Variable sensitivity of CCR5-tropic human immunodeficiency virus type 1 isolates to inhibition by RANTES analogs

Aminooxypentane (AOP)-RANTES efficiently and specifically blocks entry of non-syncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) into host cells. Inhibition appears to be mediated by increased intracellular retention of the CCR5 coreceptor- AOP-RANTES complex and/or competitive binding of AOP-RANTES with NSI R5 HIV-1 isolates for CCR5. Although AOP-RANTES and other beta-chemokine analogs are potent inhibitors, the extreme heterogeneity of the HIV-1 envelope glycoproteins (gp120 and gp41) and variable coreceptor usage may affect the susceptibility of variant HIV-1 strains to these drugs. Using the same peripheral blood mononuclear cells (PBMC) with all isolates, we observed a significant variation in AOP-RANTES inhibition of 13 primary NSI R5 isolates; 50% inhibitory concentrations (IC(50)) ranged from 0.04 nM with HIV-1(A-92RW009) to 1.3 nM with HIV-1(B-BaL). Experiments performed on the same isolate (HIV-1(B-BaL)) with PBMC from different donors revealed no isolate-specific variation in AOP-RANTES IC(50) values but did show a considerable difference in virus replication efficiency. Exclusive entry via the CCR5 coreceptor by these NSI R5 isolates suggests that variable inhibition by AOP-RANTES is not due to alternative coreceptor usage but rather differential CCR5 binding. Analysis of the envelope V3 loop sequence linked a threonine or arginine at position 319 (numbering based on the HXB2 genome) with AOP-RANTES resistance. With the exception of one isolate, A319 was associated with increased sensitivity to AOP-RANTES inhibition. Distribution of AOP-RANTES IC(50) values with these isolates has promoted ongoing screens for new CCR5 agonists that show broad inhibition of HIV-1 variants.     

The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor

Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.     

Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5- and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.     

Resistance to the CCR5 inhibitor 5P12-RANTES requires a difficult evolution from CCR5 to CXCR4 coreceptor use

Viral resistance to small molecule allosteric inhibitors of CCR5 is well documented, and involves either selection of preexisting CXCR4-using HIV-1 variants or envelope sequence evolution to use inhibitor-bound CCR5 for entry. Resistance to macromolecular CCR5 inhibitors has been more difficult to demonstrate, although selection of CXCR4-using variants might be expected. We have compared the in vitro selection of HIV-1 CC1/85 variants resistant to either the small molecule inhibitor maraviroc (MVC) or the macromolecular inhibitor 5P12-RANTES. High level resistance to MVC was conferred by the same envelope mutations as previously reported after 16-18 weeks of selection by increasing levels of MVC. The MVC-resistant mutants were fully sensitive to inhibition by 5P12-RANTES. By contrast, only transient and low level resistance to 5P12-RANTES was achieved in three sequential selection experiments, and each resulted in a subsequent collapse of virus replication. A fourth round of selection by 5P12-RANTES led, after 36 weeks, to a "resistant" variant that had switched from CCR5 to CXCR4 as a coreceptor. Envelope sequences diverged by 3.8% during selection of the 5P12-RANTES resistant, CXCR4-using variants, with unique and critical substitutions in the V3 region. A subset of viruses recovered from control cultures after 44 weeks of passage in the absence of inhibitors also evolved to use CXCR4, although with fewer and different envelope mutations. Control cultures contained both viruses that evolved to use CXCR4 by deleting four amino acids in V3, and others that maintained entry via CCR5. These results suggest that coreceptor switching may be the only route to resistance for compounds like 5P12-RANTES. This pathway requires more mutations and encounters more fitness obstacles than development of resistance to MVC, confirming the clinical observations that resistance to small molecule CCR5 inhibitors very rarely involves coreceptor switching.     

