bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simian Virus 40-Based Replication of Catalytically Inactive Human Immunodeficiency Virus Type 1 Integrase Mutants in Nonpermissive T Cells and Monocyte-Derived Macrophages
Authors: Lu Richard , Nakajima Noriko , Hofmann Wolfgang , Benkirane Monsef , Teh-Jeang Kuan , Sodroski Joseph , Engelman Alan 
Published: December 2003

ABSTRACTIntegrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into thenefposition of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.
Conserved C-Terminal Threonine of Hepatitis C Virus NS3 Regulates Autoproteolysis and Prevents Product Inhibition
Authors: Wang Wenyan , Lahser Frederick C. , Yi MinKyung , Wright-Minogue Jacquelyn , Xia Ellen , Weber Patricia C. , Lemon Stanley M. , Malcolm Bruce A. 
Published: December 2003

ABSTRACT

Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative
trans
cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.
Expansion of a Unique Region in the Marek's Disease Virus Genome Occurs Concomitantly with Attenuation but Is Not Sufficient To Cause Attenuation
Authors: Silva R. F. , Reddy S. M. , Lupiani B. 
Published: December 2003

ABSTRACT
Pathogenic Marek's disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Δ132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Δ132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Δ132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.
Neutrality of the Canonical NF-κB-Dependent Pathway for Human and Murine Cytomegalovirus Transcription and Replication In Vitro
Authors: Benedict Chris A. , Angulo Ana , Patterson Ginelle , Ha Sukwon , Huang Huang , Messerle Martin , Ware Carl F. , Ghazal Peter 
Published: December 2003

ABSTRACTCytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor κB (NF-κB) after infection of fibroblast and macrophage cells. NF-κB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-κB in transient-transfection assays. Here we investigate whether the NF-κB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-κB-alpha (IκBαM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-κB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-κB pathway and binding of NF-κB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.
The M4 Gene of Murine Gammaherpesvirus Modulates Productive and Latent Infection In Vivo
Authors: Townsley A. C. , Dutia B. M. , Nash A. A. 
Published: December 2003

ABSTRACT
Murine gammaherpesvirus 68 (MHV-68) infection of mice represents a viable small-animal model for the study of gammaherpesvirus pathogenesis. MHV-76 is a deletion mutant of MHV-68, which lacks four MHV-68-specific genes (M1 to M4) and eight viral tRNA-like sequences at the 5′ end of the genome. These genes are implicated in latency and/or immune evasion. Consequently, MHV-76 is attenuated in the acute phase of in vivo infection with respect to MHV-68. Little is known about the role of M4 in viral infection, except that it is expressed as an immediate-early/early transcript during lytic replication of MHV-68 in vitro. To elucidate the contribution M4 makes to in vivo pathogenesis, we created a novel MHV-76 mutant (MHV-76inM4), in which the region of MHV-68 coding for M4 and accompanying putative promoter elements were inserted into the 5′ region of the MHV-76 genome. The growth of MHV-76inM4 in vitro was indistinguishable from that of MHV-76 and MHV-68. However, virus titers from MHV-76inM4-infected BALB/c mice were significantly increased with respect to MHV-76 at early times in the lung. In addition, at days 17 and 21 postinfection, there was a significant elevation in latent viral load in splenocytes of MHV-76inM4-infected mice compared to MHV-76. Like MHV-76-infected mice, MHV-76inM4-infected mice display no evidence of overt splenomegaly, a finding characteristic of MHV-68 infection. M4 expression in vivo was detectable during productive infection in the lung and during the establishment of latency in the spleen, but in general M4 was not detectable during long-term latency (day 100 postinfection).
DC-SIGN from African Green Monkeys Is Expressed in Lymph Nodes and Mediates Infection intransof Simian Immunodeficiency Virus SIVagm
Authors: Ploquin Mickaël J.-Y. , Diop Ousmane M. , Sol-Foulon Nathalie , Mortara Lorenzo , Faye Abdourahmane , Soares Marcelo A. , Nerrienet Eric , Le Grand Roger , Van Kooyk Yvette , Amara Ali , Schwartz Olivier , Barré-Sinoussi Françoise , Müller-Trutwin Michaela C. 
Published: December 2003

