Biomedical subjects

I W Park

Publications and source records attributed to I W Park.

At least 19 recordsLinked to original sources

PubMed2003

Miliary tuberculosis and acute respiratory distress syndrome.

OBJECTIVE: Miliary tuberculosis is a life-threatening disease caused by the haematogenous spread of Mycobacterium tuberculosis. We evaluated the clinical manifestations of 34 patients with miliary tuberculosis. DESIGN: A retrospective case review. RESULTS: The diagnosis of miliary tuberculosis was based on the identification of miliary nodules on chest radiography and one of the three following criteria: 1) acid-fast bacilli smear and/or culture positive in clinical specimens (22/34), 2) histopathological identification of TB granuloma (6/34), and 3) radiological and clinical improvement after anti-tuberculosis treatment (6/34). The median age (+/-SD) of the patients was 42.7 +/- 21.6 years, with two peaks, in the age group 20-30 and in those over 60. There were 16 underlying diseases in 14 patients, of which liver cirrhosis was the most common. The drug sensitivity pattern was available for 17 isolates of M. tuberculosis: 14 were sensitive, while the other three were resistant to at least one anti-tuberculosis drug. Eight patients developed acute respiratory distress syndrome (ARDS), five of whom died during intensive care. Platelet count, serum albumin and liver enzyme level at the time of admission were significant factors both for ARDS development and for survival. CONCLUSION: ARDS caused by miliary TB is associated with a high fatality rate; scope remains for improvement in its management.

Adolescent↗

PubMed2001

HIV-1 Tat induces microvascular endothelial apoptosis through caspase activation.

HIV-1 Tat, in addition to its critical role in viral transcription, is secreted from infected cells and can act as a proto-cytokine. We studied the effects of HIV-1 Tat in primary human microvascular endothelial cells of lung origin and found that it caused apoptosis. This apoptosis occurred without induction of either Fas or TNF, known mediators of programmed cell death. Tat, like Fas ligand, induced cleavage of chromatin structure, as evidenced by changes in DNA laddering, incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay), and mono- or oligonucleosomes. Furthermore, Tat treatment caused cleavage of poly(A/DP)-ribose polymerase, a substrate of caspases. Caspase-3, but not caspase-9, was activated following treatment of primary human microvascular endothelial cells of lung origin with either Tat or anti-Fas agonist Ab (anti-Fas). Inhibition of caspase-3 activity markedly reduced apoptosis. Although Fas-mediated apoptosis involved changes in Bcl-2, Bax, and Bad regulatory proteins, such alterations were not observed with Tat. Taken together, these data demonstrate that HIV-1 Tat is able to activate apoptosis in microvascular endothelium by a mechanism distinct from TNF secretion or the Fas pathway.

Amino Acid Sequence↗

PubMed2001

HIV-1 Tat promotes monocyte chemoattractant protein-1 secretion followed by transmigration of monocytes.

The mechanism whereby HIV-infected cells transit from the bloodstream into tissues is not well defined. This phenomenon was addressed by studying the effects of HIV-1 Tat, a protein secreted by infected cells, on human lung microvascular endothelial cells (HMVEC-Ls). It was found that monocyte chemoattractant protein-1 (MCP-1) was released from HMVEC-Ls in a dose- and time-dependent manner after Tat treatment. MCP-1 is a potent beta-chemokine that recruits monocytes and T cells and promotes cell adhesion and transmigration across an endothelial monolayer. It was also observed that MCP-1 and the culture medium from Tat-treated HMVEC-Ls were chemotactic for CD14(+) monocytes from human peripheral blood and for THP-1, a promonocytic cell line used as a model system. To characterize the signaling pathways underlying the observed induction of MCP-1, HMVEC-Ls were treated with 2 different protein kinase inhibitors: PD98059, a MAP kinase inhibitor, and GF109203X, a protein kinase C (PKC) inhibitor. MCP-1 release was significantly reduced when PKC was inhibited, and slightly decreased when PI3 kinase was blocked; no effect on MCP-1 release was observed on MAP kinase inhibition. Similarly, transmigration of THP-1 cells was significantly impaired by the PKC inhibitor, but not by the other tested inhibitors. These data indicate that the HIV-1 Tat protein may act as a protocytokine by causing the release of MCP-1 from the endothelial monolayer, and thereby facilitating monocyte transmigration into tissues via a PKC signaling pathway.

