Cellular compartments of human immunodeficiency virus type 1 replication in vivo: determination by presence of virion-associated host proteins and impact of opportunistic infection.

Antigens derived from host cells are detectable in the envelope of human immunodeficiency virus type 1 (HIV-1) and result in a distinctive viral phenotype reflecting that of the host cell. An immunomagnetic capture assay targeting discriminatory host proteins was developed to differentiate between HIV-1 derived from macrophages and lymphocytes. HIV-1 propagated in macrophages or lymphocytes in vitro was selectively captured by monoclonal antibodies directed against the virally incorporated cell-type-specific host markers CD36 (macrophages) and CD26 (lymphocytes). Furthermore, by targeting these markers, virus of defined cellular origin was selectively captured from a mixed pool of in vitro-propagated viruses. This technique was further refined in order to determine the impact of opportunistic infection on HIV-1 expression from these cellular compartments in vivo. Analysis of cell-free virus purified from plasma of patients with HIV-1 infection suggested that in those with an opportunistic infection, viral replication occurred in activated lymphocytes. Interestingly, there was also significant replication in activated macrophages in those patients with untreated pulmonary tuberculosis. Thus, in addition to lymphocytes, the macrophage cellular pool may serve as an important source of cell-free HIV-1 in patients with opportunistic infections that lead to marked macrophage activation. This novel viral capture technique may allow researchers to address a wide range of important questions regarding virus-host dynamics.

Isoquinolinesulphonamide derivatives inhibit transcriptional elongation of human immunodeficiency virus type 1 RNA in a promyelocytic model of latency.

Using the OM-10.1 promyelocytic model of inducible human immunodeficiency virus type 1 (HIV-1) infection, we tested a panel of known protein kinase inhibitors for an ability to block tumour necrosis factor-alpha-induced HIV-1 expression. Among the compounds tested, the broad-spectrum protein kinase inhibitor H-7 uniquely blocked HIV-1 expression at the level of viral transcription, but did not inhibit nuclear factor kappaB activation or function. In structure-activity analysis this inhibitory activity of H-7 on HIV-1 expression corresponded with the known structural requirements for the interaction of H-7 with the ATP-binding region of protein kinase C, suggesting that it was indeed related to the kinase inhibitory properties of H-7. The mechanism of H-7 transcriptional inhibition did not involve chromatin remodelling at the HIV-1 long terminal repeat promoter, as shown by nuc-1 disruption, and appeared to involve HIV-1 RNA elongation but not initiation. Therefore, H-7 and related isoquinolinesulphonamide analogues are most likely inhibiting a kinase target essential for HIV-1 transcriptional elongation whose identity may provide new therapeutic targets for intervention.

Persistent zoonotic infection of a human with simian foamy virus in the absence of an intact orf-2 accessory gene.

Although foamy viruses (FVs) are endemic among nonhuman primates, FV infection among humans is rare. Recently, simian foamy virus (SFV) infection was reported in 4 of 231 individuals occupationally exposed to primates (1.8%). Secondary transmission to spouses has not been seen, suggesting that while FV is readily zoonotic, humans may represent dead-end hosts. Among different simian species, SFV demonstrates significant sequence diversity within the U3 region of the long terminal repeat (LTR) and 3' accessory open reading frames (ORFs). To examine if persistent human SFV infection and apparent lack of secondary transmission are associated with genetic adaptations in FV regulatory regions, we conducted sequence analysis of the LTR, internal promoter, ORF-1, and ORF-2 on a tissue culture isolate and peripheral blood mononuclear cell samples from a human infected with SFV of African green monkey origin (SFV-3). Compared to the prototype SFV-3 sequence, the LTR, internal promoter, and FV transactivator (ORF-1) showed sequence conservation, suggesting that FV zoonosis is not dependent on host-specific adaptation to these transcriptionally important regions. However, ORF-2 contains a number of deleterious mutations predicted to result in premature termination of protein synthesis. ORF-2 codes in part for the 60-kDa Bet fusion protein, proposed to be involved in the establishment of persistent cellular SFV infections. These results suggest that persistent human infection by SFV and reduced transmissibility may be influenced by the absence of a functional ORF-2.

Loss of inducible virus in CD45RA naive cells after human immunodeficiency virus-1 entry accounts for preferential viral replication in CD45RO memory cells.