ABSTRACTAfrican green monkeys (AGMs) infected by simian immunodeficiency virus (SIV) SIVagm are resistant to AIDS. SIVagm-infected AGMs exhibit levels of viremia similar to those described during pathogenic human immunodeficiency virus type 1 (HIV-1) and SIVmac infections in humans and macaques, respectively, but contain lower viral loads in their lymph nodes. We addressed the potential role of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN; CD209) in viral dissemination. In previous studies, it has been shown that human DC-SIGN and macaque DC-SIGN allow transmission of HIV and SIVmac to T cells. Here, we looked at the ability of DC-SIGN derived from AGM lymph nodes to interact with SIVagm. We show that DC-SIGN-expressing cells are present mainly in the medulla and often within the cortex and/or paracortex of AGM lymph nodes. We describe the isolation and characterization of at least three isoforms ofdc-signmRNA in lymph nodes of AGMs. The predicted amino acid sequence from the predominant mRNA isoform, DC-SIGNagm1, is 92 and 99% identical to the corresponding human and rhesus macaque DC-SIGN amino acid sequences, respectively. DC-SIGNagm1 is characterized by the lack of the fourth motif in the repeat domain. This deletion was also detected in thedc-signgene derived from thirteen animals belonging to five other African monkey species and from four macaques (Macaca fascicularisandM. mulatta). Despite three- to seven-amino-acid modifications compared to DC-SIGNmac, DC-SIGNagm1 allows transmission of SIVagm to T cells. Furthermore, AGM monocyte-derived dendritic cells (MDDC) expressed at least 100,000 DC-SIGN molecules and were able to transmit SIVagm to T cells. At a low multiplicity of infection (10−550% tissue culture infective doses/cell), viral transmission by AGM MDDC was mainly DC-SIGN dependent. The present study reveals that DC-SIGN from a natural host species of SIV has the ability to act as an efficient attachment and transmission factor for SIVagm and suggests the absence of a direct link between this ability and viral load levels in lymph nodes.
Role of the Ectodomain of the gp41 Transmembrane Envelope Protein of Human Immunodeficiency Virus Type 1 in Late Steps of the Membrane Fusion Process
Authors: Bär Séverine , Alizon Marc 
Published: December 2003

ABSTRACT

The membrane fusion process mediated by the gp41 transmembrane envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) was addressed by a flow cytometry assay detecting exchanges of fluorescent membrane probes (DiI and DiO) between cells expressing the HIV-1 envelope proteins (Env) and target cells. Double-fluorescent cells were detected when target cells expressed the type of chemokine receptor, CXCR4 or CCR5, matching the type of gp120 surface envelope protein, X4 or R5, respectively. Background levels of double-fluorescent cells were observed when the gp120-receptor interaction was blocked by AMD3100, a CXCR4 antagonist. The L568A mutation in the N-terminal heptad repeat (HR1) of gp41 resulted in parallel inhibition of the formation of syncytia and double-fluorescent cells, indicating that gp41 had a direct role in the exchange of fluorescent probes. In contrast, three mutations in the loop region of the gp41 ectodomain, located on either side of the Cys-(X)
5
-Cys motif (W596 M and W610A) or at the distal end of HR1 (D589L), had limited or no apparent effect on membrane lipid mixing between Env
+
and target cells, while they blocked formation of syncytia and markedly reduced the exchanges of cytoplasmic fluorescent probes. The loop region could therefore have a direct or indirect role in events occurring after the merging of membranes, such as the formation or dilation of fusion pores. Two types of inhibitors of HIV-1 entry, the gp41-derived peptide T20 and the betulinic acid derivative RPR103611, had limited effects on membrane exchanges at concentrations blocking or markedly reducing syncytium formation. This finding confirmed that T20 can inhibit the late steps of membrane fusion (post-lipid mixing) and brought forth an indirect argument for the role of the gp41 loop region in these steps, as mutations conferring resistance to RPR103611V were mapped in this region (I595S or L602H).
Nuclear Localization of Avian Polyomavirus Structural Protein VP1 Is a Prerequisite for the Formation of Virus-Like Particles
Authors: Johne Reimar , Müller Hermann 
Published: December 2003