Cell Culture Techniques↗

PubMed2000

Stromal cell-derived factor-1alpha stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C.

The stromal cell-derived factor-1 (SDF-1) is an alpha chemokine that binds to the CXCR4 receptor. Knock-out studies in mice demonstrate that this ligand-receptor pair is essential in hematopoiesis. One function of SDF-1 appears to be the regulation of migration of hematopoietic progenitor cells. We previously characterized signal transduction pathways induced by SDF-1alpha in human hematopoietic progenitors and found tyrosine phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK), the adaptor molecule p130 Cas, and the cytoskeletal protein paxillin. To better understand the functional role of signaling molecules connecting the CXCR4 receptor to the process of hematopoietic migration, we studied SDF-1alpha-mediated pathways in a model hematopoietic progenitor cell line (CTS), as well as in primary human bone marrow CD34(+) cells. We observed that several other focal adhesion components, including focal adhesion kinase (FAK) and the adaptor molecules Crk and Crk-L, are phosphorylated on SDF-1alpha stimulation. Using a series of specific small molecule inhibitors, both protein kinase C (PKC) and phosphoinositide-3 kinase (PI-3K) appeared to be required for SDF-1alpha-mediated phosphorylation of focal adhesion proteins and the migration of both CTS and primary marrow CD34(+) cells, whereas the mitogen-activated protein kinases ERK-1 and -2 were not. These studies further delineate the molecular pathways mediating hematopoietic progenitor migration and response to an essential chemokine, SDF-1alpha. (Blood. 2000;95:2505-2513)

Animals↗

PubMed1999

Multiple clonal abnormalities in the bronchial epithelium of patients with lung cancer.

BACKGROUND: Several molecular changes, including loss of heterozygosity (i.e., deletion of one copy of allelic DNA sequences) and alterations in microsatellite DNA, have been detected early in the pathogenesis of lung cancer, even in histologically normal epithelium. In the bronchial epithelium of patients with lung cancer, we have determined the frequency, size, and patterns of molecularly abnormal clonal patches. METHODS: We studied formalin-fixed, paraffin-embedded samples from 16 surgically resected lung carcinomas (five squamous cell carcinomas, four small-cell carcinomas, six adenocarcinomas, and one large-cell carcinoma). From each carcinoma, we microdissected foci (each containing about 200 cells) of tumor tissue and equivalent samples of histologically normal and abnormal epithelium. Furthermore, multiple discontinuous foci of bronchial epithelium were analyzed from methanol-fixed samples from three additional patients with lung cancer (two with squamous cell carcinoma and one with adenocarcinoma). We used two-step polymerase chain reaction-based assays involving 12 microsatellite markers at seven chromosomal regions frequently deleted in lung cancer. RESULTS: Two hundred eighteen foci of nonmalignant bronchial epithelium (195 of histologically normal or slightly abnormal epithelium and 23 of dysplastic epithelium) were studied from the 19 surgically resected lobectomy specimens. Thirteen (68%) of the 19 specimens had at least one focus of bronchial epithelium with molecular changes. At least one molecular abnormality was detected in 32% of the 195 histologically normal or slightly abnormal foci and in 52% of the 23 dysplastic foci. Extrapolating from our two-dimensional analyses, we estimate that most clonal patches contain approximately 90 000 cells. Although, in a given individual, tumors appeared homogeneous with respect to molecular changes, the clonally altered patches of mildly abnormal epithelium were heterogeneous. CONCLUSIONS: Our findings indicate that multiple small clonal or subclonal patches containing molecular abnormalities are present in normal or slightly abnormal bronchial epithelium of patients with lung cancer.

Adenocarcinoma↗

PubMed1999

Expression and utilization of co-receptors in HIV and simian immunodeficiency virus infection of megakaryocytes.