Controversy exists concerning the preferential infection and replication of human immunodeficiency virus-1 (HIV-1) within naive (CD45RA+) and memory (CD45RO+) subsets of CD4+ lymphocytes. To explore the susceptibility of these subsets to HIV-1 infection, we purified CD45RA+/CD4+ (RA) and CD45RO+/CD4+ (RO) cells from normal donors and subjected them to a novel monokine activation culture scheme. Following HIV-1 infection and interleukin-2 (IL-2) induction, viral production measured on day 13 was 19-fold greater in RO cultures compared with RA cultures. IL-2-stimulated proliferation in uninfected control cultures was equivalent. To explore the mechanisms by which RA cells were reduced in viral production capacity, RA and RO cells were exposed to HIV-1 followed by treatment with trypsin, and then phytohemagglutinin antigen (PHA)-stimulated at days 4, 7, and 10 postinfection. HIV-1 production in day 4 postinfection RA and RO cultures was analogous, indicating that viral fusion and entry had occurred in both cell types. However, whereas similarly treated day 7 and 10 postinfection RO cultures produced virus, HIV-1 was markedly reduced or lost in the corresponding RA cultures. These results suggest that a temporally labile postfusion HIV-1 complex exists in unstimulated RA cells that requires cellular activation signals beyond that provided by IL-2 alone for productive infection.

Influence of cell cycle on HIV-1 expression differs among various models of chronic infection.

Because of an inherent dependence on host cell second and third messenger signaling pathways for activation of HIV-1 expression, a potential exists for a relationship between the induction of latent HIV-1 and cell-cycle-related events. To investigate this potential relationship, cellular models of latent HIV-1 infection (OM-10.1 promyelocytes, ACH-2 T-lymphocytes, and U1 promonocytes) were chemically treated or gamma-irradiated to synchronize cultures at each cell cycle stage and then examined for constitutive and TNF-alpha-induced HIV-1 expression. Cell cycle synchronization alone had no effect on HIV-1 expression in OM-10.1 and U1 cultures; whereas enhanced constitutive HIV-1 expression was observed in ACH-2 cultures at G2 + M. A 2 hour TNF-alpha treatment of all synchronized OM-10.1 cultures activated HIV-1 expression to a similar extent as unsynchronized cultures. In contrast, the extent of TNF-alpha-induced HIV-1 expression in ACH-2 S and G2 + M cultures and in the U1 G0/G1 culture was greater than that in unsynchronized control cultures. However, no delay in the initial response was observed. Thus, the influence of cell cycle on constitutive and induced HIV-1 expression varied in each cellular model and, therefore, may further relate to the different molecular mechanisms maintaining viral latency.

Ligand passing by the p75 tumour necrosis factor receptor enhances HIV-1 activation.

Recently, a HIV-dependent upmodulation of the p75 tumour necrosis factor receptor (TNFr75) was observed using latently-infected OM-10.1 promyelocytes; although the participation of TNFr75 in HIV-1 activation remained undefined. Here, using receptor cross-linking by agonistic antibodies, no direct HIV-1 activation via TNFr75 was observed. Signalling via the p55 tumour necrosis factor receptor (TNFr55) accounted for the full extent of HIV-1 activation in OM-10.1 cultures. However, in tumour necrosis factor alpha (TNF-alpha) dose titration experiments, antibody blockade of TNFr75 decreased the dose response markedly, indicating a ligand passing function. TNFr75 blockade did not alter the dose response to agonistic TNFr55 antibody induction; verifying that the effect on the TNF-alpha dose response was not due to negative signalling or cytolysis. These results demonstrate that, although not directly involved in signal transduction resulting in HIV-1 activation, TNFr75 can serve a critical ligand passing function and permit continued HIV-1 expression during limited TNF-alpha availability.

Compounds that target novel cellular components involved in HIV-1 transcription.