Critical Amino Acids within the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein V4 N- and C-Terminals Contribute to Virus Entry

The importance of the fourth variable (V4) region of the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein (Env) in virus infection has not been well clarified, though the polymorphism of this region has been found to be associated with disease progression to acquired immunodeficiency syndrome (AIDS). In the present work, we focused on the correlation between HIV-1 gp120 V4 region polymorphism and the function of the region on virus entry, and the possible mechanisms for how the V4 region contributes to virus infectivity. Therefore, we analyzed the differences in V4 sequences along with coreceptor usage preference from CCR5 to CXCR4 and examined the importance of the amino acids within the V4 region for CCR5- and CXCR4-tropic virus entry. In addition, we determined the influence of the V4 amino acids on Env expression and gp160 processing intracellularly, as well as the amount of Env on the pseudovirus surface. The results indicated that V4 tended to have a shorter length, fewer potential N-linked glycosylation sites (PNGS), greater evolutionary distance, and a lower negative net charge when HIV-1 isolates switched from a coreceptor usage preference for CCR5 to CXCR4. The N- and C-terminals of the HIV-1 V4 region are highly conserved and critical to maintain virus entry ability, but only the mutation at position 417 in the context of ADA (a R5-tropic HIV-1 strain) resulted in the ability to utilize CXCR4. In addition, 390L, 391F, 414I, and 416L are critical to maintain gp160 processing and maturation. It is likely that the hydrophobic properties and the electrostatic surface potential of gp120, rather than the conformational structure, greatly contribute to this V4 functionality. The findings provide information to aid in the understanding of the functions of V4 in HIV-1 entry and offer a potential target to aid in the development of entry inhibitors.     

Role of the human immunodeficiency virus type 1 envelope gene in viral fitness

A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.     

Covariance of Charged Amino Acids at Positions 322 and 440 of HIV-1 Env Contributes to Coreceptor Specificity of Subtype B Viruses,and Can Be Used to Improve the Performance of V3 Sequence-Based Coreceptor Usage Prediction Algorithms

The ability to determine coreceptor usage of patient-derived human immunodeficiency virus type 1 (HIV-1) strains is clinically important, particularly for the administration of the CCR5 antagonist maraviroc. The envelope glycoprotein (Env) determinants of coreceptor specificity lie primarily within the gp120 V3 loop region, although other Env determinants have been shown to influence gp120-coreceptor interactions. Here, we determined whether conserved amino acid alterations outside the V3 loop that contribute to coreceptor usage exist, and whether these alterations improve the performance of V3 sequence-based coreceptor usage prediction algorithms. We demonstrate a significant covariant association between charged amino acids at position 322 in V3 and position 440 in the C4 Env region that contributes to the specificity of HIV-1 subtype B strains for CCR5 or CXCR4. Specifically, positively charged Lys/Arg at position 322 and negatively charged Asp/Glu at position 440 occurred more frequently in CXCR4-using viruses, whereas negatively charged Asp/Glu at position 322 and positively charged Arg at position 440 occurred more frequently in R5 strains. In the context of CD4-bound gp120, structural models suggest that covariation of amino acids at Env positions 322 and 440 has the potential to alter electrostatic interactions that are formed between gp120 and charged amino acids in the CCR5 N-terminus. We further demonstrate that inclusion of a “440 rule” can improve the sensitivity of several V3 sequence-based genotypic algorithms for predicting coreceptor usage of subtype B HIV-1 strains, without compromising specificity, and significantly improves the AUROC of the geno2pheno algorithm when set to its recommended false positive rate of 5.75%. Together, our results provide further mechanistic insights into the intra-molecular interactions within Env that contribute to coreceptor specificity of subtype B HIV-1 strains, and demonstrate that incorporation of Env determinants outside V3 can improve the reliability of coreceptor usage prediction algorithms.     

