Small-molecule inhibition of human immunodeficiency virus type 1 replication by specific targeting of the final step of virion maturation

Despite the effectiveness of currently available human immunodeficiency virus type 1 (HIV-1) therapies, a continuing need exists for new drugs to treat HIV-1 infection. We investigated the mechanism by which 3-O-[3',3'-dimethylsuccinyl]-betulinic acid (DSB) inhibits HIV-1 replication. DSB functions at a late stage of the virus life cycle but does not inhibit the HIV-1 protease in vitro or interfere with virus assembly or release. DSB specifically delays the cleavage of Gag between the capsid (CA) and p2, resulting in delayed formation of the mature viral core and reduced HIV-1 infectivity. Replication of simian immunodeficiency virus (SIV) was resistant to DSB; however, a chimeric SIV carrying CA-p2 sequences from HIV-1 was inhibited by the drug, indicating that susceptibility to DSB maps to the CA-p2 region of the HIV-1 Gag protein. A single point mutation at the CA-p2 cleavage site of HIV-1 conferred strong resistance to DSB, confirming the target of the drug. HIV-1 strains that are resistant to a variety of protease inhibitors were sensitive to DSB. These findings indicate that DSB specifically protects the CA-p2 cleavage site from processing by the viral protease during virion maturation, thereby revealing a novel mechanism for pharmacologic inhibition of HIV-1 replication.     

Small-molecule inhibition of HIV-1 Vif

The HIV-1 protein Vif, essential for in vivo viral replication, targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses and hepatitis B virus. As Vif has no known cellular homologs, it is an attractive, yet unrealized, target for antiviral intervention. Although zinc chelation inhibits Vif and enhances viral sensitivity to A3G, this effect is unrelated to the interaction of Vif with A3G. We identify a small molecule, RN-18, that antagonizes Vif function and inhibits HIV-1 replication only in the presence of A3G. RN-18 increases cellular A3G levels in a Vif-dependent manner and increases A3G incorporation into virions without inhibiting general proteasome-mediated protein degradation. RN-18 enhances Vif degradation only in the presence of A3G, reduces viral infectivity by increasing A3G incorporation into virions and enhances cytidine deamination of the viral genome. These results demonstrate that the HIV-1 Vif-A3G axis is a valid target for developing small molecule-based new therapies for HIV infection or for enhancing innate immunity against viruses.     

Biological activity of human immunodeficiency virus type 1 Vif requires membrane targeting by C-terminal basic domains.

Human immunodeficiency virus type 1 (HIV-1) encodes a Vif protein which is important for virus replication and infectivity. Vif is a cytoplasmic protein which exists in both membrane-associated and soluble forms. The membrane-associated form is an extrinsic membrane protein which is tightly associated with the cytoplasmic side of membranes. We have analyzed the mechanism of membrane targeting of Vif and its role in HIV-1 replication. Mutagenesis studies demonstrate that C-terminal basic domains are required for membrane association. Vif mutations which disrupt membrane association also inhibit HIV-1 replication, indicating that membrane localization of Vif is likely to be required for its biological activity in vivo. Membrane binding of Vif is almost completely abolished by trypsin treatment of membranes. These results demonstrate that membrane localization of Vif requires C-terminal basic domains and interaction with a membrane-associated protein(s). This interaction may serve to direct Vif to a specific cellular site, since immunofluorescence staining and plasma membrane fractionation studies show that Vif is localized predominantly to an internal cytoplasmic compartment rather than to the plasma membrane. The mechanism of membrane targeting of Vif is different in some respects from that of other extrinsic membrane proteins, such as Ras, Src, and MARCKS, which utilize a basic domain together with a lipid modification for membrane targeting. Membrane targeting of Vif is likely to play an important role in HIV-1 replication and thus may be a therapeutic target.     