ABSTRACT
Virions of polyomaviruses consist of the major structural protein VP1, the minor structural proteins VP2 and VP3, and the viral genome associated with histones. An additional structural protein, VP4, is present in avian polyomavirus (APV) particles. As it had been reported that expression of APV VP1 in insect cells did not result in the formation of virus-like particles (VLP), the prerequisites for particle formation were analyzed. To this end, recombinant influenza viruses were created to (co)express the structural proteins of APV in chicken embryo cells, permissive for APV replication. VP1 expressed individually or coexpressed with VP4 did not result in VLP formation; both proteins (co)localized in the cytoplasm. Transport of VP1, or the VP1-VP4 complex, into the nucleus was facilitated by the coexpression of VP3 and resulted in the formation of VLP. Accordingly, a mutant APV VP1 carrying the N-terminal nuclear localization signal of simian virus 40 VP1 was transported to the nucleus and assembled into VLP. These results support a model of APV capsid assembly in which complexes of the structural proteins VP1, VP3 (or VP2), and VP4, formed within the cytoplasm, are transported to the nucleus using the nuclear localization signal of VP3 (or VP2); there, capsid formation is induced by the nuclear environment.
Characterization of the Inhibition of Hepatitis C Virus RNA Replication by Nonnucleosides
Authors: Tomei Licia , Altamura Sergio , Bartholomew Linda , Bisbocci Monica , Bailey Carolyn , Bosserman Michele , Cellucci Antonella , Forte Eleonora , Incitti Ilario , Orsatti Laura , Koch Uwe , De Francesco Raffaele , Olsen David B. , Carroll Steven S. , Migliaccio Giovanni 
Published: December 2003

ABSTRACTThe RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.
Imparting Temperature Sensitivity and Attenuation in Ferrets to A/Puerto Rico/8/34 Influenza Virus by Transferring the Genetic Signature for Temperature Sensitivity from Cold-Adapted A/Ann Arbor/6/60
Authors: Jin Hong , Zhou Helen , Lu Bin , Kemble George 
Published: December 2003

ABSTRACT

The four temperature-sensitive (
ts
) loci identified in the PB1 and PB2 gene segments of cold-adapted A/Ann Arbor/6/60 influenza virus, the master donor virus for influenza A virus (MDV-A) FluMist vaccines, were introduced into a divergent A/Puerto Rico/8/34 influenza virus strain. Recombinant A/Puerto Rico/8/34 virus with these four introduced
ts
loci exhibited both
ts
and
att
phenotypes similar to those of MDV-A, which could be used as a donor virus for manufacturing large quantities of inactivated influenza virus vaccine against potential pandemic strains.
Childhood and adolescence poisoning in NSW, Australia: an analysis of age, sex, geographic, and poison types
Authors: Lam L T 
Published: December 2003

