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R Geleziunas

Publications and source records attributed to R Geleziunas.

At least 37 records · Page 2Linked to original sources
PubMed1995

Increased soluble tumor necrosis factor receptor expression and release by human immunodeficiency virus type 1 infection.

High levels of circulating soluble tumor necrosis factor receptors (sTNF-R) are associated with HIV-1 infection and disease. To understand better this association, we have investigated p55 and p75 TNF-R expression on peripheral blood mononuclear cell (PBMC) subsets and in the promonocytic cell line U937, with or without HIV infection. Using flow cytometry and monoclonal antibodies both to sTNF-R and to PBMC subsets, TNF-R were found to be expressed mostly by monocytes and in decreasing amounts and intensity in the following order: CD14+ cells > CD8+ cells > CD4+ cells. Expression of TNF-R was higher on cells obtained from HIV-infected than from noninfected subjects, and expression of p75 sTNF-R was much higher than that of p55 sTNF-R. Studying the U937 cells revealed that over 80% of the cells expressed both sTNF-R, but with greater fluorescence intensity in the HIV-1 chronically infected cells (U-937-IIIB). Treatment of the cells with PMA caused an accelerated release into the medium of both sTNF-R, with a sharp decline in their cell surface expression. Basal levels of mRNA transcripts for p75 TNF-R were higher in the U-937-IIIB cells than in the uninfected cells, but p55 TNF-R mRNA was expressed only in the HIV-1-infected cells. These findings show that HIV-1 infection is accompanied by predominant elevation of p75 TNF-R surface expression on monocytes and CD8+ lymphocytes, and results in both increased message and expression of these receptors in monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1995

The human immunodeficiency virus type 1 (HIV-1) CD4 receptor and its central role in promotion of HIV-1 infection.

Interactions between the viral envelope glycoprotein gp120 and the cell surface receptor CD4 are responsible for the entry of human immunodeficiency virus type 1 (HIV-1) into host cells in the vast majority of cases. HIV-1 replication is commonly followed by the disappearance or receptor downmodulation of cell surface CD4. This potentially renders cells nonsusceptible to subsequent infection by HIV-1, as well as by other viruses that use CD4 as a portal of entry. Disappearance of CD4 from the cell surface is mediated by several different viral proteins that act at various stages through the course of the viral life cycle, and it occurs in T-cell lines, peripheral blood CD4+ lymphocytes, and monocytes of both primary and cell line origin. At the cell surface, gp120 itself and in the form of antigen-antibody complexes can trigger cellular pathways leading to CD4 internalization. Intracellularly, the mechanisms leading to CD4 downmodulation by HIV-1 are multiple and complex; these include degradation of CD4 by Vpu, formation of intracellular complexes between CD4 and the envelope precursor gp160, and internalization by the Nef protein. Each of the above doubtless contributes to the ultimate depletion of cell surface CD4, although the relative contribution of each mechanism and the manner in which they interact remain to be definitively established.

Amino Acid Sequence↗
PubMed1994

HIV-1 associated down-modulation of CD4 gene expression is differentially restricted in lymphocytic and monocytic cell lines.

We previously demonstrated that chronic infection of a monocytic cell line (U-937) with human immunodeficiency virus type 1 (HIV-1) was not accompanied by down-modulation of CD4 transcription, unlike the situation with CD4+ T lymphocyte lines. To better understand the refractoriness of monocytes to alterations in levels of CD4 mRNA, we treated HIV-IIIB chronically infected U-937 cells with phorbol myristate acetate (PMA), a known stimulus of HIV gene expression. Although PMA caused a significant increase in HIV mRNA levels that was sustained over 7 days, no effect on CD4 transcript levels was noted. Clonal derivatives of HIV-IIIB-infected U-937 cells, which produced a variety of infectious and defective particles, were likewise not affected in ability to produce CD4 mRNA. To rule out the possibility that U-937 cells select out HIV-1 variants unable to modulate CD4 mRNA levels, we passaged infectious virus from a U-937 clonal derivative (UHC1) onto different monocytic and T lymphocytic cell lines. In monocytic cell lines (U-937, PLB-985, THP-1), we observed an avirulent infection that did not affect CD4 mRNA levels, whereas UHC1 infection of each of two T lymphocytic cell lines (CEM-T4, Jurkat) caused both cytopathic replication and reductions in CD4 mRNA levels. In one case (Jurkat), variants expressing low CD4 mRNA may have emerged, because the outgrowth no longer expressed viral products. In the other case (CEM-T4), high expression of viral genes was accompanied by CD4 mRNA down-modulation, suggesting either that low-CD4-expressing variants were selected that maintained viral gene expression or that CD4 gene expression was repressed by viral products.

