Triterpene derivatives that inhibit human immunodeficiency virus type 1 replication

Triterpene derivatives were analyzed for anti-HIV-1 activity and for cellular toxicity. Betulinic aldehyde, betulinic nitrile, and morolic acid derivatives were identified to have anti-HIV-1 activity. These derivatives inhibit a late step in virus replication, likely virus maturation.     

Identification of a mutation in editing of defective Newcastle disease virus recombinants that modulates P-gene mRNA editing and restores virus replication and pathogenicity in chicken embryos

Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5'-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5'-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9- to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5'-A(5) stretch (5'-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5'-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5'-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants.     

A mutation in alpha helix 3 of CA renders human immunodeficiency virus type 1 cyclosporin A resistant and dependent: rescue by a second-site substitution in a distal region of CA

The replication of many isolates of human immunodeficiency virus type 1 (HIV-1) is enhanced by binding of the host cell protein cyclophilin A (CypA) to the viral capsid protein (CA). The immunosuppressive drug cyclosporine A (CsA) and its nonimmunosuppressive analogs bind with high affinity to CypA and inhibit HIV-1 replication. Previous studies have identified two mutations, A92E and G94D, in the CypA-binding loop of CA that confer the ability of HIV-1 to replicate in the presence of CsA. Interestingly, CsA stimulates the replication of HIV-1 mutants containing either the A92E or G94D substitution in some human cell lines. Here, we show that substitution of alanine for threonine at position 54 of CA (T54A) also confers HIV-1 resistance to and dependence on CsA. Like the previously identified CsA-resistant/dependent mutants, infection by the T54A mutant was stimulated by CsA in a target cell-specific manner. RNA interference-mediated reduction of CypA expression enhanced the permissiveness of HeLa cells to infection by the T54A mutant. A suppressor mutation, encoding a substitution of threonine for alanine at position 105 of CA (A105T), was identified through adaptation of the T54A mutant virus for growth in CEM cells. A105T rescued the impaired single-cycle infectivity and replication defects of both T54A and A92E mutants. These results indicate that CA determinants outside the CypA-binding loop can modulate the dependence of HIV-1 infection on CypA.     

A second-site mutation at glutamate-257 that restores the function of the mutant yeast ribosomal protein L5 containing lysine-270,271-->arginine

A genetic approach was used to identify interacting regions of yeast ribosomal protein L5 (also known as L1, L1a, or YL3). Previous studies from our laboratory showed that residues K270 and K271 in protein L5 are essential for its function. The mutant L5 protein in which both residues were replaced by arginine residues (K270,271R) exhibited about 80% RNA binding capability compared to the wild-type and the mutant protein was assembled into the 60S ribosomal subunits in vivo. The yeast strain expressing this mutant protein in a homozygous form was lethal (Biochim. Biophys. Acta 1308 (1996) 133-141). In the present study, this non-functional mutant was used to select intragenic suppressors. A spontaneous, intragenic suppressor which contained an E257K substitution (in addition to the primary mutations) was identified. The suppressor protein bound about 60% of yeast 5S rRNA in vitro compared to the wild-type. To gain more insight into the nature of the intragenic suppressor, additional mutant proteins in which E257 was substituted by a variety of amino acids were produced by site-directed mutagenesis. The ability of each mutant protein to bind yeast 5S rRNA in vitro and to suppress the lethal effect of the double K270,271 mutation in vivo were examined. Results suggest communication between two non-contiguous domains on protein L5 and that several factors, such as electrostatic interaction and hydrogen bonding are likely to play a role in this global communication. Mutation studies on E257 alone also reveal that substitutions of this residue in L5 protein could affect cell growth under specified conditions, but a variety of changes could be tolerated without serious deleterious effects. We propose a working model in which E257 is located in a loop and the dynamic as well as the flexibility of this loop is important for L5 function.     

Inefficient human immunodeficiency virus replication in mobile lymphocytes

Cell-to-cell viral transfer facilitates the spread of lymphotropic retroviruses such as human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV), likely through the formation of "virological synapses" between donor and target cells. Regarding HIV replication, the importance of cell contacts has been demonstrated, but this phenomenon remains only partly characterized. In order to alter cell-to-cell HIV transmission, we have maintained cultures under continuous gentle shaking and followed viral replication in this experimental system. In lymphoid cell lines, as well as in primary lymphocytes, viral replication was dramatically reduced in shaken cultures. To document this phenomenon, we have developed an assay to assess the relative contributions of free and cell-associated virions in HIV propagation. Acutely infected donor cells were mixed with carboxyfluorescein diacetate succinimidyl ester-labeled lymphocytes as targets, and viral production was followed by measuring HIV Gag expression at different time points by flow cytometry. We report that cellular contacts drastically enhance productive viral transfer compared to what is seen with infection with free virus. Productive cell-to-cell viral transmission required fusogenic viral envelope glycoproteins on donor cells and adequate receptors on targets. Only a few syncytia were observed in this coculture system. Virus release from donor cells was unaffected when cultures were gently shaken, whereas virus transfer to recipient cells was severely impaired. Altogether, these results indicate that cell-to-cell transfer is the predominant mode of HIV spread and help to explain why this virus replicates so efficiently in lymphoid organs.     