Mutational analysis of the human immunodeficiency virus type 1 Vif protein

Lentivirus Vif proteins are potent regulators of virus infectivity. However, relatively little is known about the functional domains, peptide motifs, or residues of any Vif protein. In this report, we present the first extensive mutagenesis analysis of the 192-amino-acid human immunodeficiency virus type 1 (HIV-1) Vif protein. A large number of scanning missense (mostly alanine substitution) and deletion mutations were introduced into the HIV-1HXB3 vif gene, and the resulting proteins were evaluated for the induction of virus infectivity as well as subcellular localization. The results show that amino acids dispersed throughout Vif's linear sequence are important for function. However, because many of the inactive proteins also appear to be mislocalized, we suggest that many of them may actually be misfolded rather lacking an intracellular targeting signal. Interestingly, disruptions within an internal region spanning residues 114 to 146 give rise to mutant proteins that either retain function or are inactive but are not substantially mislocalized. We therefore speculate that this region, which harbors two essential cysteine residues and one essential serine residue, may contain aspects of a putative Vif effector domain.     

Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.     

Vif substitution enables persistent infection of pig-tailed macaques by human immunodeficiency virus type 1

Among Old World monkeys, pig-tailed macaques (Pt) are uniquely susceptible to human immunodeficiency virus type 1 (HIV-1), although the infection does not persist. We demonstrate that the susceptibility of Pt T cells to HIV-1 infection is due to the absence of postentry inhibition by a TRIM5 isoform. Notably, substitution of the viral infectivity factor protein, Vif, with that from pathogenic SIVmne enabled replication of HIV-1 in Pt T cells in vitro. When inoculated into juvenile pig-tailed macaques, the Pt-tropic HIV-1 persistently replicated for more than 1.5 to 2 years, producing low but measurable plasma viral loads and persistent proviral DNA in peripheral blood mononuclear cells. It also elicited strong antibody responses. However, there was no decline in CD4(+) T cells or evidence of disease. Surprisingly, the Pt-tropic HIV-1 was rapidly controlled when inoculated into newborn Pt macaques, although it transiently rebounded after 6 months. We identified two notable differences between the Pt-tropic HIV-1 and SIVmne. First, SIV Vif does not associate with Pt-tropic HIV-1 viral particles. Second, while Pt-tropic HIV-1 degrades both Pt APOBEC3G and APOBEC3F, it prevents their inclusion in virions to a lesser extent than pathogenic SIVmne. Thus, while SIV Vif is necessary for persistent infection by Pt-tropic HIV-1, improved expression and inhibition of APOBEC3 proteins may be required for robust viral replication in vivo. Additional adaptation of the virus may also be necessary to enhance viral replication. Nevertheless, our data suggest the potential for the pig-tailed macaque to be developed as an animal model of HIV-1 infection and disease.     

Subcellular localization of the Vif protein of human immunodeficiency virus type 1.

The Vif (viral infectivity factor) protein of human immunodeficiency virus type 1 (HIV-1) has been shown to dramatically enhance the infectivity of HIV-1 virus particles during virus production. The subcellular localization of Vif was examined to elucidate cellular pathways which may be important for Vif function. Indirect immunofluorescence staining of Vif demonstrated a diffuse cytoplasmic distribution and showed that most Vif was not associated with the Golgi complex, a proposed site of localization (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). Subcellular fractionation of transfected COS cells and HIV-1-infected Jurkat and CEM cells demonstrated that Vif is a cytoplasmic protein which exists in both a soluble cytosolic form and membrane-associated form. The membrane-associated form of Vif is a peripheral membrane protein which is tightly associated with the cytoplasmic side of cellular membranes. The C terminus of Vif was required for the stable association of Vif with membranes. The C terminus was also essential for Vif function, suggesting that the association of Vif with membranes is likely to be important for its biological activity. The highly conserved regions at residues 103 to 115 and 142 to 150 were important for Vif function but did not affect membrane association, indicating that these regions are likely to be important for other, as-yet-unknown functions.     

The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin

The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.     

