Effect of sequence polymorphism and drug resistance on two HIV-1 Gag processing sites.

The HIV-1 proteinase (PR) has proved to be a good target for antiretroviral therapy of AIDS, and various PR inhibitors are now in clinical use. However, there is a rapid selection of viral variants bearing mutations in the proteinase that are resistant to clinical inhibitors. Drug resistance also involves mutations of the nucleocapsid/p1 and p1/p6 cleavage sites of Gag, both in vitro and in vivo. Cleavages at these sites have been shown to be rate limiting steps for polyprotein processing and viral maturation. Furthermore, these sites show significant sequence polymorphism, which also may have an impact on virion infectivity. We have studied the hydrolysis of oligopeptides representing these cleavage sites with representative mutations found as natural variations or that arise as resistant mutations. Wild-type and five drug resistant PRs with mutations within or outside the substrate binding site were tested. While the natural variations showed either increased or decreased susceptibility of peptides toward the proteinases, the resistant mutations always had a beneficial effect on catalytic efficiency. Comparison of the specificity changes obtained for the various substrates suggested that the maximization of the van der Waals contacts between substrate and PR is the major determinant of specificity: the same effect is crucial for inhibitor potency. The natural nucleocapsid/p1 and p1/p6 sites do not appear to be optimized for rapid hydrolysis. Hence, mutation of these rate limiting cleavage sites can partly compensate for the reduced catalytic activity of drug resistant mutant HIV-1 proteinases.     

Altered gag polyprotein cleavage specificity of feline immunodeficiency virus/human immunodeficiency virus mutant proteases as demonstrated in a cell-based expression system

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.     

Cleavage of Human Immunodeficiency Virus Type 1 Proteinase from the N-Terminally Adjacent p6* Protein Is Essential for Efficient Gag Polyprotein Processing and Viral Infectivity

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.     

Effect of linker insertion mutations in the human immunodeficiency virus type 1 gag gene on activation of viral protease expressed in bacteria.

We have expressed the human immunodeficiency virus type 1 (HIV-1) protease (PR) in bacteria as a Gag-PR polyprotein (J. Luban and S.P. Goff, J. Virol. 65:3203-3212, 1991). The protein displays enzymatic activity, cleaving the Gag polyprotein precursor Pr55gag to the expected products. The PR enzyme is only active as a dimer, and we hypothesized that PR activation might be used as an indicator of polyprotein multimerization. We constructed 25 linker insertion mutations throughout gag and assessed the PR activity of mutant Gag-PR polyproteins by the appearance of Gag cleavage products in bacterial lysates. All mutant constructs produced stable protein in bacteria. PR activity of the majority of the Gag-PR mutants was indistinguishable from that of the wild type. Six mutants, one with an insertion in the matrix (MA), four with insertions in the capsid (CA), and one with insertions in the nucleocapsid (NC), globally disrupted polyprotein processing. When PR was provided in trans on a separate plasmid, the Gag proteins were cleaved with wild-type efficiency. These results suggest that the gag mutations identified as disruptive of polyprotein processing did not conceal the scissile bonds of the polyprotein. Rather, the mutations prevented PR activation in the context of a Gag-PR polyprotein, perhaps by preventing polyprotein dimerization.     

Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis

Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.     

Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain.

The mature human immunodeficiency virus type 1 proteinase (PR; 11 kDa) can cleave all interdomain junctions in the Gag and Gag-Pol polyprotein precursors. To determine the activity of the enzyme in its precursor form, we blocked release of mature PR from a truncated Gag-Pol polyprotein by introducing mutations into the N-terminal Phe-Pro cleavage site of the PR domain. The mutant precursor autoprocessed efficiently upon expression in Escherichia coli. No detectable mature PR was released; however, several PR-related products ranging in size from approximately 14 to 18 kDa accumulated. Products of the same size were generated when mutant precursors were digested with wild-type PR. Thus, PR can utilize cleavage sites in the region upstream of the PR domain, resulting in the formation of extended PR species. On the basis of active-site titration, the PR species generated from mutated precursor exhibited wild-type activity on peptide substrates. However, the proteolytic activity of these extended enzymes on polyprotein substrates provided exogenously was low when equimolar amounts of extended and wild-type PR proteins were compared. Mammalian cells expressing the mutated precursor produced predominantly precursor and considerably reduced amounts of mature products. Released particles consisted mostly of uncleaved or partially cleaved polyproteins. Our results suggest that precursor forms of PR can autoprocess but are less efficient in processing of the Gag precursor for formation of mature virus particles.     

Ordered processing of the human immunodeficiency virus type 1 GagPol precursor is influenced by the context of the embedded viral protease

Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.     

Mutations that alter the activity of the Rous sarcoma virus protease.

Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing "flaps," and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P,G106V, as well as the triple mutant, H65G,R105P,G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.     

Variability at human immunodeficiency virus type 1 subtype C protease cleavage sites: an indication of viral fitness?

Naturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1) subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6(gag)) exhibited moderate variation, and four sites (p2/NC, TFP/p6(pol), p6(pol)/PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6(gag) is an important phosphoprotein required for virion release, and TFP/p6(pol), a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy.     

Sequential Steps in Human Immunodeficiency Virus Particle Maturation Revealed by Alterations of Individual Gag Polyprotein Cleavage Sites

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.     