BACKGROUND: Therapeutic intervention designed to block expression of human immunodeficiency virus (HIV) at a cellular level may slow the clinical progression of HIV-1 disease. MATERIALS AND METHODS: Cellular models of latent (OM-10.1 and U1) and chronic (8E5) HIV infection were used to evaluate two benzothiophene derivatives, PD 121871 and PD 144795, for an ability to inhibit HIV activation and expression. RESULTS: The benzothiophene derivatives were effective at micromolar concentrations in preventing tumor necrosis factor alpha (TNF alpha)-induced HIV-1 expression in OM 10.1 and U1 cultures. These compounds inhibited the activation of HIV-1 transcription; however, this inhibition was selective in that another TNF alpha-induced response, the transcription of autocrine TNF alpha, was unaffected. Constitutive HIV-1 expression by chronically infected 8E5 cells was also significantly reduced when treated with these experimental compounds. In TNF alpha-treated OM-10.1 cultures, the inhibition of HIV-1 transcription by these compounds was not due to a block of nuclear factor-kappa B induction. The benzothiophene derivatives also inhibited HIV-1 activation by phorbol ester treatment of OM-10.1 promyelocytes, although no inhibition of cellular differentiation toward a macrophage-like phenotype was observed. Furthermore, these experimental compounds induced a state of HIV-1 latency in cytokine-activated OM-10.1 cultures even when maintained under constant TNF alpha stimulation. The benzothiophene derivatives did not inhibit the activity of the HIV-1 trans-activator, Tat, when evaluated in transient transfection assays. CONCLUSIONS: The benzothiophene derivatives appear to inhibit a critical cellular component, distinct from nuclear factor-kappa B, involved in HIV transcription and may serve to identify new therapeutic targets to restrict HIV expression.

Human immunodeficiency virus type 1 RNA expression by four chronically infected cell lines indicates multiple mechanisms of latency.

Recent information has suggested that posttranscriptional mechanisms, whereby human immunodeficiency virus type 1 (HIV-1) RNA exists as multiply spliced transcripts without promoting an accumulation of the larger messages, are responsible for maintaining a stable state of nonproductive viral expression or viral latency. To test the universality of these observations, we compared the patterns of viral RNA splicing and the frequencies of cells actually harboring HIV-1 RNA in four chronically HIV-1-infected cell lines (U1 [promonocytic], ACH-2 [T lymphocytic], OM-10.1 [promyelocytic], and J1.1 [T lymphocytic]). In uninduced U1 and ACH-2 cultures, a high frequency of cells (approximately one in six) contained HIV-1 RNA but mainly as multiply spliced transcripts, again supporting a posttranscriptional mechanism maintaining viral latency. In sharp contrast, only 1 in 50 cells in uninduced OM-10.1 and J1.1 cultures contained HIV-1 RNA, indicating a primary transcriptional mechanism controlling viral expression in these cells. Furthermore, those OM-10.1 and J1.1 cells that did contain viral RNA were in a state of productive HIV-1 expression marked by the presence of both spliced and unspliced transcripts. Even though the total absence of viral RNA in the majority of OM-10.1 and J1.1 cells indicated a state of absolute latency, treatment with tumor necrosis factor alpha induced transcription of HIV-1 RNA in nearly 100% of the cells in all four of the chronically infected cultures. Tumor necrosis factor alpha induction of U1, ACH-2, and OM-10.1 cultures resulted in an initial accumulation of multiply spliced HIV-1 RNA followed by a transition to the larger unspliced viral RNA transcripts. This RNA splice transition was less apparent in the J1.1 cell line. These results demonstrate that host cell-specific transcriptional and posttranscriptional mechanisms are important factors in the control of HIV-1 latency.

Association of high density lipoprotein with whole blood-associated acetaldehyde levels.

We hypothesized that while moderate drinking is associated with increasing levels of high density lipoprotein (HDL) levels, excessive drinking of ethanol might, in fact, be associated with lower HDL levels and by implication increased cardiovascular risk. We therefore performed analyses of whole blood-associated acetaldehyde (WBAA) as a measure of drinking behavior and HDL on blood samples from 2780 individuals applying for life insurance. Whole blood-associated acetaldehyde correlated positively with HDL in the entire sample set throughout the range of values (R = 0.101, p = 0.0001). The relationship held for females (N = 477, p = 0.036) but was stronger for males (N = 2277, p = 0.0001). We conclude that ethanol consumption correlates positively with HDL for both males and females and that the relationship persists through higher ranges of ethanol consumption.

Tumor necrosis factor receptor expression and signal transduction in HIV-1-infected cells.