Mechanisms of human immunodeficiency virus type 1 inhibition by hydroxyurea

Virus life cycles depend on cellular factors. Therefore, targeting cellular in combination with viral enzymes could be an effective control in virus replication. In contrast to viral proteins, cellular proteins are not prone to mutations; therefore, viral escape is not expected from drugs inhibiting cellular factors. Hydroxyurea inhibits the cellular enzyme ribonucleotide reductase, thus reducing DNA synthesis. Furthermore, this drug potentiates the activity of nucleoside analogues, inhibits the escape of A-analogue resistant mutants, and increases the phosphorylation of T-analogues. Besides its antiviral activity, hydroxyurea effects the immune system by decreasing immune activation, inhibiting the expansion of CD8 cells and the depletion of CD4 cells. Hydroxyurea has been used in medicine for 40 years, is well tolerated, and it is the least expensive available anti-HIV-1 drug. These characteristics make hydroxyurea a primary candidate for use in combination therapies for the treatment of HIV-1 infection.     

Reciprocal regulatory interaction between human herpesvirus 8 and human immunodeficiency virus type 1

Human herpesvirus 8 (HHV8) is the primary viral etiologic agent in Kaposi's sarcoma (KS). However, individuals dually infected with both HHV8 and human immunodeficiency virus type 1 (HIV-1) show an enhanced prevalence of KS when compared with those singularly infected with HHV8. Host immune suppression conferred by HIV infection cannot wholly explain this increased presentation of KS. To better understand how HHV8 and HIV-1 might interact directly in the pathogenesis of KS, we queried for potential regulatory interactions between the two viruses. Here, we report that HHV8 and HIV-1 reciprocally up-regulate the gene expression of each other. We found that the KIE2 immediate-early gene product of HHV8 interacted synergistically with Tat in activating expression from the HIV-1 long terminal repeat. On the other hand, HIV-1 encoded Tat and Vpr proteins increased intracellular HHV8-specific expression. These results provide molecular insights correlating coinfection with HHV8 and HIV-1 with an unusually high incidence of KS.     

Intravirion processing of the human immunodeficiency virus type 1 Vif protein by the viral protease may be correlated with Vif function

The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus particles through an interaction with viral genomic RNA in which it associates with the viral nucleoprotein complex. We now demonstrate for the first time that virus-associated Vif is subject to proteolytic processing by the viral protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral Pr and was sensitive to Pr inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala(150). Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also did not influence Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function, suggesting that intravirion processing of Vif is important for the production of infectious viruses.     

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.     

Cytoskeleton association and virion incorporation of the human immunodeficiency virus type 1 Vif protein.

The human immunodeficiency virus type 1 (HIV-1) Vif protein has an important role in the regulation of virus infectivity. This function of Vif is cell type specific, and virions produced in the absence of Vif in restrictive cells have greatly reduced infectivity. We show here that the intracellular localization of Vif is dependent on the presence of the intermediate filament vimentin. Fractionation of acutely infected T cells or transiently transfected HeLa cells demonstrates the existence of a soluble and a cytoskeletal form and to a lesser extent the presence of a detergent-extractable form of Vif. Confocal microscopy suggests that in HeLa cells, Vif is predominantly present in the cytoplasm and closely colocalizes with the intermediate filament vimentin. Treatment of cells with drugs affecting the structure of vimentin filaments affect the localization of Vif accordingly, indicating a close association of Vif with this cytoskeletal component. The association of Vif with vimentin can cause the collapse of the intermediate filament network into a perinuclear aggregate. In contrast, analysis of Vif in vimentin-negative cells reveals significant staining of the nucleus and the nuclear membrane in addition to diffuse cytoplasmic staining. In addition to the association of Vif with intermediate filaments, analyses of virion preparations demonstrate that Vif is incorporated into virus particles. In sucrose density gradients, Vif cosediments with capsid proteins even after detergent treatment of virus preparations, suggesting that Vif is associated with the inner core of HIV particles. We propose a model in which Vif has a crucial function as a virion component either by regulating virus maturation or following virus entry into a host cell possibly involving an interaction with the cellular cytoskeletal network.     

Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1

Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.