Antiviral properties of aminodiol inhibitors against human immunodeficiency virus and protease

A series of aminodiol inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were identified by using an in vitro peptide cleavage assay. BMS 182,193, BMS 186,318, and BMS 187,071 protected cells against HIV-1, HIV-2, and simian immunodeficiency virus infections, with 50% effective doses ranging from 0.05 to 0.33 microM, while having no inhibitory effect on cells infected with unrelated viruses. These compounds were also effective in inhibiting p24 production in peripheral blood mononuclear cells infected with HIV-1 IIIB and against the zidovudine-resistant HIV-1 strain A018C. Time-of-addition studies indicated that BMS 182,193 could be added as late as 27 h after infection and still retain its antiviral activity. To directly show that the activity of these compounds in culture was due to inhibition of proteolytic cleavage, the levels of HIV-1 gag processing in chronically infected cells were monitored by Western blot (immunoblot) analysis. All compounds blocked the processing of p55 in a dose-dependent manner, with 50% effective doses of 0.4 to 2.4 microM. To examine the reversibility of BMS 186,318, chronically infected CEM-SS cells were treated with drug and virions purified from the culture medium. Incubation of HIV-1 particles in drug-free medium indicated that inhibition of p55 proteolysis was slowly reversible. The potent inhibition of HIV-1 during both acute and chronic infections indicates that these aminodiol compounds are effective anti-HIV-1 compounds.     

Thalidomide and thalidomide analogs reduce HIV type 1 replication in human macrophages in vitro

Thalidomide is currently being evaluated for efficacy in alleviating some manifestations of HIV-1 infection. To determine whether thalidomide has any direct effects on HIV-1 infection, we investigated the effect of thalidomide and also of three structural analogs of thalidomide on HIV-1 replication in vitro in human monocyte-derived macrophages. The thalidomide analogs were previously shown to inhibit TNF-alpha production in vitro at much lower concentrations than thalidomide. In HIV-1-infected macrophages treated with thalidomide or thalidomide analogs, viral replication was reduced by 60 to 80% as determined by measuring viral RT activity in the culture supernatants. In all experiments the analogs inhibited HIV-1 replication more efficiently than did thalidomide. The drugs also reduced HIV-1 gag mRNA expression. Furthermore, the drugs caused a decrease in NF-kappaB-binding activity in nuclear extracts of HIV-1-infected macrophages. The role of NF-kappaB in the drug-induced inhibition of HIV-1 replication was confirmed using an NF-kappaB-defective mutant virus to infect macrophages.     

In vitro isolation and identification of human immunodeficiency virus (HIV) variants with reduced sensitivity to C-2 symmetrical inhibitors of HIV type 1 protease

Protease inhibitors are another class of compounds for treatment of human immunodeficiency virus (HIV)-caused disease. The emergence of resistance to the current anti-HIV drugs makes the determination of potential resistance to protease inhibitors imperative. Here we describe the isolation of an HIV type 1 (HIV-1) resistant to an HIV-protease inhibitor. Serial passage of HIV-1 (strain RF) in the presence of the inhibitor, [2-pyridylacetylisoleucylphenylalanyl-psi (CHOH)]2 (P9941), failed to yield a stock of virus with a resistance phenotype. However, variants of the virus with 6- to 8-fold reduced sensitivity to P9941 were selected by using a combination of plaque assay and endpoint titration. Genetic analysis and computer modeling of the variant proteases revealed a single change in the codon for amino acid 82 (Val-->Ala), which resulted in a protease with lower affinity and reduced sensitivity to this inhibitor and certain, but not all, related inhibitors.     

