Context surrounding processing sites is crucial in determining cleavage rate of a subset of processing sites in HIV-1 Gag and Gag-Pro-Pol polyprotein precursors by viral protease

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.     

Coassembly and complementation of Gag proteins from HIV-1 and HIV-2, two distinct human pathogens

Approximately one million people in the world are dually infected with both HIV-1 and HIV-2. To identify potential interactions between these two human pathogens, we examined whether HIV-1 and HIV-2 Gag proteins can coassemble and functionally complement each other. We generated HIV-1- and HIV-2-based vectors with mutations in Gag; compared with wild-type vectors, these mutants had drastically decreased viral titers. Coexpression of the mutant HIV-1 and HIV-2 Gag could generate infectious viruses; furthermore, heterologous complementation in certain combinations showed efficiency similar to homologous complementation. Additionally, we used bimolecular fluorescence complementation analysis to directly demonstrate that HIV-1 and HIV-2 Gag can interact and coassemble. Taken together, our results indicate that HIV-1 and HIV-2 Gag polyproteins can coassemble and functionally complement each other during virus replication; to our knowledge, this is the first demonstration of its kind. These studies have important implications for AIDS treatment and the evolution of primate lentiviruses.     

Pre-existence and emergence of drug resistance in HIV-1 infection.

Antiviral treatment of HIV-1 infection often fails because of the rapid emergence of resistant virus within weeks of the start of therapy. This raises the question of whether resistant viruses pre-exist in drug-naive patients or whether it is produced after the start of therapy. Here we compare the likelihood of pre-existence with the likelihood of production of resistant virus during therapy. We show that provided resistant virus pre-exists, then a stronger therapy may lead to a greater initial reduction of virus load, but will also cause a faster rise of resistant virus. In this case the total benefit of treatment is independent of the degree of inhibition of sensitive virus. If, on the other hand, resistant mutants do not pre-exist, then the emergence of resistance during treatment depends on the efficacy of the drug. If the drug is sufficiently potent to eradicate sensitive virus, then the probability that resistant mutants first appear during therapy is smaller than the probability that they existed before therapy. If the drug cannot eradicate the sensitive virus, then after sufficiently long time resistant mutants will appear. However, mutants that are unlikely to pre-exist may taken long time to appear.     

Inhibition of HIV-1 maturation via drug association with the viral Gag protein in immature HIV-1 particles

The small molecule 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently inhibits human immunodeficiency virus, type 1 (HIV-1) replication by interfering with proteolytic cleavage of the viral Gag protein at a specific site. Here we have demonstrated that the antiviral mechanism involves the association of DSB with Gag at a 1:1 stoichiometry within immature HIV-1 particles. The binding was specific, as mutations in Gag that confer resistance to DSB inhibited the association, which could be competed by DSB but not by the inactive compound betulinic acid. The addition of DSB to purified immature viral cores inhibited the cleavage of Gag at the CA-SP1 junction in vitro, thus reproducing the effect of the drug when present during maturation of HIV-1 particles. Based on these findings, we propose a model in which a trimer of DSB associates with the CA-SP1 junction of adjacent subunits within the Gag polymer. The model may explain the ability of highly similar compounds to specifically target the seemingly unrelated steps of HIV-1 maturation and virus entry.     

Human Endogenous Retrovirus K Gag Coassembles with HIV-1 Gag and Reduces the Release Efficiency and Infectivity of HIV-1

Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.     

Polymorphism in Gag gene cleavage sites of HIV-1 non-B subtype and virological outcome of a first-line lopinavir/ritonavir single drug regimen

Virological failure on a boosted-protease inhibitor (PI/r) first-line triple combination is usually not associated with the detection of resistance mutations in the protease gene. Thus, other resistance pathways are being investigated. First-line PI/r monotherapy is the best model to investigate in vivo if the presence of mutations in the cleavage sites (CS) of gag gene prior to any antiretroviral treatment might influence PI/r efficacy. 83 patients were assigned to initiate antiretroviral treatment with first-line lopinavir/r monotherapy in the randomised Monark trial. We compared baseline sequence of gag CS between patients harbouring B or non-B HIV-1 subtype, and between those who achieved viral suppression and those who experienced virological failure while on LPV/r monotherapy up to Week 96. Baseline sequence of gag CS was available for 82/83 isolates; 81/82 carried at least one substitution in gag CS compared to HXB2 sequence. At baseline, non-B subtype isolates were significantly more likely to harbour mutations in gag CS than B subtype isolates (p<0.0001). Twenty-three patients experienced virological failure while on lopinavir/r monotherapy. The presence of more than two substitutions in p2/NC site at baseline significantly predicted virological failure (p = 0.0479), non-B subtype isolates being more likely to harbour more than two substitutions in this specific site. In conclusion, gag cleavage site was highly polymorphic in antiretroviral-naive patients harbouring a non-B HIV-1 strain. We show that pre-therapy mutations in gag cleavage site sequence were significantly associated with the virological outcome of a first-line LPV/r single drug regimen in the Monark trial.     

