bluuebunny/crossref_metadata_2025 huggingface dataset:
Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics
Authors: Wagner Ralf , Wolff Thorsten , Herwig Astrid , Pleschka Stephan , Klenk Hans-Dieter
Published: July 2002

The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377–8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.
Glycosphingolipids Promote Entry of a Broad Range of Human Immunodeficiency Virus Type 1 Isolates into Cell Lines Expressing CD4, CXCR4, and/or CCR5
Authors: Hug Peter , Lin Han-Ming Joseph , Korte Thomas , Xiao Xiaodong , Dimitrov Dimiter S. , Wang Ji Ming , Puri Anu , Blumenthal Robert
Published: July 2002

Treatment of human osteosarcoma cells, expressing CD4 and various chemokine receptors, with the glucosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), blocked target membrane glycosphingolipid (GSL) biosynthesis and reduced the susceptibility of cells to infection and fusion mediated by envelope glycoproteins from a variety of human immunodeficiency virus type 1 (HIV-1) isolates that utilize CXCR4 and/or CCR5. PPMP treatment of the cell lines did not significantly change the cell surface expression of CD4, CXCR4, and/or CCR5, nor did it alter the chemokine receptor association with CD4. PPMP-treated cells exhibited no changes in chemokine-induced Ca2+mobilization and chemotaxis. However, massive envelope glycoprotein conformational changes triggered by CD4 and the appropriate chemokine receptor on the target membrane were inhibited when the target cells were treated with PPMP. Addition of various purified GSLs to PPMP-treated target cells showed that for all isolates tested, globotriaosylceramide (Gb3) was the most potent GSL in restoring the fusion susceptibility of target cells with cells expressing HIV-1 envelope glycoproteins; addition of the monosialoganglioside GM3 yielded a slight enhancement of fusion susceptibility. Our data are consistent with the notion that a limited number of specific GSL species serve as crucial elements in organizing gp120-gp41, CD4, and an appropriate chemokine receptor into a membrane fusion complex.
CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes
Authors: Arthos James , Rubbert Andrea , Rabin Ronald L. , Cicala Claudia , Machado Elizabeth , Wildt Kathryne , Hanbach Meredith , Steenbeke Tavis D. , Swofford Ruth , Farber Joshua M. , Fauci Anthony S.
Published: July 2002

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1α, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.
Continued Circulation in China of Highly Pathogenic Avian Influenza Viruses Encoding the Hemagglutinin Gene Associated with the 1997 H5N1 Outbreak in Poultry and Humans
Authors: Cauthen Angela N. , Swayne David E. , Schultz-Cherry Stacey , Perdue Michael L. , Suarez David L.
Published: July 2002

Since the outbreak in humans of an H5N1 avian influenza virus in Hong Kong in 1997, poultry entering the live-bird markets of Hong Kong have been closely monitored for infection with avian influenza. In March 1999, this monitoring system detected geese that were serologically positive for H5N1 avian influenza virus, but the birds were marketed before they could be sampled for virus. However, viral isolates were obtained by swabbing the cages that housed the geese. These samples, known collectively as A/Environment/Hong Kong/437/99 (A/Env/HK/437/99), contained four viral isolates, which were compared to the 1997 H5N1 Hong Kong isolates. Analysis of A/Env/HK/437/99 viruses revealed that the four isolates are nearly identical genetically and are most closely related to A/Goose/Guangdong/1/96. These isolates and the 1997 H5N1 Hong Kong viruses encode common hemagglutinin (H5) genes that have identical hemagglutinin cleavage sites. Thus, the pathogenicity of the A/Env/HK/437/99 viruses was compared in chickens and in mice to evaluate the potential for disease outbreaks in poultry and humans. The A/Env/HK/437/99 isolates were highly pathogenic in chickens but caused a longer mean death time and had altered cell tropism compared to A/Hong Kong/156/97 (A/HK/156/97). Like A/HK/156/97, the A/Env/HK/437/99 viruses replicated in mice and remained localized to the respiratory tract. However, the A/Env/HK/437/99 isolates caused only mild pathological lesions in these tissues and no clinical signs of disease or death. As a measure of the immune response to these viruses, transforming growth factor β levels were determined in the serum of infected mice and showed elevated levels for the A/Env/HK/437/99 viruses compared to the A/HK/156/97 viruses. This study is the first to characterize the A/Env/HK/437/99 viruses in both avian and mammalian species, evaluating the H5 gene from the 1997 Hong Kong H5N1 isolates in a different genetic background. Our findings reveal that at least one of the avian influenza virus genes encoded by the 1997 H5N1 Hong Kong viruses continues to circulate in mainland China and that this gene is important for pathogenesis in chickens but is not the sole determinant of pathogenicity in mice. There is evidence that H9N2 viruses, which have internal genes in common with the 1997 H5N1 Hong Kong isolates, are still circulating in Hong Kong and China as well, providing a heterogeneous gene pool for viral reassortment. The implications of these findings for the potential for human disease are discussed.
Viral Activation of Interleukin-15 (IL-15): Characterization of a Virus-Inducible Element in the IL-15 Promoter Region
Authors: Azimi Nazli , Tagaya Yutaka , Mariner Jennifer , Waldmann Thomas A.
Published: July 2002

