replication. The effect exerted by Vpr on HIV replication and CD4 expression suggests that this protein can regulate both the establishment of a productive HIV-1 infection and CD4-mediated T-cell functions.
Interaction of the Rabies Virus P Protein with the LC8 Dynein Light Chain
Authors: Raux Hélène , Flamand Anne , Blondel Danielle 
Published: July 2002

ABSTRACT
The rabies virus P protein is involved in viral transcription and replication but its precise function is not clear. We investigated the role of P (CVS strain) by searching for cellular partners by using a two-hybrid screening of a PC12 cDNA library. We isolated a cDNA encoding a 10-kDa dynein light chain (LC8). LC8 is a component of cytoplasmic dynein involved in the minus end-directed movement of organelles along microtubules. We confirmed that this molecule interacts with P by coimmunoprecipitation in infected cells and in cells transfected with a plasmid encoding P protein. LC8 was also detected in virus particles. Series of deletions from the N- and C-terminal ends of P protein were used to map the LC8-binding domain to the central part of P (residues 138 to 172). These results are relevant to speculate that dynein may be involved in the axonal transport of rabies virus along microtubules through neuron cells.
Adeno-Associated Virus RNAs Appear in a Temporal Order and Their Splicing Is Stimulated during Coinfection with Adenovirus
Authors: Mouw Matthew B. , Pintel David J. 
Published: July 2002

ABSTRACT
We have used a quantitative RNase protection assay to characterize the relative accumulation and abundance of individual adeno-associated virus type 2 (AAV) RNAs throughout the course of AAV-adenovirus coinfections and preinfections. We have demonstrated that there is a previously unrecognized temporal order to the appearance of AAV RNAs. First, unspliced P5-generated transcripts, which encode Rep78, were detectable prior to the significant accumulation of other AAV RNAs. Ultimately, as previously demonstrated, P19-generated products accumulated to levels greater than those generated from P5, and P40-generated transcripts predominated in the total RNA pool. Second, the percentage of each class of AAV RNA that was spliced increased during infection, and the degree of this increase was different for the P5/P19 products than for those generated by P40. At late times postcoinfection, approximately 90% of P40 products, but only approximately 50% of RNAs generated by P5 and P19, were seen to be spliced; thus, the AAV intron was removed to different final levels from these different RNA species. We have shown that each of the AAV RNAs is quite stable; the majority of each RNA species persisted 6 h after treatment with actinomycin D. Quantification of the accumulation of individual AAV RNAs, over intervals during which degradation was negligible, allowed us to infer that at late times during infection the relative strength of P5, P19, and P40 was approximately 1:3:18, respectively, consistent with the steady-state accumulated levels of the RNAs generated by each promoter. All AAV RNAs exited to the cytoplasm with similar efficiencies in the presence or absence of adenovirus; however, adenovirus coinfection appeared to stimulate total splicing of AAV RNAs and the relative use of the downstream intron acceptor. Our results confirm and extend previous observations concerning the appearance and processing of AAV-generated RNAs.
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

ABSTRACTFour 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACTThe 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.