Selective inhibition of syncytium-inducing and nonsyncytium-inducing HIV-1 variants in individuals receiving didanosine or zidovudine, respectively

By studying changes in the clonal composition of HIV-1 populations during the first weeks of zidovudine (ZDV) treatment before the development of ZDV resistance-conferring mutations, we demonstrated previously a selective inhibition of nonsyncytium-inducing (NSI) HIV-1, even when present as coexisting population in individuals also harboring syncytium-inducing (SI) HIV-1. In this study, we observed the opposite in individuals receiving didanosine (ddI) treatment. In these individuals (n = 7) a median -0.98 log change (range -1.55-0.08) in infectious cellular SI load was observed, whereas the coexisting NSI load was only minimally affected (median -0.15 log, range -1.27-0.50; P = 0.03). The virus phenotype-dependent treatment responses were independent of the clonal composition of HIV-1 populations at baseline. Individuals treated with a combination of ZDV and ddI revealed an equal decline of both NSI and SI infectious cellular load (n = 4; NSI: median -1.55 log, range -2.19 to -1.45; SI: median -1.47 log, range -1.81 to -0.86; P = 0.56). To test the hypothesis that the previously reported optimal activation of ZDV and ddI in activated and resting T cells, respectively, in combination with the differential T cell tropism of NSI and SI HIV-1 is the basis for the observed virus phenotype specific efficacy of nucleoside analogs, we studied the effect of treatment with a protease inhibitor that does not require intracellular activation. Individuals receiving ritonavir (n = 4) indeed showed equal declines in NSI and SI infectious cellular load (NSI: median -2.37 log, range -2.59 to -2.16; SI: median -2.82 log, range -3.14 to -2.50; P = 0.25). Our data suggest HIV-1 phenotype as an additional parameter in the design of optimal treatment regimens.     

Evolution of syncytium-inducing and non-syncytium-inducing biological virus clones in relation to replication kinetics during the course of human immunodeficiency virus type 1 infection

To investigate the temporal relationship between human immunodeficiency virus type 1 (HIV-1) replicative capacity and syncytium-inducing (SI) phenotype, biological and genetic characteristics of longitudinally obtained virus clones from two HIV-1-infected individuals who developed SI variants were studied. In one individual, the emergence of rapidly replicating SI and non-syncytium-inducing (NSI) variants was accompanied by a loss of the slowly replicating NSI variants. In the other subject, NSI variants were always slowly replicating, while the coexisting SI variants showed an increase in the rate of replication. Irrespective their replicative capacity, the NSI variants remained present throughout the infection in both individuals. Phylogenetic analysis of the V3 region showed early branching of the SI variants from the NSI tree. Successful SI conversion seemed a unique event since no SI variants were found among later-stage NSI variants. This was also confirmed by the increasing evolutionary distance between the two subpopulations. At any time point during the course of the infection, the variation within the coexisting SI and NSI populations did not exceed 2%, indicating continuous competition within each viral subpopulation.     

Lower prevalence and incidence of HIV-1 syncytium-inducing phenotype among injecting drug users compared with homosexual men

OBJECTIVE: To study the prevalence, incidence and predictive value for progression to AIDS of the HIV-1 syncytium-inducing (SI) phenotype in HIV-infected injecting drug users (IDU) compared with HIV-infected homosexual men. DESIGN: Two prospective cohort studies on HIV-1 infection among IDU and homosexual men. METHODS: HIV-infected IDU (n = 225) and homosexual men (n = 366) without AIDS were studied from March 1989 through December 1993. Data on laboratory markers, including the presence of SI variants, demographics, behavioural characteristics and clinical events were collected at every visit. RESULTS: At baseline, SI variants were detected in 4% of IDU and 17% of homosexual men. During the study period 18 IDU and 68 homosexual men switched from non-SI to SI phenotype (4-year cumulative incidence, 14.6 and 28.4%, respectively) before AIDS diagnosis. Among participants with a documented date of HIV infection the cumulative incidence of SI was lower among IDU than homosexual men (4-year cumulative incidence, 6.2 and 20.7%, respectively). At AIDS diagnosis, 21% of all AIDS cases among IDU had the SI phenotype compared with 54% among homosexual men. In both risk groups an accelerated CD4 decline was found after the non-SI-to-SI switch. The SI phenotype appeared to be a predictor of AIDS (multivariate relative hazard, 5.33), independent of CD4 cell count and p24 antigen at baseline. In the multivariate time-dependent analysis, the relative hazard of SI phenotype decreased considerably, which is consistent with the hypothesis that the effect of SI phenotype on progression to AIDS is mediated by CD4 cell count. CONCLUSION: The SI phenotype is associated with accelerated CD4 decline and progression to AIDS in both risk groups. The remarkable lower prevalence and incidence of the SI phenotype among IDU may implicate a difference in pathogenesis and natural history of HIV infection linked to transmission group.     