We identified an interferon regulatory factor motif (IRF-E) upstream of an NF-κB binding site in the interleukin-15 (IL-15) promoter. Since these two motifs are part of the virus-inducible enhancer region of the beta interferon promoter, we speculated that there might be similar responses of these two genes to stimuli such as viruses. To test this hypothesis, L929 cells were infected with Newcastle disease virus (NDV), which led to the induction of IL-15 mRNA and protein expression. Using IL-15 promoter-reporter deletion constructs, a virus-inducible region, encompassing IRF-E, NF-κB, and a 13-nucleotide sequence flanked by these two motifs, was mapped to the −295-to-−243 position relative to the transcription initiation site. Using cotransfection studies, it was demonstrated that all three motifs were essential to achieve the maximum promoter activity induced by IRF-1 and NF-κB expression plasmids. The presence of a virus-inducible region in the IL-15 promoter suggests a role for IL-15 as a component of host antiviral defense mechanisms.
E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1
Authors: Advani Sunil J. , Weichselbaum Ralph R. , Roizman Bernard
Published: July 2002

The transition from G1to S phase in the cell cycle requires sequential activation of cyclin-dependent kinase 4 (cdk4) and cdk2, which phosphorylate the retinoblastoma protein, causing the release of E2F. Free E2F upregulates the transcription of genes involved in S phase and cell cycle progression. Recent studies from this and other laboratories have shown that herpes simplex virus 1 stabilizes cyclin D3 early in infection and that early events in viral replication are sensitive to inhibitors of some cdks. On the other hand cdk2 is not activated. Here we report studies on the status of members of E2F family in cycling HEp-2 and HeLa cells and quiescent serum-starved, contact-inhibited human lung fibroblasts. The results show that (i) at 8 h postinfection or thereafter, E2F-1 and E2F-5 were posttranslationally modified and/or translocated from nucleus to the cytoplasm, (ii) E2F-4 was hyperphophorylated, and (iii) overall, E2F binding to cognate DNA sites was decreased at late times after infection. These results concurrent with those cited above indicate that late in infection activation of S-phase genes is blocked both by posttranslational modification and translocation of members of E2F family to inactive compartments and by the absence of active cdk2. The observation that E2F were also posttranslationally modified in quiescent human lung fibroblasts that were not in S phase at the time of infection suggests that specific viral gene products are responsible for modification of the members of E2F family and raises the possibility that in infected cells, activation of the S phase gene is an early event in viral infection and is then shut off at late times. This is consistent with the timing of stabilization of cyclin D3 and the events blocked by inhibitors of cdks.
Sodium-Dependent Neutral Amino Acid Transporter Type 1 Is an Auxiliary Receptor for Baboon Endogenous Retrovirus
Authors: Marin Mariana , Tailor Chetankumar S. , Nouri Ali , Kabat David
Published: July 2002