Effects of second-site mutations on dominant interference by a human immunodeficiency virus type 1 envelope glycoprotein mutant.

We have demonstrated previously that a human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein containing a Val-to-Glu substitution at the second amino acid of the transmembrane glycoprotein gp41 (termed the 41.2 mutant) dominantly interferes with wild-type envelope-mediated syncytium formation and virus infectivity. To understand the mechanism by which the 41.2 mutant exerts the dominant interfering phenotype and thereby determine further how the mutant might be used as an inhibitor of viral spread, additional mutations were made in the envelope gene, and the effects of these mutations on interference were determined. It was found that processing of the 41.2 mutant glycoprotein in gp120 and gp41 subunits and a functional CD4-binding domain are necessary for the interfering phenotype to be exhibited fully. However, neither a wild-type V3 loop nor the gp41 cytoplasmic tail is necessary for efficient interference. In addition, it was determined that the dominant interfering phenotype is not conferred exclusively by the glutamate substitution at amino acid 2 of gp41, since a substitution with a basic residue at this position also results in a dominant interfering envelope glycoprotein.     

Inhibition of human immunodeficiency virus replication by cell membrane-crossing oligomers

Although rapidly becoming a valuable tool for gene silencing, regulation or editing in vitro, the direct transfer of small interfering ribonucleic acids (siRNAs) into cells is still an unsolved problem for in vivo applications. For the first time, we show that specific modifications of antisense oligomers allow autonomous passage into cell lines and primary cells without further adjuvant or coupling to a cell-penetrating peptide. For this reason, we termed the specifically modified oligonucleotides "cell membrane-crossing oligomers" (CMCOs). CMCOs targeted to various conserved regions of human immunodeficiency virus (HIV)-1 were tested and compared with nontargeting CMCOs. Analyses of uninfected and infected cells incubated with labeled CMCOs revealed that the compounds were enriched in infected cells and some of the tested CMCOs exhibited a potent antiviral effect. Finally, the CMCOs did not exert any cytotoxicity and did not inhibit proliferation of the cells. In vitro, our CMCOs are promising candidates as biologically active anti-HIV reagents for future in vivo applications.     

A recombinant vesicular stomatitis virus bearing a lethal mutation in the glycoprotein gene uncovers a second site suppressor that restores fusion

Vesicular stomatitis virus (VSV), a prototype of the Rhabdoviridae family, contains a single surface glycoprotein (G) that is responsible for attachment to cells and mediates membrane fusion. Working with the Indiana serotype of VSV, we employed a reverse genetic approach to produce fully authentic recombinant viral particles bearing lethal mutations in the G gene. By altering the hydrophobicity of the two fusion loops within G, we produced a panel of mutants, W72A, Y73A, Y116A, and A117F, that were nonfusogenic. Propagation of viruses bearing those lethal mutations in G completely depended on complementation by expression of the glycoprotein from the heterologous New Jersey serotype of VSV. The nonfusogenic G proteins oligomerize and are transported normally to the cell surface but fail to mediate acid pH-triggered membrane fusion. The nonfusogenic G proteins also interfered with the ability of wild-type G to mediate fusion, either by formation of mixed trimers or by inhibition of trimer function during fusion. Passage of one recombinant virus, A117F, identified a second site suppressor of the fusion block, E76K. When analyzed in the absence of the A117F substitution, E76K rendered G more sensitive to acid pH-triggered fusion, suggesting that this compensatory mutation is destabilizing. Our work provides a set of authentic recombinant VSV particles bearing lethal mutations in G, confirms that the hydrophobic fusion loops of VSV G protein are critical for membrane fusion, and underscores the importance of the sequence elements surrounding the hydrophobic tips of the fusion loops in driving fusion. This study has implications for understanding dominant targets for inhibition of G-mediated fusion. Moreover, the recombinant viral particles generated here will likely be useful in dissecting the mechanism of G-catalyzed fusion as well as study steps of viral assembly.     