Combining classifiers for HIV-1 drug resistance prediction

This paper applies and studies the behavior of three learning algorithms, i.e. the Support Vector machine (SVM), the Radial Basis Function Network (the RBF network), and k-Nearest Neighbor (k-NN) for predicting HIV-1 drug resistance from genotype data. In addition, a new algorithm for classifier combination is proposed. The results of comparing the predictive performance of three learning algorithms show that, SVM yields the highest average accuracy, the RBF network gives the highest sensitivity, and k-NN yields the best in specificity. Finally, the comparison of the predictive performance of the composite classifier with three learning algorithms demonstrates that the proposed composite classifier provides the highest average accuracy.     

Flu channel drug resistance: a tale of two sites

The M2 proteins of influenza A and B virus, AM2 and BM2, respectively, are transmembrane proteins that oligomerize in the viral membrane to form proton-selective channels. Proton conductance of the M2 proteins is required for viral replication; it is believed to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. In addition to the role of M2 in proton conductance, recent mutagenesis and structural studies suggest that the cytoplasmic domains of the M2 proteins also play a role in recruiting the matrix proteins to the cell surface during virus budding. As viral ion channels of minimalist architecture, the membrane-embedded channel domain of M2 has been a model system for investigating the mechanism of proton conduction. Moreover, as a proven drug target for the treatment of influenza A infection, M2 has been the subject of intense research for developing new anti-flu therapeutics. AM2 is the target of two anti-influenza A drugs, amantadine and rimantadine, both belonging to the adamantane class of compounds. However, resistance of influenza A to adamantane is now widespread due to mutations in the channel domain of AM2. This review summarizes the structure and function of both AM2 and BM2 channels, the mechanism of drug inhibition and drug resistance of AM2, as well as the development of new M2 inhibitors as potential anti-flu drugs.     

Amino acid substitutions in Gag protein at non-cleavage sites are indispensable for the development of a high multitude of HIV-1 resistance against protease inhibitors.

Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.     

Interactions of HIV-1 Gag with assembly cofactors

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.     

Role of myristylation in HIV-1 Gag assembly

Assembly of the human immunodeficiency virus type 1 (HIV-1) first occurs on the plasma membrane of host cells where binding is driven by strong electrostatic interactions between the N-terminal matrix (MA) domain of the structural precursor polyprotein, Gag, and the membrane. MA is also myristylated, but the exact role this modification plays is not clear. In this study, we compared the protein oligomerization and membrane binding properties of Myr(+) and Myr(-) Gag(MA) expressed in COS-1 cells. Sedimentation studies in solution showed that both the myristylated Gag precursor and the mature MA product were detected in larger complexes than their unmyristylated counterparts, and the myristylated MA protein bound liposomes with approximately 3-fold greater affinity than unmyristylated MA. Aromatic residues near the N-terminal region of the MA protein were more accessible to chymotrypsin in the unmyristylated form and, consistent with this, an epitope in the N-terminal region was more exposed. Moreover, the cyclophilin binding site in the CA domain downstream of MA was more accessible in the unmyristylated Gag protein, while the Tsg101 binding site in the C-terminal region was equally available in the unmyristylated and myristylated Gag proteins. Taken together, our results suggest that myristylation promotes assembly by inducing conformational changes and facilitating MA multimerization. This observation offers a novel role for myristylation.     

The stoichiometry of Gag protein in HIV-1

The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the immature virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete immature HIV particle contains approximately 5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the immature virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.     

The effect of HIV-1 Gag myristoylation on membrane binding

During the viral life cycle, an HIV protein, Gag, assembles at the host membrane, specifically at lipid raft regions, at very high concentrations leading to viral particle budding. Gag is post-translationally modified with an N-terminal myristate group which is thought to target Gag to lipid rafts thus aiding in assembly. Here we have analyzed the membrane binding of myristoylated HIV-1 Gag and a non-myristoylated form of HIV-1 Gag to various membrane models. After assessing the extent of myristoylation by HPLC and radiometric assays, we compared membrane binding using fluorescence methods. We found that myristoylated Gag shows a greater than twofold increase in binding affinity to model rafts. A structural model to explain these results is presented.     

Molecular dynamics studies on HIV-1 protease drug resistance and folding pathways

Drug resistance to HIV-1 protease involves the accumulation of multiple mutations in the protein. We investigate the role of these mutations by using molecular dynamics simulations that exploit the influence of the native-state topology in the folding process. Our calculations show that sites contributing to phenotypic resistance of FDA-approved drugs are among the most sensitive positions for the stability of partially folded states and should play a relevant role in the folding process. Furthermore, associations between amino acid sites mutating under drug treatment are shown to be statistically correlated. The striking correlation between clinical data and our calculations suggest a novel approach to the design of drugs tailored to bind regions crucial not only for protein function, but for folding as well.     

Gag induces the coalescence of clustered lipid rafts and tetraspanin-enriched microdomains at HIV-1 assembly sites on the plasma membrane

The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominately at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.