The baboon endogenous retrovirus (BaEV) belongs to a large, widely dispersed interference group that includes the RD114 feline endogenous virus and primate type D retroviruses. Recently, we and another laboratory independently cloned a human receptor for these viruses and identified it as the human sodium-dependent neutral amino acid transporter type 2 (hASCT2). Interestingly, mouse and rat cells are efficiently infected by BaEV but only become susceptible to RD114 and type D retroviruses if the cells are pretreated with tunicamycin, an inhibitor of protein N-linked glycosylation. To investigate this host range difference, we cloned and analyzed NIH Swiss mouse ASCT2 (mASCT2). Surprisingly, mASCT2 did not mediate BaEV infection, which implied that mouse cells might have an alternative receptor for this virus. In addition, elimination of the two N-linked oligosaccharides from mASCT2 by mutagenesis, as substantiated by protein
N
-glycosidase F digestions and Western immunoblotting, did not enable it to function as a receptor for RD114 or type D retroviruses. Based on these results, we found that the related ASCT1 transporters of humans and mice are efficient receptors for BaEV but are relatively inactive for RD114 and type D retroviruses. Furthermore, elimination of the two N-linked oligosaccharides from extracellular loop 2 of mASCT1 by mutagenesis enabled it to function as an efficient receptor for RD114 and type D retroviruses. Thus, we infer that the tunicamycin-dependent infection of mouse cells by RD114 and type D retroviruses is caused by deglycosylation of mASCT1, which unmasks previously buried sites for viral interactions. In contrast, BaEV efficiently employs the glycosylated forms of mASCT1 that occur normally in untreated mouse cells.
Induction of CD8 T Cells by Vaccination with Recombinant Adenovirus Expressing Human Papillomavirus Type 16 E5 Gene Reduces Tumor Growth
Authors: Liu Dai-Wei , Tsao Yeou-Ping , Hsieh Chang-Hsun , Hsieh Jer-Tsong , Kung John T. , Chiang Chia-Lien , Huang Shyh-Jer , Chen Show-Li
Published: July 2002

The potential of the E5 protein as a tumor vaccine candidate has not been explored yet. In this study, we evaluate the human papillomavirus type 16 (HPV-16) E5 protein delivered by an adenovirus vector as a tumor vaccine for cervical lesions. The results demonstrate that a single intramuscular injection of a recombinant adenovirus carrying the HPV-16 E5 gene into syngeneic animals can reduce the growth of tumors which contain E5 gene expression. Moreover, the E5 vaccine-induced tumor protection occurs through CD8 T cells but not through CD4 T cells in in vitro assays. In addition, our studies using knockout mice with distinct T-cell deficiencies confirm that cytotoxic T-lymphocyte-induced tumor protection is CD8 dependent but CD4 independent. Hence, HPV-16 E5 can be regarded as a tumor rejection antigen.
Lentivirus Vector Gene Expression during ES Cell-Derived Hematopoietic Development In Vitro
Authors: Hamaguchi Isao , Woods Niels-Bjarne , Panagopoulos Ioannis , Andersson Elisabet , Mikkola Hanna , Fahlman Cecilia , Zufferey Romain , Carlsson Leif , Trono Didier , Karlsson Stefan
Published: July 2002

The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.
Molecular Evolution of a Type 1 Wild-Vaccine Poliovirus Recombinant during Widespread Circulation in China
Authors: Liu Hong-Mei , Zheng Du-Ping , Zhang Li-Bi , Oberste M. Steven , Pallansch Mark A. , Kew Olen M.
Published: July 2002

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3′-terminal sequences of VP1 (115 nt) and the 5′ half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3′ half of 2A were more distantly related (<90% nucleotide sequence match) to those of other contemporary wild polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of ∼3.7 × 10
−2
substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection.
Enrichment of Immediate-Early 1 (m123/pp89) Peptide-Specific CD8 T Cells in a Pulmonary CD62LloMemory-Effector Cell Pool during Latent Murine Cytomegalovirus Infection of the Lungs
Authors: Holtappels Rafaela , Pahl-Seibert Marcus-Folker , Thomas Doris , Reddehase Matthias J.
Published: July 2002