HIV-1 Env C2-V4 Diversification in a Slow-Progressor Infant Reveals a Flat but Rugged Fitness Landscape

Human immunodeficiency virus type-1 (HIV-1) fitness has been associated with virus entry, a process mediated by the envelope glycoprotein (Env). We previously described Env genetic diversification in a Zambian, subtype C infected, slow-progressor child (1157i) in parallel with an evolving neutralizing antibody response. Because of the role the Variable-3 loop (V3) plays in transmission, cell tropism, neutralization sensitivity, and fitness, longitudinally isolated 1157i C2-V4 alleles were cloned into HIV-1_NL4-3-eGFP and -DsRed2 infectious molecular clones. The fluorescent reporters allowed for dual-infection competitions between all patient-derived C2-V4 chimeras to quantify the effect of V3 diversification and selection on fitness. ‘Winners’ and ‘losers’ were readily discriminated among the C2-V4 alleles. Exceptional sensitivity for detection of subtle fitness differences was revealed through analysis of two alleles differing in a single synonymous amino acid. However, when the outcomes of N = 33 competitions were averaged for each chimera, the aggregate analysis showed that despite increasing diversification and divergence with time, natural selection of C2-V4 sequences in this individual did not appear to be producing a ‘survival of the fittest’ evolutionary pattern. Rather, we detected a relatively flat fitness landscape consistent with mutational robustness. Fitness outcomes were then correlated with individual components of the entry process. Env incorporation into particles correlated best with fitness, suggesting a role for Env avidity, as opposed to receptor/coreceptor affinity, in defining fitness. Nevertheless, biochemical analyses did not identify any step in HIV-1 entry as a dominant determinant of fitness. Our results lead us to conclude that multiple aspects of entry contribute to maintaining adequate HIV-1 fitness, and there is no surrogate analysis for determining fitness. The capacity for subtle polymorphisms in Env to nevertheless significantly impact viral fitness suggests fitness is best defined by head-to-head competition.     

Evolution of CCR5 use before and during coreceptor switching

The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.     

Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 loop library

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1(JR-FL) proviral DNA. The constructed HIV-1 library contained 6.6 x 10(6) independent clones containing a set of 0-10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 microM in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure.     

Novel small-molecule inhibitors of hepatitis C virus entry block viral spread and promote viral clearance in cell culture

Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection.     

Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.     

Phenotypic studies on recombinant human immunodeficiency virus type 1 (HIV-1) containing CRF01_AE env gene derived from HIV-1-infected patient,residing in central Thailand

Human immunodeficiency virus type 1 (HIV-1) env genes were cloned from blood samples of HIV-1-infected Thai patients, and 35 infectious CRF01_AE envelope glycoprotein (Env)-recombinant viruses were established. In this report, we examined the neutralization susceptibility of these viruses to human monoclonal antibodies, 2G12, IgG1 b12, 2F5 and 4E10, pooled patient plasma, coreceptor antagonists and fusion inhibitor, T-20. The neutralization susceptibility of CRF01_AE Env-recombinant viruses to 2F5, 4E10, patient plasma, coreceptor antagonists and T-20 varied, while most viruses showed low susceptibility to 2G12 and IgG1 b12. Several dual-tropic viruses showed lower susceptibility to 2F5 and 4E10 than CXCR4- or CCR5-tropic viruses. Neutralization susceptibility of the CRF01_AE Env-recombinant virus to pooled patient plasma was negatively correlated with the length of the V1/V2 region or the number of potential N-linked glycosylation sites in conserved regions of gp120. No correlation was found between the coreceptor usage and neutralization susceptibility of the virus to T-20, whereas several dual-tropic viruses showed higher susceptibility to coreceptor antagonists than CXCR4- or CCR5-tropic viruses. We propose that these CRF01_AE Env-recombinant viruses are useful to further study the molecular mechanism of the susceptibility of CRF01_AE Env to neutralizing antibodies and viral entry inhibitors.     