Antifungal activity of interleukin-2-activated natural killer (NK1.1+) lymphocytes against Candida albicans

Protease inhibitors are currently the most effective antiviral agents against human immunodeficiency virus type 1 (HIV-1). In this study we determined the effect of four HIV-1 protease inhibitors on human T cell leukemia virus type 1 (HTLV-I). Rhesus monkey cells infected with HTLV-I were treated with different concentrations of indinavir, saquinavir, ritonavir, or nelfinavir. The effect of these inhibitors was monitored through their effect on the processing efficiency of the viral Gag protein in cells, the natural substrate for the viral protease. These inhibitors failed to block processing of HTLV-I Gag. To confirm these findings, human cells were cotransfected with plasmids encoding infectious copies of HIV-1 and HTLV-I, and the cells were subsequently treated with these same HIV-1 protease inhibitors. At concentrations between 5 and 50 times the IC50 for inhibition of HIV-1 replication, inhibition of HIV-1 Gag cleavage was apparent. In contrast, no effect on HTLV-I Gag processing was seen. At higher concentrations, HIV-1 Gag processing was essentially completely inhibited whereas HTLV-I Gag cleavage was still unaffected. Thus, these inhibitors are not effective inhibitors of HTLV-I Gag processing. Sequence alignments of the HIV-1 and HTLV-I viral proteases and processing sites suggest that the active site of the HTLV-I protease may have subtle differences in substrate recognition compared with the HIV-1 protease.     

Kinetic characterization of human immunodeficiency virus type-1 protease-resistant variants

Passage of human immunodeficiency virus type-1 (HIV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in sequential accumulation of mutations in HIV-1 protease. We have characterized recombinant HIV-1 proteases that contain these mutations either individually (L10F, M46I, I47V, I50V) or in combination (the double mutant L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic properties and affinities for sulfonamide inhibitors and other classes of inhibitors were determined. For the I50V mutant, the efficiency (kcat/Km) of processing peptides designed to mimic cleavage junctions in the HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple mutant had a 2-fold higher processing efficiency than the I50V single mutant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory. The effects of mutation on processing efficiency were used in conjunction with the inhibition constant (Ki) to evaluate the advantage of the mutation for viral replication in the presence of drug. These analyses support the virological observation that the addition of M46I and I47V mutations on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478. Crystal structures and molecular models of the active site of the HIV-1 protease mutants suggest that changes in the active site can selectively affect the binding energy of inhibitors with little corresponding change in substrate binding.     

Kinetic properties of saquinavir-resistant mutants of human immunodeficiency virus type 1 protease and their implications in drug resistance in vivo

In order to study the basis of resistance of human immunodeficiency virus, type 1 (HIV-1), to HIV-1 protease inhibitor saquinavir, the catalytic and inhibition properties of the wild-type HIV-1 protease and three saquinavir resistant mutants, G48V, L90M, and G48V/L90M, were compared. The kinetic parameter kcat/Km was determined for these proteases using eight peptide substrates whose sequences were derived from the natural processing site sequences of HIV-1. The kcat/Km values were determined using conventional steady-state kinetics as well as initial velocities of mixed substrate cleavages under the condition where the substrate concentrations [S]o < Km. The independently determined kcat and Km values for some of the substrates confirmed the accuracy of the mixed-substrate method and also permitted the calculation in all cases of true rather than relative kcat/Km values. The Ki values were also determined. Using a previously described kinetic model [Tang, J., & Hartsuck, J. A. (1995) FEBS Lett. 367, 112-116], the relative processing activities of HIV-1 protease variants were estimated in the saquinavir concentration range of 0-10(-7) M. Although the protease activity of G48V, L90M, and G48V/L90M are only about 10, 7, and 3% of that of the wild-type HIV-1 protease in the absence of inhibitor, the resistance tendencies of the three mutants are clearly manifest by relatively less activity loss as inhibitor concentration becomes higher. Also, the ratios of the activities of the four protease species at certain saquinavir concentrations appear to correlate with the population ratios of the four protease species at different time points of clinical trials. This correlation suggests that the population ratio of the protease species is driven by in vivo saquinavir concentration, which appears to be in the range 10(-10)-10(-9) M during the clinical trials.     

Upregulation of HIV-1 replication in chronically infected cells by ingenol derivatives

We have previously reported that ingenol derivatives are highly potent inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in acutely infected cells. In this study, however, we have found that some ingenol derivatives strongly enhance the replication of HIV-1 in chronically infected cells at nanomolar concentrations. One of the derivatives could activate nuclear factor kappa B (NF-kappa B), a potent inducer of HIV-1 replication, through the activation of protein kinase C (PKC). Whereas another derivative, which affected neither PKC nor NF-kappa B, significantly enhanced HIV-1 replication, suggesting that a PKC-independent mechanism may also exist in ingenol derivative-induced HIV-1 upregulation.     