Interstitial cytomegalovirus (CMV) pneumonia is a clinically relevant complication in recipients of bone marrow transplantation (BMT). Recent data for a model of experimental syngeneic BMT and concomitant infection of BALB/c mice with murine CMV (mCMV) have documented the persistence of tissue-resident CD8 T cells after clearance of productive infection of the lungs (J. Podlech, R. Holtappels, M.-F. Pahl-Seibert, H.-P. Steffens, and M. J. Reddehase, J. Virol. 74:7496–7507, 2000). It was proposed that these cells represent antiviral “standby” memory cells whose functional role might be to help prevent reactivation of latent virus. The pool of pulmonary CD8 T cells was composed of two subsets defined by the T-cell activation marker L-selectin (CD62L): a CD62Lhisubset of quiescent memory cells, and a CD62Llosubset of recently resensitized memory-effector cells. In this study, we have continued this line of investigation by quantitating CD8 T cells specific for the three currently published antigenic peptides of mCMV: peptide YPHFMPTNL processed from the immediate-early protein IE1 (pp89), and peptides YGPSLYRRF and AYAGLFTPL, derived from the early proteins m04 (gp34) and M84 (p65), respectively. IE1-specific CD8 T cells dominated in acute-phase pulmonary infiltrates and were selectively enriched in latently infected lungs. Notably, most IE1-specific CD8 T cells were found to belong to the CD62Llosubset representing memory-effector cells. This finding is in accordance with the interpretation that IE1-specific CD8 T cells are frequently resensitized during latent infection of the lungs and may thus be involved in the maintenance of mCMV latency.
Interaction of Recombinant Norwalk Virus Particles with the 105-Kilodalton Cellular Binding Protein, a Candidate Receptor Molecule for Virus Attachment
Authors: Tamura Masaru , Natori Katsuro , Kobayashi Masahiko , Miyamura Tatsuo , Takeda Naokazu
Published: July 2002

Norwalk virus(NV), responsible for outbreaks of acute gastroenteritis, comprises the species of the genus Norwalk-like viruses in the familyCaliciviridae. Although the study of the molecular biology of NV has been hampered by a lack of culture systems or small experimental animal models, virus-like particles (VLPs) generated with recombinant baculoviruses harboring the capsid protein gene of NV provide a useful tool for investigating NV-cell interactions. In this study, the attachment of the recombinant VLPs derived from the Ueno virus (UEV), a strain belonging to the genogroup II NVs, to mammalian and insect cells was examined. Kinetic analyses of the binding of the recombinant VLPs of the UEV (rUEVs) to Caco-2 cells demonstrated that the binding was specific and occurred in a dose-dependent manner. Approximately 7.5% of the prebound rUEVs were internalized into the Caco-2 cells. Enzymatic and chemical modification of Caco-2 cell surface molecules suggested that the binding was directly mediated by a protein-protein interaction. A virus overlay protein-binding assay (VOPBA) indicated that rUEVs appeared to bind to a 105-kDa molecule, designated as the NV attachment (NORVA) protein. Furthermore, the assay indicated that its native conformational structure was indispensable for the binding activity. In Caco-2 cells, the NORVA protein was detected when VOPBA was carried out with the VLPs from Seto and Funabashi viruses, which are serologically different NVs from UEV, used as probes. The binding of rUEVs to NORVA protein was also observed in six mammalian cell lines other than Caco-2. These data suggest that the attachment of NV to mammalian cells is mediated by NORVA protein, which is ubiquitously expressed in the mammalian cells. The present study is the first report on the role of the cellular molecule in the binding of recombinant VLPs of NV.
Nectin2α (PRR2α or HveB) and Nectin2δ Are Low-Efficiency Mediators for Entry of Herpes Simplex Virus Mutants Carrying the Leu25Pro Substitution in Glycoprotein D
Authors: Lopez Marc , Cocchi Francesca , Menotti Laura , Avitabile Elisa , Dubreuil Patrice , Campadelli-Fiume Gabriella
Published: July 2002