Impact of mutations in the coreceptor binding site on human immunodeficiency virus type 1 fusion, infection, and entry inhibitor sensitivity

An increasingly large number of antiviral agents that prevent entry of human immunodeficiency virus (HIV) into cells are in preclinical and clinical development. The envelope (Env) protein of HIV is the major viral determinant that affects sensitivity to these compounds. To understand how changes in Env can impact entry inhibitor sensitivity, we introduced six mutations into the conserved coreceptor binding site of the R5 HIV-1 strain YU-2 and measured the effect of these changes on CD4 and coreceptor binding, membrane fusion levels and rates, virus infection, and sensitivity to the fusion inhibitors enfuvirtide (T-20) and T-1249, the CCR5 inhibitor TAK-779, and an antibody to CD4. The mutations had little effect on CD4 binding but reduced CCR5 binding to various extents. In general, reductions in coreceptor binding efficiency resulted in slower fusion kinetics and increased sensitivity to TAK-779 and enfuvirtide. In addition, low CCR5 binding usually reduced overall fusion and infection levels. However, one mutation adjacent to the bridging sheet beta21 strand, P438A, had little effect on fusion activity, fusion rate, infectivity, or sensitivity to enfuvirtide or T-1249 despite causing a marked reduction in CCR5 binding and a significant increase in TAK-779 sensitivity. Thus, our findings indicate that changes in the coreceptor binding site of Env can modulate its fusion activity, infectivity, and entry inhibitor sensitivity by multiple mechanisms and suggest that reductions in coreceptor binding do not always result in prolonged fusion kinetics and increased sensitivity to enfuvirtide.     

A replication-competent, neutralization-sensitive variant of simian immunodeficiency virus lacking 100 amino acids of envelope

Coding sequences for the first two variable loops of the gp120 envelope glycoprotein were removed from simian immunodeficiency virus (SIV) strain 239 (SIVmac239). This deletion encompassed 100 amino acids. The resulting virus replicated poorly after transfection into immortalized T-cell lines, with peak replication occurring only after 25 to 30 days. Limited passaging of SIVmac239DeltaV1V2 in cultures gave rise to a variant which had significantly improved replication kinetics but which retained the original 100-amino-acid deletion in gp120. Cloning and sequencing revealed 11 changes in the envelope, including amino acid substitutions in both gp120 (5 substitutions) and gp41(6 substitutions). Four of the five changes in gp120 are predicted to lie within and around the putative coreceptor binding domain, a region which is believed to be covered by the V1 and V2 loops in the native envelope complex. Analysis of recombinant clones surprisingly revealed that the changes in gp41 were sufficient to overcome the replication deficiency created by deletion of the V1 and V2 loops from gp120. The SIVmac239DeltaV1V2 envelope displayed a significant reduction in its ability to mediate cell-cell fusion, and the infectious titer of SIVmac239DeltaV1V2 was approximately four- to eightfold lower than that of parental SIVmac239. Although SIVmac239 is strongly dependent on both CD4 and a coreceptor for entry, envelope protein lacking the V1 and V2 loops was able to mediate fusion with CD4(-) CCR5(+) cells at 60% the level observed with CD4(+) CCR5(+) cells. Plasma from SIVmac239-infected monkeys was at least 100 to 1,000 times more effective at neutralizing SIVmac239DeltaV1V2 than SIVmac239. These results demonstrate the dispensability of the V1-V2 sequences of SIVmac239 for viral replication, a role for V1 and V2 in shielding the coreceptor binding region of the envelope, and the extreme sensitivity of a SIV lacking these sequences to antibody-mediated neutralization.     

HIV-1 Envelope Glycoprotein Variable Loops Are Indispensable for Envelope Structural Integrity and Virus Entry

