A single polymorphism in HIV-1 subtype C SP1 is sufficient to confer natural resistance to the maturation inhibitor bevirimat

3-O-(3',3'-Dimethylsuccinyl) betulinic acid (DSB), also known as PA-457, bevirimat (BVM), or MPC-4326, is a novel HIV-1 maturation inhibitor. Unlike protease inhibitors, BVM blocks the cleavage of the Gag capsid precursor (CA-SP1) to mature capsid (CA) protein, resulting in the release of immature, noninfectious viral particles. Despite the novel mechanism of action and initial progress made in small-scale clinical trials, further development of bevirimat has encountered unexpected challenges, because patients whose viruses contain genetic polymorphisms in the Gag SP1 (positions 6 to 8) protein do not generally respond well to BVM treatment. To better define the role of amino acid residues in the HIV-1 Gag SP1 protein that are involved in natural polymorphisms to confer resistance to the HIV-1 maturation inhibitor BVM, a series of Gag SP1 chimeras involving BVM-sensitive (subtype B) and BVM-resistant (subtype C) viruses was generated and characterized for sensitivity to BVM. We show that SP1 residue 7 of the Gag protein is a primary determinant of SP1 polymorphism-associated drug resistance to BVM.     

Phenotypic Susceptibility to Bevirimat in Isolates from HIV-1-Infected Patients without Prior Exposure to Bevirimat

Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient''s ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.With the continued advancement of antiretroviral therapeutic options, the treatment of HIV-1 infection has greatly improved over the years. However, the development of drug-resistant variants of HIV-1 in infected patients often leads to treatment failure and underscores the need for new therapies for these patients. The naturally occurring medicinal compound betulinic acid and some of its derivatives have been shown to inhibit HIV-1 in novel ways (4-6). Bevirimat (BVM; also known as PA-457, DSB, and MPC-4326) is a derivative of betulinic acid that is currently in phase 2 clinical trials for the treatment of HIV-1 infection (3, 15) and is the first member of a novel class of maturation inhibitors of HIV-1 (8). Evidence has shown that BVM acts by inhibiting the last cleavage of the HIV-1 Gag polyprotein (Fig. ​(Fig.1)1) (8, 20, 22), preventing the release of the Gag small peptide 1 (SP1) from the Gag capsid (CA or p24) protein. This in turn leads to the formation of an immature p25 CA protein-SP1 instead of the expected p24 CA protein, which cannot condense in a timely fashion to form the capsid core and which gives rise to noninfectious virion progeny. Interestingly, BVM activity in an in vitro cell-free system could be detected only when Gag was able to assemble into an immature particle-like structure, strongly suggesting that the presumed binding of BVM near the CA protein-SP1 cleavage site is highly dependent on the surrounding Gag tertiary structure (8, 13, 21).Open in a separate windowFIG. 1.Schematic of HIV-1 Gag polyprotein showing amino acid residues associated with bevirimat susceptibility. MA, matrix; CA, capsid; SP1, small peptide 1; NC, nucleocapsid; SP2, small peptide 2.In vitro drug resistance selection experiments with BVM have been conducted and identified several mutations (Fig. ​(Fig.1)1) in the vicinity of the Gag CA protein-SP1 cleavage site. These included the mutations at the P1′ and P1 residues of the cleavage site, A364V (SP1-A1V) and L363F or L363M (CA protein-L231F or CA protein-L321M), respectively, as well as A366T or A366V (SP1-A3T or SP1-A3V) and H358Y (CA protein-H226Y) (1, 8, 22). Resistance to BVM in these mutants was characterized by their ability to cleave the p25 CA protein-SP1 efficiently in the presence of the drug (up to 3 μM) without significantly affecting their ability to replicate (1). In a phase 2 clinical trial of BVM with antiretroviral (ARV)-experienced patients, about half of the patients treated with BVM did not respond to treatment (mean HIV-1 load reduction, 0.05 log₁₀ copies/ml), while the other half showed a strong response to treatment (mean HIV-1 load reduction, 1.26 log₁₀ copies/ml) after 2 weeks of dosing (7, 12). Genotypic analyses of these patients'' HIV-1 isolates have identified three key polymorphic sites in SP1 (Q369, V370, and T371) that are associated with innate resistance to BVM (7, 12), and baseline phenotypic analyses of patient samples from BVM clinical trials have confirmed the presence of >100-fold resistance to BVM among isolates from patients with polymorphisms at these sites (16).The aim of the present study was to investigate the phenotypic susceptibility to BVM of HIV-1 isolates from HIV-1-infected patients who had not previously been exposed to BVM, in order to shed light on the observed lack of response to BVM in certain patient populations. We hypothesized that prior ARV treatment and, more specifically, treatment with protease inhibitors (PIs) may have led to the presence of adaptive mutations in Gag that could confer resistance to BVM.     