Marked difference between angiotensin-converting enzyme and neutral endopeptidase inhibition in vivo by a dual inhibitor of both enzymes

Dual inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) offers the potential for improved therapy of hypertension and cardiac failure. S 21402-1 [(2S)-2-[(2S,3R)-2-thiomethyl-3-phenylbutanamido] propionic acid] is a sulfhydryl-containing potent inhibitor of both NEP (Ki = 1.7 nM) and ACE (Ki = 4.5 nM). S 21402-1 and the sulfhydryl-containing ACE inhibitor captopril were administered to rats by intraperitoneal injection (0, 0.3, 3, 30, 300 mg/kg). Urine was collected for 4 h; then plasma and kidneys were collected. The difference in NEP and ACE inhibition by S 21402-1 in vivo was greater than 1000-fold. All doses of S 21402-1 inhibited NEP, as indicated by plasma NEP activity, radioinhibitor binding to kidney sections, urinary sodium excretion and bradykinin-(1-7)/bradykinin-(1-9) ratio. However, only 300 mg/kg S 21402-1 inhibited ACE, as indicated by plasma angiotensin II/angiotensin I ratio, renin and angiotensinogen levels. Although S 21402-1 (30 and 300 mg/kg) inhibited renal NEP, as indicated by the bradykinin-(1-7)/bradykinin-(1-9) ratio in kidney, S 21402-1 had no effect on renal ACE, as indicated by the angiotensin II/angiotensin I ratio in kidney. Moreover, captopril was greater than 10-fold more potent than S 21402-1 as an ACE inhibitor in vivo. In separate experiments, the pressor response of anesthetized rats to angiotensin I showed more rapid decay in ACE inhibition by S 21402-1 than by captopril. These studies indicated that in vivo modification of S 21402-1 caused a much greater decrease in potency of ACE inhibition than NEP inhibition. Consequently, effective ACE inhibition by S 21402-1 required doses much higher than those required for NEP inhibition.     

Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules

To replicate, HIV-1 must integrate a cDNA copy of the viral RNA genome into a chromosome of the host. The integration system is a promising target for antiretroviral agents, but to date no clinically useful integration inhibitors have been identified. Previous screens for integrase inhibitors have assayed inhibition of reactions containing HIV-1 integrase purified from an Escherichia coli expression system. Here we compare action of inhibitors in vitro on purified integrase and on subviral preintegration complexes (PICs) isolated from lymphoid cells infected with HIV-1. We find that many inhibitors active against purified integrase are inactive against PICs. Using PIC assays as a primary screen, we have identified three new anthraquinone inhibitors active against PICs and also against purified integrase. We propose that PIC assays are the closest in vitro match to integration in vivo and, as such, are particularly appropriate for identifying promising integration inhibitors.     

The potential of various HIV-1 mutants to inhibit the replication of wild-type virus

Transluminal placement of a stent graft in patients with an abdominal aortic aneurysm is a new endovascular technique that offers a potentially less invasive and less risky alternative to open surgery. Complications after stent graft placement are not infrequent, but in most cases secondary endovascular intervention is successful. We describe a late major leak in the aneurysmal sac caused by a distal migration of the iliac limb of a bifurcated graft. This late complication was successfully treated by covered stent placement, excluding and thrombosing completely the reformed aneurysm.     

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor zeta chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.     

Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes

Trans- and cis-1,2-dichloroethylene (DCE) isomers inhibit their own metabolism in vivo by inactivation of the metabolizing enzyme, presumably the cytochrome P450 isoform, CYP2E1. In this study, we examined cytochrome P450 isoform-specific inhibition by three chloroethylenes, cis-DCE, trans-DCE, and trichloroethylene (TCE), and evaluated several kinetic mechanisms of enzyme inhibition with physiological models of inhibition. Trans-DCE was more potent than cis-DCE, and both were much more effective than TCE in inhibiting CYP2E1. The kinetics of in vitro loss of p-nitrophenol hydroxylase (pNP-OH) activity (a marker of CYP2E1) in microsomal incubations and of the in vivo gas uptake results were most consistent with a mechanism in which inhibition of the metabolizing enzyme (CYP2E1) was presumed to be related to interaction of a reactive DCE metabolite with remaining substrate-bound, active CYP2E1. The kinetics of inhibition by TCE, a weak inhibitor in vitro, were very different from that of the dichloroethylenes. With TCE, parent compound concentrations influenced enzyme loss. Trans-DCE was a more potent inhibitor of CYP2E1 than cis-DCE based on both in vivo and in vitro studies. Quantitative differences in the inhibitory properties of the 1,2-DCE isomers may be due to the different stability of epoxides formed from bioactivation by CYP2E1. Epoxide intermediates of DCE metabolism, reacting by water addition, would yield dialdehyde, a potent cross-linking reagent.     