OBJECTIVE: To analyse the expression and specificity of co-receptors for the entry of HIV and simian immunodeficiency virus (SIV) into megakaryocytes. DESIGN AND METHODS: The expression of co-receptors was determined by flow cytometric analysis in combination with reverse transcription-polymerase chain (RT-PCR) reaction. The specificity of co-receptors in virus entry was determined by the infection of HIV-1 pseudotyped with X4- (HXB2), R5- (YU2), or R5X4-tropic (89.6) envelope proteins of HIV-1 or with envelope proteins of SIVpbj1.9. RESULTS: The model human erythroleukemia (HEL) cell line, exhibiting megakaryocytic-like properties, expressed CCR5, CCR3, CXCR4, and CPR15/BOB, and all viruses except YU2 (R5) efficiently entered the cells. The blocking of virus entry with specific chemokines showed that the entry of HXB2 (X4) was impaired by SDF-1beta but not by other chemokines, indicating that CXCR4 was a major co-receptor for the entry of HXB2. Primary human bone marrow megakaryocytes displayed a different repertoire of co-receptor expression from that of HEL cells, as all viruses except YU2 efficiently entered these cells. However, chemokine blocking experiments showed that the entry of HXB2 into primary bone marrow megakaryocytes was insufficiently blocked by SDF-1beta compared with the entry into HEL cells, suggesting that alternative co-receptors could be employed for the entry of X4 virus into bone marrow cells. CONCLUSION: These data suggest that cells of megakaryocytic lineage are susceptible to infection by X4 viruses, with less marked susceptibility to R5 isolates, and that SDF-1beta efficiently blocks the infection of HEL cells but not of primary bone marrow megakaryocytes. Our data reveal that novel co-receptors are probably utilized for the entry of X4 virus into megakaryocytes.

Animals↗

PubMed1999

CD4 receptor-dependent entry of human immunodeficiency virus type-1 env-pseudotypes into CCR5-, CCR3-, and CXCR4-expressing human alveolar macrophages is preferentially mediated by the CCR5 coreceptor.

Alveolar macrophages (AM) are important host-defense cells and targets of human immunodeficiency virus type 1 (HIV-1) infection. However, the receptors mediating HIV-1 entry into AM are not completely characterized. We observed that, in addition to CD4 receptors, AM from healthy adults expressed low levels of CCR5, CCR3, and CXCR4 chemokine receptors by flow cytometry, and specific messenger RNA was detected for all three receptors by reverse transcriptase/polymerase chain reaction. The macrophage monocytotropic (M-tropic; YU2) and dual-tropic (89.6) HIV-1 env-pseudotypes entered AM efficiently, as expected given CCR3 and CCR5 expression. However, the T-lymphocytotropic (T-tropic; HXB2) pseudotype did not enter AM despite expression of the appropriate chemokine coreceptor CXCR4. Incubation of AM with regulated on activation, normal T cells expressed and secreted (RANTES) significantly impaired entry of the M-tropic (YU2) HIV-1 pseudotype, whereas SDF-1beta or eotaxin did not impair entry. The entry of simian immunodeficiency virus (SIV) pbj1.9 env-pseudotype into AM was not blocked by RANTES, SDF-1beta, or eotaxin. The competence of these chemokine receptors for virus entry was confirmed in Cf2Th canine thymocytes cotransfected with the human CD4 and chemokine receptors. Entry of the M-tropic (YU2) HIV-1 pseudotype was shown to be mediated by either CCR3 or CCR5, the T-tropic (HXB2) HIV-1 pseudotype by CXCR4, and the dual-tropic (89.6) HIV-1 or the SIVpbj1. 9 pseudotype by CCR5, CCR3, or CXCR4. Our data indicate that the mechanisms for HIV-1 entry are both receptor-specific and cell type-specific, and that chemokine receptor expression on AM does not fully explain cell susceptibility to different virus isolates.

Adult↗

PubMed1997

Prospective study of corticosteroid as an adjunct in the treatment of endobronchial tuberculosis in adults.