CD4 Antigens↗
PubMed1994

Cell surface down-modulation of CD4 after infection by HIV-1.

Entry of HIV-1 into host cells is generally mediated by the cell surface CD4 receptor after specific interaction with the viral envelope glycoprotein gp120. Infection by HIV-1 commonly leads to the disappearance of CD4 from the plasma membrane, a phenomenon referred to as receptor down-modulation. This, in turn, renders cells refractory to subsequent infection by the same or other viruses that use the CD4 receptor for entry, creating a state of superinfection immunity. CD4 down-modulation is a complex process involving a variety of viral gene products, the effects of which may be manifest at different stages within the viral replication cycle. CD4 disappearance from the cell surface occurs in each of the CD4+ lymphocytes, T-cell lines, monocytic cell lines, and monocyte-derived macrophages. Internalization of CD4 can occur after binding of either gp120 alone or gp120 antigen-antibody complexes, and may also be mediated by the HIV-1 Nef gene. Other factors that cause cell surface CD4 depletion include reductions in CD4 transcript levels, impaired translation of CD4 mRNA, formation of CD4-gp160 intracellular complexes, and degradation of CD4 mediated by the HIV-1 Vpu gene.

Animals↗
PubMed1994

Correlation between high level gp160 expression and reduced CD4 biosynthesis in clonal derivatives of human immunodeficiency virus type 1-infected U-937 cells.

We have compared cytoplasmic CD4 mRNA accumulation, CD4 biosynthesis and steady-state levels of both CD4 protein and mRNA in a variety of clonal derivatives of U-937 cells, chronically infected with human immunodeficiency virus type 1 IIIB (HIV-1), that express various cellular and viral phenotypes. These phenotypes included defective processing of either gp160 or Gag-Pol, viruses with severely limited host-range, and inability to generate viral products. All clones, with the exception of the one that failed to generate viral mRNA and proteins, did not express cell surface CD4. Furthermore, each of these clones had steady-state levels of CD4 mRNA which were either equivalent to or higher than those of the parental U-937 cell line. Patterns of cytoplasmic CD4 mRNA levels resembled those of total RNA, suggesting that CD4 mRNA transport from the nucleus to the cytoplasm was unaffected by HIV-1 infection. Profiles of steady-state levels of the CD4 protein resembled those of CD4 mRNA in the UHC clones, but CD4 biosynthesis was reduced in all clones with the exception of that which failed to express viral products. This report is the first demonstration that steady-state CD4 biosynthesis is reduced in HIV-1-infected cells. In general, there was a good correlation between high levels of expression of gp160 and reduced CD4 biosynthesis. These results suggest that HIV-1 env gene products may contribute to the observed reduction in levels of CD4 biosynthesis.

Blotting, Northern↗
PubMed1993

Infection of human monocyte-derived macrophages by human immunodeficiency virus mediated by cell-to-cell transmission.

We have infected ten-day-old primary cultures of human monocyte-derived macrophages (MDM) with HIV-1 by cocultivation with chronically infected monocytic cell lines. This work has involved the U-937 monocytoid cell line, chronically infected with the HIV-IIIB strain of HIV-1 (U-937HIV IIIB) as well as a number of cell clones, termed UHC, which were derived from U-937HIV IIIB by limiting dilution. Cell-free virus, derived from each of U-937HIV IIIB cells and the UHC1 clone were noninfectious for MDM, as determined by failure to express viral p24 antigen (Ag). In contrast, viral p24 Ag production was detected in MDM that had been cocultivated with U-937HIV IIB, and with each of three UHC clones that produced infectious virus. Infection, in each case, was confirmed by polymerase chain reaction detection via the amplification of proviral DNA. In contrast, cocultivation with the UHC15.7 clone, which fails to cleave viral gp160 to its gp120 and gp41 products or the UHC8 clone, which lacks functional reverse transcriptase, did not lead to infection of MDM. Pretreatment of MDM for 2 hr with 1 microM AZT completely prevented infection by culture fluids containing HIVada, a macrophage-tropic virus, but did not affect infection mediated by cocultivation. These results suggest that cell-to-cell transmission of HIV-1, among monocyte-derived macrophages, can be mediated by proviral DNA. Moreover, gp120 at the surface of infected cells may play an important role in this process, since cell-to-cell HIV transmission could not be demonstrated with the UHC clone that is defective in cleavage of the viral envelope glycoprotein gp160.