HIV-1 envelope (Env) glycoprotein is a trimer of heterodimer of gp120 and gp41, and derives from a trimeric glycoprotein precursor, gp160. Gp120 contains five conserved regions that are interspersed with 5 variable loop regions (V1–V5). Env variations in variable loop length and amino acid composition may associate with virus pathogenesis, virus sensitivity to neutralizing antibodies (nAbs) and disease progression. To investigate the role of each variable loop in Env function, we generated a panel of JRFL gp160 loop deletion mutants and examined the effects of each loop deletion on Env expression, Env cell surface display and Env-mediated virus entry into permissive cells. We found that deletion of V1 and V2 (ΔV1V2), or loop D (ΔlpD) abolished virus entry, the same effect as deletion of V3 (ΔV3), while deletion of V3 crown (ΔV3C) significantly enhanced virus assembly and entry. We further found that deletion of V4 (ΔV4) or V5 (ΔV5), or replacement of V4 or V5 with flexible linkers of the same lengths knocked out the receptor and coreceptor binding sites in gp120, but significantly enhanced the exposure of the N-trimer structure and the membrane proximal external region (MPER) in gp41. Although deletion of V4 or V5 did not affect Env expression, they negatively affected Env cell surface display, leading to the failure in virus assembly and subsequent entry. Taken together, we found that Env variable loops were indispensable for Env structural integrity and virus entry. Our findings may have implications for development of HIV-1 vaccine immunogens and therapeutics.     

Involvement in K+ access of Leu318 at the extracellular domain flanking M3 and M4 of the Na+,K+-ATPase alpha-subunit

The effect of point mutation in the sequence 316TWLE319, which occurs in the extracellular loop flanking the third (M3) and the fourth (M4) transmembrane segment (L3/4) of the Na+,K+-ATPase alpha-subunit, was examined. Mutation of Glu319 to Asp yielded an enzyme with full activity, whereas substituting Glu319 to Ala resulted in a severe loss of activity. A negative charge was introduced along the sequence, one residue at a time, from Thr316 to Leu318 (by E-scanning) in the mutant construct with Glu319 already mutated to Gln. The activity that had been reduced to 60% by the mutation of Glu319 to Gln was restored upon the introduction of a negative charge by E-scanning. When Leu318 was replaced by Glu in a series of scanning experiments, the K+ sensitivity of the ATPase activity was lowered. The lowering of K+ sensitivity was further demonstrated when a mutation of Leu318 to Glu was introduced into the wild-type enzyme. Furthermore, mutants with Leu318 to Gln, Arg, and Phe displayed lower K+ sensitivity similar to that of Leu318 to Glu mutant. Leu318 may be in access path for K+, and any substitution at this position may interfere with access of K+ from outside the cell.     

Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120

T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a trans-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC(50)) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log(10) higher than the mean IC(50) for CXCR4 (X4) isolates (P = 0. 0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC(50) for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log(10) higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC(50) for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log(10) lower than the IC(50) for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.     

Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates.     

Clinical resistance to enfuvirtide does not affect susceptibility of human immunodeficiency virus type 1 to other classes of entry inhibitors

The clinical use of the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide (ENF) can select for drug-resistant HIV-1 strains bearing mutations in the HR1 region of the viral envelope (Env) protein. We analyzed the properties of multiple Env proteins isolated from five patients who experienced an initial decline in viral load after ENF therapy followed by subsequent rebound due to emergence of ENF-resistant HIV-1. Prior to ENF therapy, each patient harbored genetically and phenotypically diverse Env proteins that used CCR5 and/or CXCR4 to elicit membrane fusion. Coreceptor usage patterns of the Envs isolated from two patients underwent homogenization following ENF therapy, whereas in the other three patients, recombination appeared to allow the introduction of a single HR1 sequence with ENF resistance mutations into phenotypically distinct Env proteins. Analysis of individual Env clones also revealed that prior to ENF therapy, there was sometimes marked heterogeneity in the susceptibility of individual Env proteins to coreceptor inhibitors. After virologic failure, all Envs acquired resistance to ENF but exhibited no consistent change in their sensitivity to the fusion inhibitor T-1249 or to coreceptor inhibitors. In summary, using patient-derived Env proteins, we found that ENF failure was associated with emergence of high-level resistance to ENF due largely to mutations in HR1 but that susceptibility to other entry inhibitors was unaffected, that in these late-stage patients there was greater clonal variability to coreceptor than to fusion inhibitors, and that recombination events in vivo could sometimes restore Env genotypic and phenotypic heterogeneity by introducing drug-resistant gp41 sequences into heterologous gp120 backgrounds.