Although endobronchial tuberculosis frequently causes bronchial stenosis, there are no specific therapies to prevent the sequelae. The use of corticosteroids remains controversial and there have been no prospective comparative studies about the effectiveness of corticosteroids. This study was undertaken in order to determine the effectiveness of corticosteroids in the prevention of complications of endobronchial tuberculosis. Thirty-four patients with endobronchial tuberculosis who were admitted to Chung-Ang University hospital from March 1991 to December 1995 were evaluated prospectively to determine the effect of corticosteroid in the treatment of endobronchial tuberculosis. All patients were randomly divided into two groups: group 1 (n=17, anti-tuberculosis chemotherapy only) and group 2 (n=17, combining anti-tuberculosis chemotherapy with oral corticosteroid). Serial bronchoscopies, pulmonary function tests and chest roentgenograms were analyzed every 2 months until the complete resolution of endobronchial tuberculosis. Before treatment commenced there were no significant differences between the two groups with respect to sex, mean age, pulmonary function, chest roentgenogram and morphologic patterns of endobronchial lesion. After treatment, the healing rate of bronchoscopic findings and changes in pulmonary function showed no significant differences between the two groups. Radiologic improvements were observed in all eight patients (five in group 1 and three in group 2) with segmental atelectasis on chest roentgenograms after 2 months of treatment. This study suggests that corticosteroid therapy would not influence the outcome of endobronchial tuberculosis and that prompt treatment with early diagnosis, before formation of fibrosis would be necessary to prevent complications of endobronchial tuberculosis, such as bronchostenosis.

Adolescent↗

PubMed1997

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys.

In vivo passage of a chimeric simian-human immunodeficiency virus (SHIV-89.6) expressing the human immunodeficiency virus type 1 (HIV-1) tat, rev, vpu, and env genes generated pathogenic viruses (SHIV-89.6P) inducing rapid CD4+ lymphocyte depletion and AIDS-like illness in rhesus monkeys (K. Reimann, J. T. Li, R. Veazey, M. Halloran, I.-W. Park, G. B. Karlsson, J. Sodroski, and N. L. Letvin, J. Virol. 70:6922-6928, 1996). To characterize the molecular changes responsible for this increase in virulence, infectious proviral clones of SHIV-89.6P isolates were derived. Viruses generated from some of these clones caused a rapid and profound decline of CD4+ lymphocytes in a high percentage of inoculated monkeys. Nucleotide changes potentially responsible for the increased virulence of SHIV-89.6P were limited to the env, tat, or long terminal repeat sequences, with most of the observed changes in env. Nucleotide changes in env altered 12 amino acids in the gp120 and gp41 exterior domains, and a 140-bp deletion in env resulted in the substitution of the carboxyl terminus of the SIVmac gp41 glycoprotein for that of the HIV-1 gp41 glycoprotein. The availability of pathogenic proviral clones should facilitate dissection of the molecular determinants of SHIV-89.6P virulence.

Animals↗

PubMed1996

Effects of human immunodeficiency virus type 1 infection on programmed cell death in the presence or absence of Bcl-2.

The effect of human immunodeficiency virus (HIV-1) infection on the programmed cell death of CD4+ lymphocytes was studied by using Jurkat cells stably expressing high levels of the Bcl-2 protein (Jurkat-Bcl2) or control cells (Jurkat-P). Both Jurkat-Bcl2 and Jurkat-P cells exhibited surface CD4 expression adequate to support HIV-1 infection. We observed no differences between HIV-1-infected Jurkat Bcl2 cells and control cells with respect to kinetics of virus replication, protein expression, and processing. Severe cytopathic effects, which were typical of acute HIV-1 infection and consisted of syncytium formation followed by single-cell lysis, were observed in both cell types. However, several lines of evidence, such as cell viability analysis by trypan blue dye exclusion, chromosomal DNA laddering, and morphologic analysis by acridine orange/ethidium bromide or Giemsa staining, indicated that HIV-1 did not induce a significant amount of programmed cell death in either cell type. These results suggest that apoptosis is at most a minor element in HIV-1-induced cytopathicity in Jurkat lymphocytes.

Apoptosis↗

PubMed1996

Targeting a foreign protein into virion particles by fusion with the Vpx protein of simian immunodeficiency virus.