The receptors for entry of herpes simplex viruses 1 and 2 (HSV-1 and -2), widely expressed in human cell lines, are members of a subset of the immunoglobulin superfamily exemplified by herpesvirus entry mediator C (HveC) and the herpesvirus immunoglobulin-like receptor (HIgR). This report focuses on two members of this subset, herpesvirus entry mediator B (HveB), recently designated nectin2/PRR2α, and its splice variant isoform, nectin2/PRR2δ. Nectin2α and -δ share the ectodomain but differ in the transmembrane and cytoplasmic regions. HveB was reported to enable entry of HSV-1 carrying mutations in glycoprotein D (gD) and of HSV-2, but not of wild-type (wt) HSV-1. We report that (i) both nectin2α and -δ served as receptors for the entry of HSV-1 mutant viruses HSV-1(U10) and -(U21) and AP7
r
that carry the Leu25Pro substitution in gD but not for HSV-1 mutants U30 and R5000 that carry the Ser140 or Ala185 substitution in gD. All of these mutants were able to overcome the block to entry mediated by expression of wt gD. (ii) Infection of cells expressing nectin2α or -δ required exposure to multiplicities of infection about 100-fold higher than those required to infect cells expressing HveC or HIgR. (iii) gD from HSV-1(U21) bound in vitro soluble forms of nectin2. The association was weaker than that to the soluble form of HveC/HIgR. Binding of wt HSV-1 gD to soluble nectin2 was not detectable. (iv) A major region of nectin2 functional in virus entry mapped to the V domain, located at the N terminus.
Partial Rescue of the Vif-Negative Phenotype of Mutant Human Immunodeficiency Virus Type 1 Strains from Nonpermissive Cells by Intravirion Reverse Transcription
Authors: Dornadula Geethanjali , Yang Shicheng , Pomerantz Roger J. , Zhang Hui
Published: July 2002

Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus type I (HIV-1) and is essential for viral replication. It appears that Vif functions in the virus-producing cells and affects viral assembly. Viruses with defects in thevifgene (vif−) generated from the “nonpermissive cells” are not able to complete reverse transcription. In previous studies, it was demonstrated that defects in thevifgene also affect endogenous reverse transcription (ERT) when mild detergents were utilized to permeabilize the viral envelope. In this report, we demonstrate that defects in thevifgene have much less of an effect on ERT if detergent is not used. When ERT was driven by addition of deoxyribonucleoside triphosphates (dNTPs) at high concentrations, certain levels of plus-strand viral DNA could also be achieved. Interestingly, ifvif− viruses, generated from nonpermissive cells and harboring large quantities of viral DNA generated by ERT, were allowed to infect permissive cells, they could partially bypass the block at intracellular reverse transcription, through whichvif− viruses without dNTP treatment could not pass. Consequently, viral infectivity can be partially rescued from thevif− phenotype. Based on our observations, we suggest thatvifdefects may cause the reverse transcription complex (RT complex) to become sensitive to mild detergent treatments within HIV-1 virions and become unstable in the target cells, such that the process of reverse transcription cannot be efficiently supported. Further dissection of RT complexes ofvif− viruses may be key to uncovering the molecular mechanism(s) of Vif in HIV-1 pathogenesis.
Subcellular Redistribution of Pit-2 P
i
Transporter/Amphotropic Leukemia Virus (A-MuLV) Receptor in A-MuLV-Infected NIH 3T3 Fibroblasts: Involvement in Superinfection Interference
Authors: Jobbagy Zsolt , Garfield Susan , Baptiste Lisa , Eiden Maribeth V. , Anderson Wayne B.
Published: July 2002