The murine endogenous retrovirus MIA14 encodes an active aspartic proteinase that is functionally similar to proteinases from D-type retroviruses

Murine intracisternal A-type particles (IAPs) are endogenous retroviruses showing sequence homologies to B/D- and avian C-type retroviruses and a gene expression strategy similar to that of D-type retroviruses. These viruses form immature particles in the endoplasmic reticulum and do not release extracellular virions, but are competent for retrotransposition within the virus-producing cell. It had been assumed that lack of polyprotein processing and maturation is due to a defect in the viral proteinase (PR), but recent experiments have shown that polyprotein processing occurs when assembly of the mouse IAP MIA14 is artificially directed to the plasma membrane. We have expressed and purified recombinant MIA14 PR and show that it undergoes N- and C-terminal autoprocessing at defined sites. Using peptide cleavage and inhibition assays and in vitro cleavage of recombinant HIV-1 and MIA14 Gag polyproteins, we show that MIA14 PR is a catalytically competent enzyme comparable in its efficiency to PRs from type D exogenous retroviruses. MIA14 PR is related to the PR of Mason-Pfizer monkey virus both functionally and with respect to its expression strategy, and is distinct from HIV-1 PR with respect to substrate specificity and catalytic efficiency. These findings reveal a functional and possibly evolutionary relationship between MIA14 and D-type retroviruses and imply that a functional PR may be relevant for intracellular retrotransposition even in the case of an endogenous retrovirus that does not produce extracellular virus.     

HIV- 1 Protease Inhibits Cap- and Poly(A)-Dependent Translation upon eIF4GI and PABP Cleavage

A number of viral proteases are able to cleave translation initiation factors leading to the inhibition of cellular translation. This is the case of human immunodeficiency virus type 1 protease (HIV-1 PR), which hydrolyzes eIF4GI and poly(A)-binding protein (PABP). Here, the effect of HIV-1 PR on cellular and viral protein synthesis has been examined using cell-free systems. HIV-1 PR strongly hampers translation of pre-existing capped luc mRNAs, particularly when these mRNAs contain a poly(A) tail. In fact, HIV-1 PR efficiently blocks cap- and poly(A)-dependent translation initiation in HeLa extracts. Addition of exogenous PABP to HIV-1 PR treated extracts partially restores the translation of polyadenylated luc mRNAs, suggesting that PABP cleavage is directly involved in the inhibition of poly(A)-dependent translation. In contrast to these data, PABP cleavage induced by HIV-1 PR has little impact on the translation of polyadenylated encephalomyocarditis virus internal ribosome entry site (IRES)-containing mRNAs. In this case, the loss of poly(A)-dependent translation is compensated by the IRES transactivation provided by eIF4G cleavage. Finally, translation of capped and polyadenylated HIV-1 genomic mRNA takes place in HeLa extracts when eIF4GI and PABP have been cleaved by HIV-1 PR. Together these results suggest that proteolytic cleavage of eIF4GI and PABP by HIV-1 PR blocks cap- and poly(A)-dependent initiation of translation, leading to the inhibition of cellular protein synthesis. However, HIV-1 genomic mRNA can be translated under these conditions, giving rise to the production of Gag polyprotein.     

The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity.

Morphogenesis of retroviruses involves ordered assembly of the structural Gag- and Gag-Pol polyproteins, with subsequent budding from the plasma membrane and proteolytic cleavage by the viral proteinase (PR). Two cleavage sites exist between the capsid (CA) and nucleocapsid (NC) domains of the human immunodeficiency virus (HIV) type 1 Gag polyprotein which are separated by a 14-amino-acid spacer peptide of unknown function. To analyze the role of the two cleavage sites and the spacer peptide, both sites were individually mutated and a deletion mutation that precisely removes the spacer peptide was constructed. Following transfection of proviral DNA carrying the point mutations, mutant polyproteins were synthesized and assembled like wild-type polyprotein, and release of particles was not significantly altered. Both mutations abolished cleavage at the respective site and reduced or abolished viral infectivity. Deletion of the spacer peptide severely affected ordered assembly and reduced particle release. The extracellular particles that were released exhibited normal density but were heterogeneous in size. Electron micrographs revealed large electron-dense plaques underneath the plasma membrane of transfected cells which appeared like confluent ribonucleoprotein complexes arrested early in the budding process. Extracellular particles exhibited very aberrant and heterogeneous morphology and were incapable of inducing viral spread. These particles may correspond to membrane vesicles sequestered by the rigid structures underneath the cell membrane and not released by a regular budding process.     

Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein.

The envelope glycoprotein of the human immunodeficiency virus type 2 (HIV-2) is synthesized as a polyprotein precursor which is proteolytically processed to produce the mature surface and transmembrane envelope glycoproteins. The processed envelope glycoprotein species are responsible for the fusion between the viral envelope and the host cell membrane during the infection process. The envelope glycoprotein also induces syncytium formation between envelope-expressing cells and receptor-bearing cells. To characterize domains of the HIV-2 envelope glycoprotein involved in membrane fusion and in proteolytic processing, we introduced single amino acid mutations into the region of the HIV-2 surface glycoprotein corresponding to the principal neutralizing determinant (the V3 loop) of HIV-1, the putative HIV-2 envelope precursor-processing sequence, and the hydrophobic amino terminus of the HIV-2 transmembrane envelope glycoprotein. The effects of these mutations on syncytium formation, virus infectivity, envelope expression, envelope processing, and CD4 binding were analyzed. Our results suggest that the V3-like region of the HIV-2 surface glycoprotein and the hydrophobic amino terminus of the transmembrane glycoprotein are HIV-2 fusion domains and characterize the effects of mutations in the HIV-2 envelope glycoprotein precursor-processing sequence.     

Structural analysis of HIV-1 maturation using cryo-electron tomography

HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA.     

Ty1 proteolytic cleavage sites are required for transposition: all sites are not created equal

The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.