The 3-O-(3',3'-dimethylsuccinyl) derivative of betulinic acid (DSB) inhibits the assembly of virus-like particles in HIV-1 Gag precursor-expressing cells

BACKGROUND: The 3-O-(3',3'-dimethylsuccinyl) derivative of betulinic acid (DSB) blocks HIV-1 maturation by interfering with viral protease (PR) at the capsid (CA)-SP1 cleavage site, a crucial region in HIV-1 morphogenesis. METHODS: We analysed the effect of DSB on the assembly of HIV-1 Gag precursor (Pr55Gag(HIV)) into membrane-enveloped virus-like particles (VLP) in baculovirus-infected cells expressing Pr55Gag(HIV), in a cellular context devoid of viral PR. RESULTS: DSB showed a dose-dependent negative effect on VLP assembly, with an IC50 approximately 10 microM. The DSB inhibitory effect was p6-independent and was also observed for intracellular assembly of non-N-myristoylated Gag core-like particles. HIV-1 VLP assembled in the presence of DSB exhibited a lower stability of their inner cores upon membrane delipidation compared with control VLP, suggesting weaker Gag-Gag interactions. DSB also inhibited the assembly of simian immunodeficiency virus SLVmac251 VLP, although with a twofold lower efficacy (IC50 approximately 20 microM). No detectable inhibitory activity was observed for murine leukaemia virus (MLV) VLP; however, fusion of the SP1-NC-p6 domains from HIV-1 to the matrix (MA)-CA domains from MLV conferred DSB sensitivity to the chimaeric Gag precursor Pr72Gag(MLV-HIV) (IC50 = 30 microM). This observation suggested that the main DSB target on Pr55Gag was the SP1 domain, but the higher degree of DSB resistance for Pr72Gag(MLV-HIV) compared with Pr55Gag(HIV) implied that other upstream Gag region(s) might contribute to DSB reactivity. CONCLUSIONS: Sequence alignment and three-dimensional modelling by homology of the CA-SP1-NC junction in HIV-1, SLVmac251 and Pr72Gag(MLV-HIV) suggested that a higher hydrophilic character of the CA region immediately upstream to the HIV-1 CA-SP1 junction, as occurred in Pr72Gag(MLV-HIV), correlated with a lower DSB sensitivity.     

New Small-Molecule Inhibitor Class Targeting Human Immunodeficiency Virus Type 1 Virion Maturation