Thioltransferase (glutaredoxin) is detected within HIV-1 and can regulate the activity of glutathionylated HIV-1 protease in vitro

Previous studies have suggested that the two conserved cysteines of the HIV-1 protease may be involved in regulating protease activity. Here, we examined diglutathionylated wild type protease (Cys-67-SSG, Cys-95-SSG) and the monoglutathionylated protease mutants (C67A, Cys-95-SSG and C95A, Cys-67-SSG) as potential substrates for thioltransferase (glutaredoxin). Time-dependent changes in the extent of deglutathionylation of each protein were assayed by reverse phase-high performance liquid chromatography. Glutathione alone was not an effective reductant, whereas thioltransferase displayed differential catalysis toward the Cys-95-SSG and Cys-67-SSG sites. At low thioltransferase concentrations (5 nM), deglutathionylation occurred almost exclusively at Cys-95-SSG. With substantially more thioltransferase (100 nM) Cys-67-SSG was partially deglutathionylated but only at 20% of the rate of Cys-95-SSG reduction. Treatment of the diglutathionylated protease with thioltransferase not only restored protease activity but generated an enzyme preparation that had a 3- to 5-fold greater specific activity relative to the fully reduced form. Immunoblot analysis of HIV-1MN virus with an antibody to thioltransferase detected a band co-migrating with recombinant thioltransferase that persisted following subtilisin treatment, indicating the presence of thioltransferase within HIV-1. Our results implicate thioltransferase in the regulation and/or maintenance of protease activity in HIV-1 infected cells.     

Accumulation of unintegrated circular viral DNA in monocytes and growth-arrested T cells following infection with HIV-1

Cytocidal retrovirus infection is characterized by rapid accumulation of unintegrated viral DNA forms. These are thought to be generated by multiple rounds of reinfection and have been suggested to play a central role in cytopathogenesis. Here we have reviewed the work done in this area with HIV-1, mostly using acutely and chronically infected T cell and monocytic cell lines and in some cases T cells blocked at S phase of the cell cycle by aphidicolin treatment. To these studies, we have compared our findings with HIV-1 infected primary peripheral blood monocyte-derived macrophages and untreated and growth-arrested MT-2 cells, two biologically disparate cell populations. Using 1- and 2-long terminal repeat (LTR) circular forms as indicators of unintegrated viral DNA, we found similar rapid accumulation in both untreated and growth-arrested MT-2 cells. In contrast, we found much lower levels in monocyte/macrophages. Our findings suggest that accumulation of unintegrated viral DNA does not require virus production and reinfection in growth-arrested T cells. The significantly lower levels found in monocyte/macrophages may reflect superinfection resistance, allowing the maintenance of a persistent infection.     

Rapid, transient changes at the env locus of plasma human immunodeficiency virus type 1 populations during the emergence of protease inhibitor resistance

Plasma human immunodeficiency virus type 1 (HIV-1) populations were genetically analyzed at their most variable locus, the envelope gene, during the rapid emergence of resistance to protease inhibitor monotherapy. Plasma virus populations remained genetically constant prior to drug treatment and during the 1 to 2 weeks following initiation of therapy, while viremia fell 10- to 100-fold. Concomitant with rapid plasma viremia rebounds associated with the emergence of drug-resistant virus, marked alterations were then detected at the env locus. Plasma population changes lasted only a few weeks before the reappearance of the pretreatment envelope variants. The emergence of resistance to single protease inhibitors was therefore associated with major but transient changes at a nonselected locus. Selection for resistance to single protease inhibitors thus appears to be more complex than the continued replication of a large, random, and therefore genetically representative sampling of the pretreatment plasma population. The possibility that drug-privileged anatomical sites containing distinct envelope variants and/or a small effective HIV-1 population size account for these results is discussed.