Crystal structures of complexes of a peptidic inhibitor with wild-type and two mutant HIV-1 proteases

Crystal structures of the protease of human immunodeficiency virus type 1 (HIV-1) and two mutant proteases, V82D and V82N, have been determined. In all three cases the enzyme forms a complex with the peptidic inhibitor U-89360E. All structures have been determined to 2.3 A resolution and have satisfactory agreement factors: 0.173 for wild type, 0.175 for V82D, and 0.182 for V82N. Comparison of the three crystal structures provides explanations which are consistent with the known kinetic properties of these mutant enzymes with the U-89360E inhibitor [Lin, Y., Lin, X., Hong, L., Foundling, S., Heinrikson, R. L., Thaisrivongs, S., Leelamanit, W., Raterman, D., Shah, M., Dunn, B.M., & Tang, J. (1995) Biochemistry 34, 1143-1152]. Unfavorable van der Waals interactions between the inhibitor and the mutated side chains at position 82 are consistent with diminished affinity for the inhibitor by the mutant enzymes. If a mutation is potentially resistant to an inhibitor, the mutant enzyme should not only have an increased Ki for the inhibitor but should also preserve considerable catalytic capability. The V82D mutant possesses these qualities. In the V82D crystal structure, a water molecule, which connects the protease flap to the inhibitor, is missing or of low occupancy. Absence of this bridge may be important in determining catalytic capability. Moreover, mutation at position 82 induces change in two polypeptide backbone regions, 35-41 and 67-68, which may be related to protease flap mobility.     

Inhibition of HIV-1 expression by HIV-2

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.     

gp 120s derived from four syncytium-inducing HIV-1 strains induce different patterns of CD4 association with lymphocyte surface molecules

This work extends our previous finding that lymphocyte treatment with gp120IIIB specifically induces CD4 association with several surface molecules to other molecules and to three other gp120s from different HIV-1 strains. The ability to induce this association was displayed by the four gp120s employed, i.e. gp120IIIB, gp120SF2, gp120MN and gp120(451), and the association patterns were different, as shown by both co-capping and immunoprecipitation. Co-capping showed that all four gp120s significantly potentiated CD4 association with CD3, CD45RA, CD45RB, CD38, CD26, CD59 and class I MHC molecules. By contrast, CD4 association with CD95 was induced only by gp120(451) and gp120MN; that with CD11a only by gp120SF2 and gp120MN; and that with CD27 and CD45RO only by gp120MN and gp120(451) respectively. All gp120s induced significant CD4 association with CD49d, but gp120SF2 displayed a significantly weaker effect than gp120IIIB. Induction of association was not mediated by inside-out signaling via the CD4-associated tyrosine kinase p58lck, since it was not inhibited by the tyrosine kinase inhibitors herbymicin and genistein, nor by CD45 bridging between CD4 and the associating molecule, since similar patterns of association were detected IN cells expressing different CD45 isoform patterns. Moreover, it was not mediated by chemokine receptors interacting with the gp120 V3 loop, since RANTES did not alter the gp120-induced CD4 association pattern. By contrast, the observation that gp120s from four HIV-1 strains induce different CD4 association patterns suggests that gp120 directly interacts with the associating molecules, possibly via their hypervariable regions.     

Endogenous production of beta-chemokines by CD4+, but not CD8+, T-cell clones correlates with the clinical state of human immunodeficiency virus type 1 (HIV-1)-infected individuals and may be responsible for blocking infection with non-syncytium-inducing HIV-1 in vitro

Recent studies have demonstrated that the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of beta-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global beta-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of beta-chemokines in nonprogressors and AIDS patients by examination of beta-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4+ and CD8+ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4+ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4+ clones from nonprogressors and CD8+ clones from AIDS patients secreted high levels of RANTES, MIP1alpha, and MIP-1beta. In contrast, CD4+ clones from AIDS patients produced no RANTES and little or no MIP-1alpha or MIP-1beta. The infection of CD4+ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to beta-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4+ clones from nonprogressors could be partially reversed by treatment with anti-beta-chemokine antibodies. These results indicate that CD4+ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including beta-chemokines, and that beta-chemokine production by CD4+, but not CD8+, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.