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.Despite the wide-ranging success of highly active retroviral therapies against human immunodeficiency virus type 1 (HIV-1), there still remain several powerful drivers to discover and develop new classes of HIV inhibitors. First and foremost is the continued acquisition of HIV-1 resistance to currently administered antiretroviral drugs. The discovery of compounds that inhibit the replication of HIV-1 via new mechanisms offers the best hope of generating drugs that are active against all HIV-1 variants in the clinic. In principle, viral mutations conferring resistance to any existing drug classes would not confer cross-resistance to drugs targeting a new mechanism. Also, as the current standard of care requires lifelong therapy for HIV-infected individuals, it is imperative that highly active retroviral therapy-related adverse effects and toxicity are minimized with new drugs.Recently, two new classes of antiretroviral medications have been approved for use in patients for whom previous HIV treatment regimens have failed; the CCR5 [chemokine (C-C motif) receptor 5] inhibitor maraviroc (9, 11) and the HIV-1 integrase (IN) inhibitor raltegravir (22). Additional novel mechanism drug classes will add similar value to the treatment of HIV-1 by further expanding the possibilities for combination regimens and providing a wider range of options for treatment of experienced patients failing first- and second-line therapies.Virion maturation represents one new antiviral mechanism that is currently being evaluated in the clinic (20, 21). During maturation of the HIV-1 viral particle, the Gag polyprotein undergoes several cleavage events mediated by the viral protease that yield the individual structural proteins, matrix (MA), capsid (CA), nucleocapsid (NC), and the p6 protein. In addition, there are spacer peptides that must be excised from between CA and NC (spacer peptide 1 [SP1]) and from between NC and p6 (SP2). Although the functions of the spacer peptides have not been clearly delineated, recent evidence suggests that SP1 stabilizes CA hexamers (25). Gag cleavage events occur in a sequential fashion as dictated by the relative kinetics of proteolysis at each site. The last of these proteolytic events cleaves at the CA-SP1 junction to convert p25 Gag to the mature p24 CA protein. This is a critical step in the replication cycle of HIV-1, as only in its mature form is CA capable of forming the higher-order complexes that comprise the mature viral core and in the absence of this maturation, viral particles remain noninfectious. Currently, two HIV-1 inhibitors [3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB/PA-457/bevirimat) and MPC-9055] targeting HIV-1 virion maturation are being evaluated in clinical trials (3, 4, 14, 16). The hallmark of this class of inhibitors is the accumulation of the CA/SP1 (p25) precursor in cells and HIV-1 viral particles in the presence of the compounds. However, to date, all of the Gag maturation inhibitors reported belong to the same general chemical class of betulinic acid derivatives.In this report, we describe a specific small-molecule inhibitor of HIV-1 Gag maturation, PF-46396 {1-[2-(4-tert-butylphenyl)-2-(2,3-dihydro-1H-inden-2-ylamino)ethyl]-3-(trifluoromethyl)pyridin-2(1H)-one}. This pyridone-based compound was first discovered as a hit from our HIV-1 high-throughput full-replication screen that incorporates all of the HIV-1 targets required for viral replication in cell culture (8). This approach allowed us to screen for multiple targets in the context of a full replication cycle and to identify compounds directed against new HIV-1 mechanisms. We demonstrate that PF-46396 is a late stage (postintegration) HIV-1 inhibitor by showing that the compound inhibits the production of infectious viral particles by HIV-1 producer cells and that the compound does not inhibit early events in HIV-1 replication. We present mechanism-of-action studies demonstrating that PF-46396 does not inhibit HIV protease but interferes specifically with the cleavage of the CA/SP1 (p25) Gag precursor to the mature CA (p24) protein and show a correlation between this mechanism and the antiretroviral activity of the compound. Consistent with these mechanistic data, we show that selection of HIV-1 isolates that are resistant to PF-46396 yields a mutation in the C-terminal region of HIV-1 CA. This single mutation is sufficient to cause significant loss of susceptibility to the compound and displays cross-resistance to the previously reported HIV-1 Gag maturation inhibitor, DSB. Conversely, we demonstrate that a mutation selected for resistance to DSB is cross-resistant to PF-46396.Our data show that PF-46396 is a specific Gag maturation inhibitor that is clearly differentiated from previously reported compounds that share this mechanism of action. The structure and, in particular, the low molecular weight of PF-46396 show that this mechanism of action can be prosecuted through a more diverse set of chemical structures than previously represented in the literature.     

Crippling HIV one mutation at a time

Accumulating data suggest that not all human immunodeficiency virus (HIV)-1-specific immune responses are equally effective at controlling HIV-1 replication. A new study now demonstrates that multiple immune-driven sequence polymorphisms in the highly conserved HIV-1 Gag region of transmitted viruses are associated with reduced viral replication in newly infected humans. These data suggest that targeting these and other conserved viral regions may be the key to developing an effective HIV-1 vaccine.     