In vitro suppression of human immunodeficiency virus type 1 replication by measles virus

During the acute phase of measles, human immunodeficiency virus type 1 (HIV-1)-infected children have a transient, but dramatic, decrease in plasma HIV-1 RNA levels (W. J. Moss, J. J. Ryon, M. Monze, F. Cutts, T. C. Quinn, and D. E. Griffin, J. Infect. Dis. 185:1035-1042, 2002). To determine the mechanism(s) by which coinfection with measles virus (MV) decreases HIV-1 replication, we established an in vitro culture system that reproduces this effect. The addition of MV to CCR5- or CXCR4-tropic HIV-1-infected human peripheral blood mononuclear cells (PBMCs) decreased HIV-1 p24 antigen production in a dose-dependent manner. This decrease occurred with the addition of MV before or after HIV-1. The inhibition of HIV-1 p24 antigen production was decreased when UV-inactivated MV or virus-free supernatant fluid from MV-infected PBMCs was used. Inhibition was not due to increased production of chemokines known to block coreceptor usage by HIV-1, a decrease in the percentage of CD4+ T cells, or a decrease in chemokine receptor expression by CD4+ T cells. Viability of PBMCs was decreased only 10 to 20% by MV coinfection; however, lymphocyte proliferation was decreased by 60 to 90% and correlated with decreased production of p24 antigen. These studies showed that an in vitro system of coinfected PBMCs could be used to dissect the mechanism(s) by which MV suppresses HIV-1 replication in coinfected children and suggest that inhibition of lymphocyte proliferation by MV may play a role in the suppression of HIV-1 p24 antigen production.     

Suppression of simian immunodeficiency virus replication by human immunodeficiency virus type 1 trans-dominant negative rev mutants.

We demonstrate that trans-dominant negative rev mutants are able to suppress simian immunodeficiency virus provirus replication in both transient cotransfection assays and stably transduced HUT 78 cells. These studies suggest that the efficacy of trans-dominant rev strategies in reducing viral burden may be evaluated in a simian immunodeficiency virus-rhesus macaque animal model.     

Nitric oxide synthesis enhances human immunodeficiency virus replication in primary human macrophages

Macrophages are suspected to play a major role in human immunodeficiency virus (HIV) infection pathogenesis, not only by their contribution to virus dissemination and persistence in the host but also through the dysregulation of immune functions. The production of NO, a highly reactive free radical, is thought to act as an important component of the host immune response in several viral infections. The aim of this study was to evaluate the effects of HIV type 1 (HIV-1) Ba-L replication on inducible nitric oxide synthase (iNOS) mRNA expression in primary cultures of human monocyte-derived macrophages (MDM) and then examine the effects of NO production on the level of HIV-1 replication. Significant induction of the iNOS gene was observed in cultured MDM concomitantly with the peak of virus replication. However, this induction was not accompanied by a measurable production of NO, suggesting a weak synthesis of NO. Surprisingly, exposure to low concentrations of a NO-generating compound (sodium nitroprusside) and L-arginine, the natural substrate of iNOS, results in a significant increase in HIV replication. Accordingly, reduction of L-arginine bioavailability after addition of arginase to the medium significantly reduced HIV replication. The specific involvement of NO was further demonstrated by a dose-dependent inhibition of viral replication that was observed in infected macrophages exposed to N(G)-monomethyl L-arginine and N(G)-nitro-L-arginine methyl ester (L-NAME), two inhibitors of the iNOS. Moreover, an excess of L-arginine reversed the addition of L-NAME, confirming that an arginine-dependent mechanism is involved. Finally, inhibitory effects of hemoglobin which can trap free NO in culture supernatants and in biological fluids in vivo confirmed that endogenously produced NO could interfere with HIV replication in human macrophages.     

Mechanistic effects of kijimicin on inhibition of human immunodeficiency virus replication

Kijimicin represents an important type of ionophore compound. In veterinary medicine, it is becoming important as anticoccidiostatic agent and feed supplement. We examined Kijimicin for its HIV inhibitory activity. The compound exhibited concentration-dependent inhibition of HIV replication in primary infected cultures of human T-lymphoblastoid H9 cells. Substantial inhibition of viral replication was observed at concentrations of Kijimicin that showed little cytotoxicity. The ratio of IC₅₀ values for the MTT to RT assays was 40. Anti HIV activity was also observed in cultures of monocytic lineage U937 cells chronically infected with HIV. Moreover, in attempting to define the inhibitory mechanism of Kijimicin, we investigated its effect on each step of HIV replication. The infectivity of progeny viral particles was reduced by Kijimicin treatment. This decrease may be due to incompletely glycosylated forms of gp120.