The Vpx and Vpr proteins of the primate immunodeficiency viruses are stoichiometrically incorporated into virion particles. The chloramphenicol acetyltransferase (CAT) enzyme, when fused to a sufficient portion of the simian immunodeficiency virus (SIVmac239) Vpx protein, was incorporated into virions and retained enzymatic activity. An analysis of the replication of this virus compared with the replication of revertants and control viruses encoding nonpackageable Vpx-CAT fusion proteins suggested that the observed delay in replication was due to cis-acting effects of the CAT gene insertion rather than to the presence of the Vpx-CAT fusion protein in the virions. These studies indicate that, in host cells where Vpx and Vpr function is not required for efficient SIVmac replication, functional enzymes can be incorporated into virions by fusion with the Vpx protein. This approach could be utilized for study of the function and localization of Vpx and/or Vpr proteins during virus replication and for attempts to disrupt virus replication by the incorporation of foreign proteins.

Animals↗

PubMed1996

A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys.

The utility of the simian immunodeficiency virus of macaques (SIVmac) model of AIDS has been limited by the genetic divergence of the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and the SIVs. To develop a better AIDS animal model, we have been exploring the infection of rhesus monkeys with chimeric simian/human immunodeficiency viruses (SHIVs) composed of SIVmac239 expressing HIV-1 env and the associated auxiliary HIV-1 genes tat, vpu, and rev. SHIV-89.6, constructed with the HIV-1 env of a cytopathic, macrophage-tropic clone of a patient isolate of HIV-1 (89.6), was previously shown to replicate to a high degree in monkeys during primary infection. However, pathogenic consequences of chronic infection were not evident. We now show that after two serial in vivo passages by intravenous blood inoculation of naive rhesus monkeys, this SHIV (SHIV-89.6P) induced CD4 lymphopenia and an AIDS-like disease with wasting and opportunistic infections. Genetic and serologic evaluation indicated that the reisolated SHIV-89.6P expressed envelope glycoproteins that resembled those of HIV-1. When inoculated into naive rhesus monkeys, SHIV-89.6P caused persistent infection and CD4 lymphopenia. This chimeric virus expressing patient isolate HIV-1 envelope glycoproteins will be valuable as a challenge virus for evaluating HIV-1 envelope-based vaccines and for exploring the genetic determinants of HIV-1 pathogenicity.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4-positive lymphocytes is accompanied by acute cytopathic effects, i.e., syncytium formation and single-cell lysis. Syncytium formation involves cell-cell fusion mediated by viral envelope glycoproteins on the surface of infected cells and by CD4 glycoproteins on adjacent cells. The molecular basis for the lysis of single-HIV-1 infected cells is unclear. Here we report that the expression of functional envelope glycoproteins from primary and laboratory-adapted HIV-1 isolates resulted in the lysis of single CD4-positive lymphocytes. As was previously observed in HIV-1 infected cultures, single-cell lysis in this system primarily involved necrosis and was not inhibited by soluble CD4. Binding of the viral envelope glycoproteins to the CD4 glycoprotein facilitated, but was not sufficient for, cytolysis. Importantly, the ability of the HIV-1 envelope glycoproteins to mediate membrane fusion was essential for single-cell killing. By contrast, the long cytoplasmic tail of the gp41 transmembrane envelope glycoprotein was neither necessary nor sufficient for single-cell lysis. These results suggest that intracellular envelope glycoprotein-CD4 interactions initiate autofusion events that disrupt cell membrane integrity, leading to single-cell lysis by HIV-1.

CD4-Positive T-Lymphocytes↗

PubMed1995

Amino acid sequence requirements for the incorporation of the Vpx protein of simian immunodeficiency virus into virion particles.

To investigate the amino acid sequence determinants for the incorporation of Vpx protein into virion particles, several mutations were introduced into the vpx gene of SIVmac239 proviral DNA, and the effects of mutations on the expression and assembly of the Vpx protein were studied. The results show that deletions of amino acids from 78 to 80 or from 82 to 87 abolished the incorporation of the expressed Vpx protein into virion particles. Other Vpx mutants, including a full-length Vpx-Vpr fusion protein and a mutant with a deletion of the C-terminal polyproline tract, were packaged efficiently. This study suggests that amino acids from 78 to 80 and 82 to 87 are important for the assembly of the Vpx protein into virus particles.

Amino Acid Sequence↗

PubMed1995

Functional analysis of the vpx, vpr, and nef genes of simian immunodeficiency virus.