Base Sequence↗
PubMed1993

Expression, purification, and RNA-binding properties of HIV-1 p15gag nucleocapsid protein.

We have cloned and expressed HIV-1 gag p15 nucleocapsid protein (NCp15) in the form of a 41-kDa fusion polypeptide with glutathione-S-transferase (GST-NCp). The recombinant protein was rapidly degraded in bacterial lysates unless Zn2+ and Cd2+ were present in the extraction buffer. Inclusion of these metals stabilized the protein, allowing facile purification of GST-NCp by affinity chromatography. The native NCp15 was readily prepared from GST-NCp by proteolytic cleavage with thrombin. Both GST-NCp and the processed NCp15 were able to bind RNA containing sequences from the 5'-end of the HIV-1 genome. This binding was unaffected by the absence or the presence of Zn2+; however, the binding of RNA was absolutely dependent on the presence of K+. The GST-NCp fusion protein was nonselective in the binding of RNA, with all transcripts, including antisense and non-HIV RNA, binding with equal efficiency. In contrast, NCp15 was highly selective in binding of RNA. Sequences within nucleotides 1244-1412 of the HIV-1 proviral genome were found necessary for maximal binding of RNA to NCp15.

Base Sequence↗
PubMed1993

Identification of a novel glucocorticoid response element within the genome of the human immunodeficiency virus type 1.

We have shown that (HIV-1) replication can be regulated by interaction between glucocorticoid hormones and the viral genome; treatment of acutely infected lymphoid and monocytoid cell lines with cortisol and dexamethasone increased HIV-1 production in culture. The magnitude of this response correlated with glucocorticoid receptor (GR) and GR message in responder and non-responder cell lines. Furthermore, treatment of each of two HIV-infected cell lines with glucocorticoids led to enhancement of HIV-1 gene expression. We have identified a novel intragenic glucocorticoid response element (GRE) within the genome of HIV-1 at position +5002 in the vif open reading frame, as well as a second potential GRE, previously identified by other researchers at position -257 in the HIV-1 negative regulatory element (LTR-NRE). Our data indicate that only the motif at position +5002 represents a true GRE that confers glucocorticoid inducibility to a MMTV-luciferase reporter gene construct. The GRE located at -257 lacked significant functional activity in its native configuration in that it could bind GR but did not transactivate reporter gene constructs. However, this sequence was able to impart glucocorticoid inducibility when inverted or dimerized. These results suggest that as in the case of other retroviruses, HIV-1 has evolved to interface with the GR signal transduction pathway to gain replicative advantage in target cells.

Animals↗
PubMed1993

Effect of 3'-azido-3'-deoxythymidine on human immunodeficiency virus type 1 replication in human fetal brain macrophages.

We investigated whether cells derived from the fetal central nervous system can support productive infection by a human immunodeficiency virus type 1 (HIV-1) isolate termed UHC-1, produced by a cellular clone derived from HIV-1 strain HIV-IIIB chronically infected U-937 promonocytic cells, and what the effect of nucleoside analogs might be on viral replication in this system. Fractionation of human fetal brain tissue into two different populations, enriched for either astrocytes or macrophages, showed that only the latter were able to support productive UHC-1 replication and generation of detectable progeny virus. Pretreatment of fetal brain macrophages with either of two nucleoside analogs, 3'-azido-3'-deoxythymidine (AZT) or the (-) enantiomer of 2'-deoxy-3'-thiacytidine, efficiently blocked production of progeny virus. Generation of unintegrated proviral DNA and HIV-1 transcripts were inhibited by pretreatment of fetal brain macrophages with 1 microM AZT. Administration of AZT at 24 h postinfection led to a slight reduction in viral transcript levels and viral progeny production by day 15 postinfection; however, brain macrophages under these conditions did not contain detectable amounts of unintegrated viral DNA. These results suggest that AZT may interfere with the accumulation of unintegrated HIV-1 DNA in brain macrophages. This is the first demonstration that nucleoside analogs are able to block HIV-1 replication in primary cultures of brain cells.