OBJECTIVE: To examine the inter-relationship between HIV-1 infection and the cell surface receptors for tumor necrosis factor (TNF)-alpha, an immunoregulatory cytokine that can enhance HIV-1 replication. DESIGN: Infected promyelocytic and promonocytic cells were examined because they normally express both types of TNF receptors. METHODS: TNF receptor surface expression was determined by specific monoclonal antibody recognition and flow cytometry, and signal transduction was detected by gel shift analysis. HIV-1 activation and expression was quantitated by reverse transcriptase assay. RESULTS: In the OM-10.1 promyelocytic model of chronic infection, TNF-alpha-induced HIV-1 expression also resulted in a substantial increase in 75 kd TNF receptor (TR75) expression although 55 kD TNF receptor (TR55) levels were not dramatically altered. A series of uninfected parental HL-60 subclones all reduced TR75 surface expression in response to TNF-alpha treatment. Enhanced TR75 expression on OM-10.1 cells followed the same TNF-alpha-dose dependency as that observed for HIV-1 production. An increase in TR75 expression was also evident during the peak of an acute HIV-1 infection of HL-60 promyelocytes. Although TR55 expression was unaltered during TNF-alpha-induced HIV activation, this receptor was still involved in the viral activation process. Antibody cross-linking of TR55, in the absence of exogenous TNF-alpha, induced maximal HIV-1 expression, an up-modulation of surface TR75, and nuclear NF-kappa B activity in OM-10.1 cultures. Surprisingly, this was the case even when an antagonistic anti-TR55 antibody was used. Anti-TR55 antibody cross-linking in chronically infected U1 promonocytic cultures could only partially substitute for TNF-alpha-induced HIV-1 expression. CONCLUSIONS: Our results demonstrated that HIV-1 infection can selectively influence the surface expression of TNF receptors, potentially influencing its own expression and altering normal immunoregulatory signal transduction.

Evaluation of an immunoblot assay for serological confirmation and differentiation of human T-cell lymphotropic virus types I and II.

The confirmation of infection with human T-cell lymphotropic virus type I (HTLV-I) and type II (HTLV-II) currently involves multiple assays. These include Western blot (immunoblot) (WB) and/or radioimmunoprecipitation assay for detection of antibodies to HTLV-specific viral proteins and polymerase chain reaction and/or peptide-based enzyme immunoassays for differentiating between the two viruses. We undertook an evaluation of a modified WB assay that includes native HTLV-I viral proteins from MT-2 cells spiked with an HTLV-I transmembrane glycoprotein (recombinant p21e) and the HTLV-I- and HTLV-II-specific recombinant proteins MTA-1 and K55. The test panel consisted of well-characterized sera from U.S. blood donors, American Indians, intravenous drug users, and patients seen in sexually transmitted disease clinics. Of 158 HTLV-I/II-seropositive serum specimens tested, 156 (98.7%) were confirmed and typed as HTLV-I or HTLV-II. Of 82 HTLV-I/II-seroindeterminate or -seronegative serum specimens, only 1 was classified as HTLV-II positive: the sample had weak gag p19 and strong gag p24 and env p21e reactivity and was radioimmunoprecipitation assay negative for env gp61/68 but polymerase chain reaction positive for HTLV-II. The specificity of the modified WB for confirming and typing serum samples was therefore 100%. We conclude that this WB assay is useful for confirming and typing HTLV infection and can help simplify HTLV-I/II testing algorithms.

Regulation of HIV-1 expression by cytokine networks in a CD4+ model of chronic infection.

Using the CD4+ model of chronic HIV-1 infection, OM-10.1, we investigated the influence of TNF-alpha regulatory networks on induced viral expression. Previously, OM-10.1 cultures were characterized to respond to exogenous TNF-alpha, as nearly 100% of the cells were activated to express HIV-1 within 24 h. In this study, OM-10.1 cells were pulse-treated, by applying exogenous factors for short periods of time and then washing, to determine if autocrine TNF-alpha could sustain HIV-1 activation in the absence of additional exogenous stimulation. After a TNF-alpha pulse treatment, the progressive increase of HIV-1-expressing OM-10.1 cells was prevented by the continuous presence of anti-TNF-alpha mAb. The inductive activity of supernatant from TNF-alpha pulse-treated OM-10.1 cultures was completely removed by absorption on immobilized anti-TNF-alpha mAb. In addition, TNF-alpha pulse-treated OM-10.1 cells activated HIV-1 expression in untreated OM-10.1 cells when cultured across a permeable membrane indicating paracrine effects. Interestingly, if TNF-alpha pulse-treated OM-10.1 cells were further pulse-treated with anti-TNF-alpha mAb, a marked reduction in autocrine TNF-alpha was observed although the level of newly synthesized TNF-alpha mRNA remained unaffected. A similar degree of inhibition over autocrine TNF-alpha production was observed when soluble TNF receptors were used as the second pulse treatment in these experiments. Although the applicability of these results to in vivo chronically HIV-1-infected cells remains to be realized, these results do indicate that activated HIV-1 expression can be influenced by self-perpetuating mechanisms during periods of limited exogenous stimulation. Furthermore, physiologic mechanisms involving soluble cytokine receptors that counteract autocrine and paracrine activation of HIV-1 expression are shown here to play a regulatory role.