Amphotropic murine leukemia virus (A-MuLV) utilizes the Pit-2 sodium-dependent phosphate transporter as a cell surface receptor to infect mammalian cells. Previous studies established that infection of cells with A-MuLV resulted in the specific down-modulation of phosphate uptake mediated by Pit-2 and in resistance to superinfection with A-MuLV. To study the mechanisms underlying these phenomena, we constructed plasmids capable of efficiently expressing ɛ epitope- and green fluorescent protein (GFP)-tagged human Pit-2 proteins in mammalian cells. Overexpression of ɛ-epitope-tagged Pit-2 transporters in NIH 3T3 cells resulted in a marked increase in sodium-dependent P
i
uptake. This increase in P
i
uptake was specifically blocked by A-MuLV infection but not by infection with ecotropic MuLV (E-MuLV) (which utilizes a cationic amino acid transporter, not Pit-2, as a cell surface receptor). These data, together with the finding that the tagged Pit-2 transporters retained their A-MuLV receptor function, indicate that the insertion of epitope tags does not affect either retrovirus receptor or P
i
transporter function. The overexpressed epitope-tagged transporters were detected in cell lysates, by Western blot analysis using both ɛ-epitope- and GFP-specific antibodies as well as with Pit-2 antiserum. Both the epitope- and GFP-tagged transporters showed almost exclusive plasma membrane localization when expressed in NIH 3T3 cells, as determined by laser scanning confocal microscopy. Importantly, when NIH 3T3 cells expressing these proteins were productively infected with A-MuLV, the tagged transporters and receptors were no longer detected in the plasma membrane but rather were localized to a punctate structure within the cytosolic compartment distinct from Golgi, endoplasmic reticulum, endosomes, lysosomes, and mitochondria. The intracellular Pit-2 pool colocalized with the virus in A-MuLV-infected cells. A similar redistribution of the tagged Pit-2 proteins was not observed following infection with E-MuLV, indicating that the redistribution of Pit-2 is not directly attributable to general effects associated with retroviral infection but rather is a specific consequence of A-MuLV–Pit-2 interactions.
Induction of Indolamine 2,3-Dioxygenase in Primary Human Macrophages by Human Immunodeficiency Virus Type 1 Is Strain Dependent
Authors: Grant Ross S. , Naif Hassan , Thuruthyil Sophie J. , Nasr Najla , Littlejohn Tamantha , Takikawa Osamu , Kapoor Vimal
Published: July 2002

Increased kynurenine pathway metabolism has been implicated in the etiology of AIDS dementia complex (ADC). The rate-limiting enzyme for this pathway is indolamine 2,3-dioxygenase (IDO). We tested the efficacy of different strains of human immunodeficiency virus type 1 (HIV1-BaL, HIV1-JRFL, and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain-derived HIV-1 isolates, laboratory-adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the highly replicating macrophage-tropic LA strain HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day 8 postinfection. Abundant HIV-1 replication did not reduce the ability of exogenous gamma interferon (IFN-γ) to induce IDO and kynurenine synthesis in HIV-infected MDM. The addition of anti-IFN-γ antibody to MDM infected with HIV1-JRFL resulted in an absence of detectable IDO protein after 48 h and a decrease of 64% ± 1% in supernatant kynurenine concentration. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent kynurenine metabolism in MDM. The induction of IDO, while apparently independent of replication capacity, appears to be mediated by a transient production of IFN-γ in MDM responding to the initial infection with selected strains of HIV-1.
Virus Entry Is a Major Determinant of Cell Tropism of Edmonston and Wild-Type Strains of Measles Virus as Revealed by Vesicular Stomatitis Virus Pseudotypes Bearing Their Envelope Proteins
Authors: Tatsuo Hironobu , Okuma Kazu , Tanaka Kotaro , Ono Nobuyiki , Minagawa Hiroko , Takade Akemi , Matsuura Yoshiharu , Yanagi Yusuke
Published: July 2002

The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVΔG*). MV glycoproteins were efficiently incorporated into VSVΔG*, producing the VSV pseudotypes. VSVΔG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVΔG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVΔG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVΔG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role.
A Single Deletion in the Membrane-Proximal Region of the Sindbis Virus Glycoprotein E2 Endodomain Blocks Virus Assembly
Authors: Hernandez Raquel , Lee Heuiran , Nelson Christine , Brown Dennis T.
Published: July 2002