Acquired Immunodeficiency Syndrome↗
PubMed1993

The role and fate of the CD4 molecule in lymphocytes and monocytes infected by HIV-1.

Infection by HIV-1 of monocyte cell lines, in contrast to T lymphocytes, did not lead to decreased steady-state levels of CD4 mRNA. Similar results were also obtained using clonal derivatives of infected U-937 cells that produced either competent, highly replicative progeny viruses or defective non-infectious particles. In each case, the infected U-937 cells or clonal derivatives were found to be significantly deficient with regard to surface representation of CD4 protein, in spite of the presence of high levels of CD4 mRNA. However, both HIV-1-infected U-937 cells, as well as clonal derivatives which produced high levels of viral env mRNA and non-infectious viral structures that lacked envelope glycoproteins, contained diminished levels of OKT4-immunoprecipitable CD4 protein, in comparison with uninfected U-937 cells. Thus, expression of viral env mRNA but neither the efficient synthesis or packaging of viral glycoproteins or viral assembly is required for disappearance of cell surface CD4 to occur. Furthermore, viral gp160 co-precipitated with CD4 in both the parental and cloned cell lines. We have also shown that the generation of intracellular complexes of gp160 and CD4 is directly responsible for the disappearance of cell surface CD4 in HIV-1-infected U-937 cells. In this system, expression of gp160 was both necessary and sufficient to result in CD4 receptor down-modulation. Finally, in vitro co-translation studies revealed that the presence or synthesis of viral gp160 led to a failure to efficiently generate CD4 protein.

Blotting, Northern↗
PubMed1993

Clinical correlates and molecular basis of HIV drug resistance.

It has been widely reported that zidovudine (ZDV)-resistant variants of human immunodeficiency virus type 1 (HIV-1) can be isolated from patients undergoing prolonged therapy with this drug. At the same time, treatment of HIV-infected individuals with ZDV and other forms of nucleotide therapy, including didanosine (ddI), have enabled patients to live longer than would otherwise be the case and to enjoy improved quality of life. HIV resistance to ZDV, ddI, and other nucleosides is attributable to a series of point mutations within the pol gene of HIV-1 that encodes the viral enzyme, reverse transcriptase (RT). This is not surprising as the virus is known to replicate at high rates in infected individuals; moreover the RT that mediates transcription of proviral DNA from viral genomic RNA is known to be highly error prone. Thus, mutants of HIV-1, which possess a drug-resistance phenotype and genotype, may be expected to emerge under the selective pressure of long-term antiviral chemotherapy. This article describes a novel mutation at site 184 within the pol gene that accounts for resistance against both ddI and zalcitibine (ddC). HIV drug resistance occurs most commonly in individuals with low CD4 cell counts who have progressed to more serious forms of disease. Moreover, viruses obtained from patients with AIDS generally display higher levels of resistance, relative to pretreatment isolates, than do viruses from patients with more-limited illness. Although observations of drug resistance can be correlated with disease progression and a weakened immune system, it is still unclear whether a cause-and-effect relationship exists. Because of the error-prone nature of viral RT and the fact that the HIV-1 genome can mutate efficiently, it can be anticipated that viral drug resistance may emerge for all forms of nucleotide therapy to be offered in the future. In addition, resistance may also become apparent with regard to drugs that block HIV replication by acting at sites within the viral replication cycle other than RT.

CD4-Positive T-Lymphocytes↗
PubMed1992

Differential susceptibilities of U-937 cell clones to infection by human immunodeficiency virus type 1.