Objective:This study aims to investigate whether there is any association between the types of poison substances and geographic locations for different age groups and sex.Design:This is a population based epidemiological study utilising routinely collected inpatient statistics.Setting:Data are collected as part of the routine vital health information system via all hospitals in New South Wales (NSW), Australia.Patients:All patients aged between 0–19 years who were admitted to a hospital because of poisoning by the four major types of substances that were defined in the study in NSW in 2000.Main results:The standardised incidence ratios of poisoning related hospitalisation between metropolitan and rural areas varied across different poison types when compared with the NSW average. While there are few differences between metropolitan and rural areas for analgesic and chemical related poisoning admissions across different age groups and sex, differences in the standardised incidence ratios between geographic locations for psychotropic and venom related poisoning admissions were found. No significant difference in standardised mortality ratios were found between metropolitan and rural areas except for females in the 10–14 years age group (standardised mortality ratio 3.24, 95% confidence interval 1.69 to 6.21).Conclusions:The results obtained in this study, on the whole, provide some evidence for an association between poison types and geographic locations for psychotropic and venom related poisoning.
EWI-2 modulates lymphocyte integrin α4β1 functions
Authors: Kolesnikova Tatiana V. , Stipp Christopher S. , Rao Ravi M. , Lane William S. , Luscinskas Francis W. , Hemler Martin E. 
Published: December 2003

AbstractThe most prominent cell-surface integrin α4β1 partner, a 70-kDa protein, was isolated from MOLT-4 T leukemia cells, using anti–α4β1 integrin antibody-coated beads. By mass spectrometry, this protein was identified as EWI-2, a previously described cell-surface partner for tetraspanin proteins CD9 and CD81. Wild-type EWI-2 overexpression had no effect on MOLT-4 cell tethering and adhesion strengthening on the α4β1 ligand, vascular cell adhesion molecule-1 (VCAM-1), in shear flow assays. However, EWI-2 markedly impaired spreading and ruffling on VCAM-1. In contrast, a mutant EWI-2 molecule, with a different cytoplasmic tail, neither impaired cell spreading nor associated with α4β1 and CD81. The endogenous wild-type EWI-2–CD81–α4β1 complex was fully soluble, and highly specific as seen by the absence of other MOLT-4 cell-surface proteins. Also, it was relatively small in size (0.5 × 106 Da to 4 × 106 Da), as estimated by size exclusion chromatography. Overexpression of EWI-2 in MOLT-4 cells caused reorganization of cell-surface CD81, increased the extent of CD81-CD81, CD81-α4β1, and α4β1-α4β1 associations, and increased the apparent size of CD81-α4β1 complexes. We suggest that EWI-2–dependent reorganization of α4β1-CD81 complexes on the cell surface is responsible for EWI-2 effects on integrin-dependent morphology and motility functions. (Blood. 2004;103: 3013-3019)
The epidemiology of chronic hepatitis C infection in survivors of childhood cancer: an update of the St Jude Children's Research Hospital hepatitis C seropositive cohort
Authors: Castellino Sharon , Lensing Shelly , Riely Caroline , Rai Shesh N. , Davila Rene , Hayden Randall T. , Fleckenstein Jackie , Levstik Mark , Taylor Shari , Dean Patrick J. , Kippenbrock Sarah , Pope Jennifer , Carr Jeanne , Strickland Donald K. , Hudson Melissa M. 
Published: December 2003

AbstractChildhood cancer survivors transfused before 1992 are at risk for chronic hepatitis C (HCV) infection. In 1995, St Jude Children's Research Hospital initiated an epidemiologic study of childhood cancer survivors with transfusion-acquired HCV. Of the 148 survivors with HCV confirmed by second-generation enzyme immunoassay, 122 consented to participate in the study. Their current median age is 29 years (range, 9 to 47 years). At enrollment, polymerase chain reaction (PCR) testing indicated chronic infection in 81.1%; genotype 1 was the most common viral genotype. Liver biopsy in 60 patients at a median of 12.4 years from the diagnosis of malignancy showed mild (28.8%) or moderate (35.6%) fibrosis; 13.6% had cirrhosis. Elevated body mass index was associated with histologic findings of increased steatosis (P = .008). Antimetabolite chemotherapy exposure was associated with early progression of fibrosis. Significant quality-of-life deficits were observed in noncirrhotic adult survivors. Antiviral therapy resulted in clearance of infection in 17 (44%) of 38 patients to date. Six patients have died; 1 patient with decompensated cirrhosis died of variceal bleeding. Despite a young age at HCV infection, the progression of liver disease in childhood cancer survivors is comparable to that seen in adults.