The envelopment of the Sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390–400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531–541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, ΔK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, ΔK391 produced wild-type levels of virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow.
Dominance of Virus over Host Factors in Cross-Species Activation of Human Cytomegalovirus Early Gene Expression
Authors: Garcı́a-Ramı́rez José J. , Ruchti Franziska , Huang Huang , Simmen Kenneth , Angulo Ana , Ghazal Peter
Published: July 2002

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential
cis
-acting sequences when complemented in
trans
. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced (∼30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.
The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles
Authors: Heljasvaara Ritva , Rodrı́guez Dolores , Risco Cristina , Carrascosa José L. , Esteban Mariano , Rodrı́guez Juan Ramón
Published: July 2002

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the
Escherichia coli lacI
operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-β-
d
-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.
Relative Dominance of Epitope-Specific Cytotoxic T-Lymphocyte Responses in Human Immunodeficiency Virus Type 1-Infected Persons with Shared HLA Alleles
Authors: Day Cheryl L. , Shea Amy K. , Altfeld Marcus A. , Olson Douglas P. , Buchbinder Susan P. , Hecht Frederick M. , Rosenberg Eric S. , Walker Bruce D. , Kalams Spyros A.
Published: July 2002

Cytotoxic T lymphocytes (CTL) target multiple epitopes in human immunodeficiency virus (HIV)-infected persons, and are thought to influence the viral set point. The extent to which HLA class I allele expression predicts the epitopes targeted has not been determined, nor have the relative contributions of responses restricted by different class I alleles within a given individual. In this study, we performed a detailed analysis of the CTL response to optimally defined CTL epitopes restricted by HLA class I A and B alleles in individuals who coexpressed HLA A2, A3, and B7. The eight HIV-1-infected subjects studied included two subjects with acute HIV infection, five subjects with chronic HIV infection, and one long-term nonprogressor. Responses were heterogeneous with respect to breadth and magnitude of CTL responses in individuals of the same HLA type. Of the 27 tested epitopes that are presented by A2, A3, and B7, 25 were targeted by at least one person. However, there was wide variation in the number of epitopes targeted, ranging from 2 to 17. The A2-restricted CTL response, which has been most extensively studied in infected persons, was found to be narrowly directed in most individuals, and in no cases was it the dominant contributor to the total HIV-1-specific CTL response. These results indicate that HLA type alone does not predict CTL responses and that numerous potential epitopes may not be targeted by CTL in a given individual. These data also provide a rationale for boosting both the breadth and the magnitude of HIV-1-specific CTL responses by immunotherapy in persons with chronic HIV-1 infection.
Viral Replicase Gene Products Suffice for Coronavirus Discontinuous Transcription
Authors: Thiel Volker , Herold Jens , Schelle Barbara , Siddell Stuart G.
Published: July 2002

We have used vaccinia virus as a vector to clone a 22.5-kbp cDNA that represents the 5′ and 3′ ends of the human coronavirus 229E (HCoV 229E) genome, the HCoV 229E replicase gene, and a single reporter gene (coding for green fluorescent protein [GFP]) located downstream of a regulatory element for coronavirus mRNA transcription. When RNA transcribed from this cDNA was transfected into BHK-21 cells, a small percentage of cells displayed strong fluorescence. A region of the mRNA encoding GFP was amplified by PCR and shown to have the unique mRNA leader-body junction indicative of coronavirus-mediated transcription. These data show that the coronavirus replicase gene products suffice for discontinuous subgenomic mRNA transcription.
Transcription Pattern of Human Herpesvirus 8 Open Reading Frame K3 in Primary Effusion Lymphoma and Kaposi's Sarcoma
Authors: Rimessi Paola , Bonaccorsi Angela , Stürzl Michael , Fabris Marina , Brocca-Cofano Egidio , Caputo Antonella , Melucci-Vigo Gianna , Falchi Mario , Cafaro Aurelio , Cassai Enzo , Ensoli Barbara , Monini Paolo
Published: July 2002