Transmission of HIV-1 Gag immune escape mutations is associated with reduced viral load in linked recipients

In a study of 114 epidemiologically linked Zambian transmission pairs, we evaluated the impact of human leukocyte antigen class I (HLA-I)-associated amino acid polymorphisms, presumed to reflect cytotoxic T lymphocyte (CTL) escape in Gag and Nef of the virus transmitted from the chronically infected donor, on the plasma viral load (VL) in matched recipients 6 mo after infection. CTL escape mutations in Gag and Nef were seen in the donors, which were subsequently transmitted to recipients, largely unchanged soon after infection. We observed a significant correlation between the number of Gag escape mutations targeted by specific HLA-B allele-restricted CTLs and reduced VLs in the recipients. This negative correlation was most evident in newly infected individuals, whose HLA alleles were unable to effectively target Gag and select for CTL escape mutations in this gene. Nef mutations in the donor had no impact on VL in the recipient. Thus, broad Gag-specific CTL responses capable of driving virus escape in the donor may be of clinical benefit to both the donor and recipient. In addition to their direct implications for HIV-1 vaccine design, these data suggest that CTL-induced viral polymorphisms and their associated in vivo viral fitness costs could have a significant impact on HIV-1 pathogenesis.     

HIV-1 proprotein processing as a target for gene therapy

The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native alpha(1) antitrypsin (alpha(1)AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of alpha(1)AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1(NL4-3). As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1(NL4-3) at challenge dose of 40 000 TCID(50) (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID(50) (MOI = 0.04). By Western blot analyses, the delivered alpha(1)AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55(Gag) as well. Furthermore, high levels of uncleaved p55(Gag) protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering alpha(1)AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55(Gag). alpha(1)AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.     

Structure-activity relationship studies on new DABOS: effect of substitutions at pyrimidine C-5 and C-6 positions on anti-HIV-1 activity.

Several 5-alkyl, 5-alkenyl, 5-iso-alkyl, 5-halo, 5-aminomethyl and 5-carboxy derivatives of S-DABOs (dihydro-alkyl (or cyclo-alkyl)thio-benzyloxopyrimidines), DATNOs (dihydro-alkylthionaphthylmethyl-oxopyrimidines) and F2-S-DABOs (dihydro-alkyl (or cyclo-alkyl)thio-2,6-difluorobenzyl-oxopyrimidines) have been prepared and tested as anti-HIV-1 agents. S-DABO derivatives bearing at C-6 position monosubstituted phenylmethyl or heteroarylmethyl units have also been synthesized. 2-Alkylthio-3,4-dihydropyrimidin-4(3H)-one derivatives of F2-S-DABO series bearing small alkyl groups at C-5 proved to be potent inhibitors of HIV-1 replication in vitro with selectivity indexes ranging from 250 to >2,500.     

Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo

Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.     

Bifunctional anti-human immunodeficiency virus type 1 small molecules with two novel mechanisms of action

A class of betulinic acid derivatives was synthesized to target two critical steps in the human immunodeficiency virus type 1 (HIV-1) replication cycle, entry and maturation. Each mechanism of HIV-1 inhibition is distinct from clinically available anti-HIV therapeutics. The viral determinants of the antientry and antimaturation activities are the bridging sheet of HIV-1 gp120 and the P24/p2 cleavage site, respectively.     

Comparison of human immunodeficiency virus type 1 Pr55(Gag) and Pr160(Gag-pol) processing intermediates that accumulate in primary and transformed cells treated with peptidic and nonpeptidic protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) produces two polyproteins, Pr55(Gag) and Pr160(Gag-Pol), that are cleaved into mature functional subunits by the virally encoded protease. Drugs that inhibit this protease are an important part of anti-HIV therapy. We studied the ordered accumulation of Gag and Gag-Pol processing intermediates by variably blocking the protease with HIV-1 protease inhibitors (PIs). Variable protease inhibition caused accumulation of a complex pattern of processing intermediates, which was the same after incubating HIV-1-infected cells with increasing concentrations of either one of the peptidomimetic inhibitors indinavir, saquinavir (SQV), ritonavir (RTV), nelfinavir, and SC-52151 or one of the nonpeptidomimetic inhibitors DMP450, DMP323, PNU-140135, and PNU-109112 for 3 days. The patterns of Gag and Gag-Pol processing intermediate accumulation were nearly identical when the following were compared: cell- versus virion-associated proteins, HIV-1-infected transformed cell lines versus primary human peripheral blood mononuclear cells (PBMCs) and HIV-1(MN) versus HIV-1(IIIB) virus strains. RTV was a more potent inhibitor of p24 production in PBMCs than SQV by approximately 7-fold, whereas SQV was a more potent inhibitor in transformed cells than RTV by approximately 30-fold. Although the antiretroviral potency of HIV-1 PIs may change as a function of cell type, the polyprotein intermediates that accumulate with increasing drug concentrations are the same. These results support sequential processing of Gag and Gag-Pol polyproteins by the HIV-1 protease and may have important implications for understanding common cross-resistance pathways.     