Single-cell clones derived from the U-937 monocytic cell line were studied for susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). Of four such clones, we found that three (UC12, UC14, and UC18) supported replication of HIV-1 more efficiently than parental U-937 cells, as measured by reverse transcriptase activity and p24 core antigen production. In contrast, another clone (UC11) showed only baseline infection throughout an 8-week culture period, before finally becoming positive for expression of viral antigen. This differential susceptibility to infection directly correlated with accumulation of intracellular viral DNA. Furthermore, the UC11 clone expressed lower levels of Sendai virus-inducible tumor necrosis factor alpha mRNA than did the UC12 or UC18 clones. Susceptibility to infection did not correlate with expression of cell surface CD4, since all clones expressed similar levels of CD4 mRNA and surface membrane CD4 protein. Prior exposure of both susceptible UC18 and resistant UC11 clones to Leu3a antibody completely blocked infection by HIV-1, suggesting that no other independent receptors were recognized by the virus.

Blotting, Southern↗
PubMed1991

Increased DNA synthesis and repair-enzyme expression in lymphocytes from patients with chronic lymphocytic leukemia resistant to nitrogen mustards.

Resistance to the nitrogen mustards in patients with chronic lymphocytic leukemia (CLL) correlates with an enhanced removal of melphalan-induced DNA interstrand cross-links. This finding suggests that DNA repair enzymes may be involved in this process. The activity of 3-methyladenine-DNA glycosylase, which can release altered bases, including adducts at the N-7 position of guanine, was increased significantly in lymphocytes from patients with resistant CLL compared with those from untreated CLL patients. Since glycosylase activity varies with cell proliferation, the amount of [3H]thymidine incorporated into DNA was determined and found to be elevated almost threefold in lymphocytes from patients with resistant CLL. The ratio of glycosylase activity to level of thymidine incorporation did not differ between these two groups of patients. Northern blot analysis of ERCC1 gene (a putative DNA repair enzyme involved in nucleotide excision repair) expression in lymphocytes from patients with CLL revealed multiple gene transcripts (1.1, 3.4, and 3.8 kilobases). In addition, analysis of two samples revealed the presence of a 2.6-kilobase transcript. The 2.6-kilobase transcript was recognized by specific RNA probes that hybridize to antisense ERCC1 transcripts. Levels of expression of the 1.1-kilobase protein encoding transcript in lymphocytes from patients with resistant CLL were increased twofold to threefold above those of untreated patients with CLL. These results indicate that increased expression of ERCC1 and increased activity of 3-methyladenine-DNA glycosylase occur with the development of resistance to the nitrogen mustards in patients with CLL, suggesting a role for enhanced DNA repair in this process.

Aged↗
PubMed1991

Diminution of CD4 surface protein but not CD4 messenger RNA levels in monocytic cells infected by HIV-1.

As expected, the productive infection of several monocytic cell lines by HIV-1 led to a diminution of cell-surface CD4 antigen. However, unlike findings reported for HIV-1-infected T cells, this decrease was not accompanied by a similar reduction in levels of CD4 transcripts. OKT4 monoclonal antibodies (MAbs) to CD4 were used in immunoprecipitation experiments to show that intracellular CD4 levels were diminished in U-937 monocytic cells that had been infected by HIV-1. These MAbs also coprecipitated viral gp120, indicating that CD4-gp120 complexes are present in infected monocytes. Our results therefore demonstrate that cell-surface down-modulation of CD4 is exclusively a post-transcriptional event in HIV-1 infected monocytic cells. These data suggest that HIV-1-mediated depletion of cell-surface CD4 in monocytes does not involve transcript down-modulation as has been reported in T lymphocytes.

Antibodies, Monoclonal↗
PubMed1990

High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells.

Limiting-dilution techniques were employed to derive single-cell clones from U-937 cells that had been chronically infected with human immunodeficiency virus type 1. All clones thus obtained were positive for the presence of viral antigens; however, not all of the clones produced infectious progeny virus, as detected by the presence of reverse transcriptase (RT) activity in culture fluids. Six of these clones were monitored over time to determine whether their phenotype of human immunodeficiency virus type 1 expression was stable. Three clones maintained production of RT activity at a high level and showed a very high percentage of cells positive for viral p24 antigen, as determined by indirect immunofluorescence. The other three clones showed variations in either their levels of RT activity or the number of cells positive for p24, after which they stabilized. Infectious virus could be recovered from only three clones, as assessed by coculture experiments with different cell types. Two other clones were shown to produce noninfectious viruses. Molecular analyses at the DNA, RNA, and protein levels showed extensive variations between the viral isolates recovered from each clone.

Blotting, Northern↗