Human herpesvirus 8 (HHV-8) is found in immunoblastic B cells of patients with multicentric Castleman's disease (MCD) and, predominantly in a latent form, in primary effusion lymphoma (PEL) cells and Kaposi's sarcoma (KS) spindle cells. Recent studies have shown that upon reactivation, HHV-8 expresses factors that downregulate major histocompatibility class I proteins and coactivation molecules and that may enable productively infected cells to escape cytotoxic T lymphocytes and natural killer cell responses. One of these viral factors is encoded by open reading frame (ORF) K3. Here we show that in PEL cells, ORF K3 is expressed through viral transcripts that are induced very early upon virus reactivation, including bicistronic RNA molecules containing coding sequences from viral ORFs K3 and 70. Specifically, we found that a bicistronic transcript was expressed in the absence of de novo protein synthesis, thereby identifying a novel HHV-8 immediate-early gene product. Several features of the RNA molecules encoding the K3 product, including multiple transcriptional start sites, multiple donor splicing sites, and potential alternative ATG usage, suggest that there exists a finely tuned modulation of ORF K3 expression. By contrast, ORF K3 transcripts are not detected in the majority of cells present in KS lesions that are latently infected by the virus, suggesting that there are other, as-yet-unknown mechanisms of immune evasion for infected KS spindle cells. Nevertheless, because HHV-8 viremia precedes the development of KS lesions and is associated with the recrudescence of MCD symptoms, the prompt expression of ORF K3 in productively infected circulating cells may be important for virus pathogenesis. Thus, molecules targeting host or viral factors that activate ORF K3 expression or inactivate the biological functions of the K3 product should be exploited for the prevention or treatment of HHV-8-associated diseases in at-risk individuals.
Simultaneous Positive and Purifying Selection on Overlapping Reading Frames of thetatandvprGenes of Simian Immunodeficiency Virus
Authors: Hughes Austin L. , Westover Kristi , da Silva Jack , O'Connor David H. , Watkins David I.
Published: July 2002

Tat-specific cytotoxic T cells have previously been shown to exert positive Darwinian selection favoring amino acid replacements of an epitope of simian immunodeficiency virus (SIV). The region of thetatgene encoding this epitope falls within a region of overlap between thetatandvprreading frames, and nonsynonymous nucleotide substitutions in thetatreading frame were found to occur disproportionately in such a way as to cause synonymous changes in thevprreading frame. Comparison of published complete SIV genomes showed Tat to be the least conserved at the amino acid level of nine proteins encoded by the virus, while Vpr was one of the most conserved. Numerous parallel amino acid changes occurred within the Tat epitope independently in different monkeys, and purifying selection on thevprreading frame, by limiting acceptable nonsynonymous substitutions in thetatreading frame, evidently has enhanced the probability of parallel evolution.
Interleukin-8 Stimulates Human Immunodeficiency Virus Type 1 Replication and Is a Potential New Target for Antiretroviral Therapy
Authors: Lane Brian R. , Lore Karin , Bock Paul J. , Andersson Jan , Coffey Michael J. , Strieter Robert M. , Markovitz David M.
Published: July 2002

Production of the C-X-C chemokines interleukin-8 (IL-8) and growth-regulated oncogene alpha (GRO-α) in macrophages is stimulated by exposure to human immunodeficiency virus type 1 (HIV-1). We have demonstrated previously that GRO-α then stimulates HIV-1 replication in both T lymphocytes and macrophages. Here we demonstrate that IL-8 also stimulates HIV-1 replication in macrophages and T lymphocytes. We further show that increased levels of IL-8 are present in the lymphoid tissue of patients with AIDS. In addition, we demonstrate that compounds which inhibit the actions of IL-8 and GRO-α via their receptors, CXCR1 and CXCR2, also inhibit HIV-1 replication in both T lymphocytes and macrophages, indicating potential therapeutic uses for these compounds in HIV-1 infection and AIDS.