Effect of natural polymorphisms in the HIV-1 CRF02_AG protease on protease inhibitor hypersusceptibility

Hypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibitor HS, namely, 17E and 64M in CRF02_AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02_AG molecular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did increase susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02_AG. In direct head-to-head virus competitions, CRF02_AG harboring the 17E/64M polymorphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02_AG clone. These findings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mutations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.     

Gag non-cleavage site mutations contribute to full recovery of viral fitness in protease inhibitor-resistant human immunodeficiency virus type 1

It is well documented that human immunodeficiency virus type 1 (HIV-1) Gag cleavage site mutations (CSMs) emerge in conjunction with various HIV-1 mutations for protease inhibitor (PI) resistance and improve viral replication capacity, which is reduced by acquisition of the resistance. However, CSMs are not the only mutations that emerge in Gag during treatment; many mutations other than CSMs (non-CSMs) have been found to accumulate in the Gag region. In the present study we demonstrate the important role of Gag non-CSMs with regard to viral fitness recovery. We selected three Gag-protease sequences with different PI resistance-associated mutations and CSMs from patients with antiretroviral treatment failure. To clarify the significance of CSMs and non-CSMs, four types of recombinant viruses with different patterns in each sequence were constructed. These were the GP type (patient-derived Gag and protease), the P type (HXB2 Gag and patient-derived protease), the GP(-c) type (CSMs removed from the GP type), and the P(+c) type (CSMs in the HXB2 Gag frame and patient-derived protease). By comparison of these four types of recombinant viruses in each patient-derived Gag-protease sequence, we found that non-CSMs, which had no systematic pattern, make a significant contribution to viral fitness recovery. Our findings demonstrate a delicate interaction between the in vivo evolution of Gag and protease to evade drug selective pressure and the importance of Gag in evaluating drug-resistant viruses.     

Inhibition of HIV-1 replication by simian restriction factors, TRIM5alpha and APOBEC3G

Old World monkey TRIM5alpha targets incoming human immunodeficiency virus type 1 (HIV-1) viral capsid, whereas the apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC)3 family hypermutate/degrade viral sequences. Here, we show the potentials of simian TRIM5alpha and APOBEC3G as therapeutic sequences for AIDS gene therapy. Both rhesus and African green monkey (agm) TRIM5alpha efficiently restrict HIV-1 vectors with divergent Gag from different HIV-1 subtypes. Human T cells genetically engineered to express agm-TRIM5alpha block or delay HIV-1 replication. Although agm-APOBEC3G expression alone only transiently suppresses HIV-1 replication, co-expression of agm-APOBEC3G and agm-TRIM5alpha successfully block the virus replication for more than 5 weeks.     

env chimeric virus technology for evaluating human immunodeficiency virus susceptibility to entry inhibitors

We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1) env genes gp120, gp41, and gp160 for evaluation of the susceptibilities of HIV to entry inhibitors. This env CVT allows the recombination of env sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding env gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its gp120 gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.3/T20 has mutations in both gp120 and gp41. Phenotypic analysis revealed that NL4.3/AMD3100 lost its susceptibility to dextran sulfate, AMD3100, AMD2763, T134, and T140 but not its susceptibility to T20, whereas NL4.3/T20 lost its susceptibility only to the inhibitory effect of T20. The recombination of gp120 of NL4.3/AMD3100 and gp41 of NL4.3/T20 or recombination of the gp160 genes of both strains into a wild-type background reproduced the phenotypic (cross-)resistance profiles of the corresponding strains selected in vitro. These data imply that mutations in gp120 alone are sufficient to reproduce the resistance profile of NL4.3/AMD3100. The same can be said for gp41 in relation to NL4.3/T20. In conclusion, we demonstrate the use of env CVT as a research tool in the delineation of the region important for the phenotypic (cross-)resistance of HIV strains to entry inhibitors. In addition, we obtained a proof of principle that env CVT can become a helpful diagnostic tool in assessments of the phenotypic resistance of clinical HIV isolates to HIV entry inhibitors.