Characterization of a B-cell immunodominant epitope of human T-lymphotropic virus type 1 (HTLV-I) envelope gp46.

The immune response elicited by a synthetic peptide derived from an immunodominant external envelope region (Env-5, amino acids 242-257) of human T-lymphotropic virus type 1 (HTLV-I) was tested in a rabbit model of HTLV-I infection. The synthetic peptide elicited a strong antibody response to the HTLV-I envelope protein gp46; however, these antibodies failed to inhibit HTLV-I-mediated cell fusion. Immunized rabbits were not protected from HTLV-I infection as determined by seroconversion to viral core proteins by immunoblot, HTLV-I p24 antigen detection in lymphocyte cultures and polymerase chain reaction for the HTLV-I provirus in lymphocyte DNA. Env-5 peptide immunization failed to induce T-cell lymphocyte proliferative responses in rabbits, but induced antibody responses in T-cell deficient Balb c nu/nu mice suggesting that the antigenic determinant represented by the Env-5 peptide is primarily a B-cell epitope. These results further define an immunodominant epitope of the HTLV-I envelope protein and suggest that potential synthetic peptide vaccines against HTLV-I infection must contain multiple antigens that induce both humoral and cellular immune reactivity.

Isolation of human T-cell lymphotropic virus type 2 from Guaymi Indians in Panama.

Human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia/lymphoma and with a chronic degenerative myelopathy. However, another major type of HTLV, HTLV-II, has been isolated only sporadically, and little is known of disease associations, transmission routes, and risk factors for HTLV-II infection. Recent studies indicate that a high percentage of certain groups of i.v. drug users and blood donors are infected with HTLV-II. Seroepidemiologic studies have found an elevated rate of seroreactivity to HTLV among Guaymi Indians from Bocas del Toro Province, Panama. To identify the cause of seroreactivity among this unique population we used HTLV-II-specific polymerase chain reaction techniques to detect HTLV genetic sequences from blood leukocytes of three seropositive Guaymi Indians. The HTLV-II primer-amplified polymerase chain reaction products from two of these subjects were partially sequenced and matched published HTLV-II nucleotide sequences in both p24 gag (94% of 107 bases) and pol (98% of 112 bases) regions. A CD4+ T-lymphocyte line established from one of these same subjects produced HTLV-II-specific proteins when tested in antigen-capture and immunoblot assays, as well as mature HTLV particles. The demonstration of HTLV-II infection in this geographically and culturally isolated Central American Indian population without typical risk factors for HTLV infection suggests that HTLV-II infection is endemic in this population and provides an important clue to potential natural reservoir for this virus.

Steady state enzyme kinetics: experimental design and data analysis by microcomputer.

One of the most time-consuming, yet essential, operations involved in the steady state kinetic study of enzymes is the design and optimization of experimental conditions. A computer program was developed for the Sinclair ZX-81 (or TS-1000, 1500) microcomputer which will optimize substrate concentration for preliminary and subsequently more refined kinetic analysis of one, two or three substrate systems. This program also analyzes the data collected from these studies by linear regression, weighted linear regression or weighted non-linear regression. In addition to the above program several of the enzyme kinetic statistical analysis programs of Cleland (1979) have been translated from FORTRAN into BASIC and implemented on the ZX-81 and the TRS-80 model II. Inexpensive commercially available software was used to overcome the inability of the ZX-81 to read data files from magnetic tape making the data analysis programs easier to use.