The role of the vpx, vpr, and nef genes in the replication of simian immunodeficiency virus (SIV) was investigated using point and deletion mutations in these genes. The effects on replication kinetics of single or combined mutants--vpx, vpr, vpx-vpr, vpx-nef, vpr-nef, and vpx-vpr-nef--in established lymphoid CEMx174 and MT-4 cells were negligible, except that the postinfection appearance of vpx-nef, vpr-nef, and vpx-vpr-nef progeny virus was slightly delayed in MT-4 cells. The vpx, but not the vpr, point mutation reverted to wild-type sequences within 12 days after infection, suggesting that stronger selection pressure for Vpx than for Vpr expression might exist in these established cell lines. In contrast to growth in the lymphoid cell lines, replication of vpx-deleted viruses in macaque peripheral blood mononuclear cells (PBMC) was severely impaired, indicating that Vpx is necessary for efficient replication in PBMC. In contrast, the vpr mutant exhibited different degrees of impairment depending on the donor animal used as a source of PBMC. A virus encoding a Vpx-Vpr fusion protein replicated in PBMC comparably to a vpr deletion mutant virus, whereas a frameshift deletion at the vpx-vpr junction of this mutant eliminated virus replication, suggesting that deletion of the C-terminal half of Vpx was partially compensated by the presence of the large Vpr portion in the fusion protein. Deletion of the nef gene did not affect SIVmac replication in PBMC. The Vpx and Vpr proteins expressed in COS-1 cells were detected in the extracellular medium and did not crossreact with Vpr- and Vpx-specific antisera, in spite of extensive amino acid similarity between these proteins. These studies indicate the importance of Vpx and Vpr in SIVmac infection and suggest that these proteins are antigenically and functionally distinct.

Animals↗

PubMed1994

Effects of vif mutations on cell-free infectivity and replication of simian immunodeficiency virus.

To investigate the function of the Vif protein of the simian immunodeficiency virus (SIV), mutations were introduced into the SIVmac239 vif gene without affecting the reading frames of other overlapping genes. The phenotypes of these mutant viruses were examined with respect to viral replication and the expression and processing of viral proteins. Transfection of vif-mutant proviral DNA into established T cell lines resulted in a significant delay in the onset of virus replication compared to that seen with the wild-type provirus. The efficiency of replication of the vif-mutant virus was dependent on cell type. MT-4 cells were permissive for replication of the vif mutant, while replication in CEMx174 cells was severely restricted. Little or no virus replication was observed following cell-free infection of the CEMx174 cell line and macaque peripheral blood mononuclear cells (PBMC). These results indicate that the requirement for vif during the replication of SIVmac239 is dependent on cell type, as has been observed for HIV-1. Following cell-free infection, mutant viruses containing combined deletions in vif and the other regulatory genes (vpx, vpr, and nef) displayed replication kinetics similar to that of viruses containing the deletion of vif alone. Viral protein expression and processing in MT-4 cells of vif-deleted viruses were indistinguishable from those of the wild-type virus. The effects of two different point mutations in vif were examined. One point mutant in vif reverted to the genetic sequence of the wild-type virus within 2 weeks.2 +

Animals↗

PubMed1991

Characterization of multiple mRNA species of simian immunodeficiency virus from macaques in a CD4+ lymphoid cell line.

Cytoplasmic poly(A)+ RNA was isolated from CEMX721.174 cells 5 to 10 days after infection with molecularly cloned simian immunodeficiency virus SIVmac239. Expression of SIV RNA was analyzed by Northern (RNA) blot hybridization and by sequencing of cDNA clones. As expected, a splice donor site was demonstrated in the untranslated leader sequence outside the left long terminal repeat. The region between pol and env was found to contain at least two splice donor and six splice acceptor sites. Splice acceptor and donor sites in the intergenic region were suitably positioned for expression of vpx, vpr, tat, and rev. Splice acceptor sites at nucleotides 8802 and 8805 were demonstrated in singly and doubly spliced RNAs with the potential of expressing nef and the second exons of tat and rev. Our results demonstrate a complex pattern of alternative splicing of SIV mRNAs. The results are very similar to what has been observed in human immunodeficiency virus type 1-infected cells, suggesting that both human and simian immunodeficiency viruses are subject to multiple levels of regulation.

Amino Acid Sequence↗