Proteolytic processing of the p2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.     

Increasing rate of cleavage at boundary between non-structural proteins 4B and 5A inhibits replication of hepatitis C virus

In hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. Although proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However, it is not known whether slow cleavage is important for viral replication or a consequence of restrictions on sequences that can be tolerated at the cleaved ends of non-structural proteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary, and their effect on replication and polyprotein processing was examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codon degeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall, the data indicate that slow cleavage of the NS4B5A boundary is important and identify a previously unrecognized role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time.     

Importance of protease cleavage sites within and flanking human immunodeficiency virus type 1 transframe protein p6* for spatiotemporal regulation of protease activation

The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors.     

Type C viral gag gene expression in chicken embryo fibroblasts and avian sarcoma virus-transformed mammalian cells.

Sensitive radioimmunoassays were developed for avian type C viral gag gene-coded proteins. These assays were used to examine the restriction to virus production by avian embryo cells and mammalian cells transformed by avian sarcoma viruses. The results indicate that although a high-molecular-weight primary translational product of the gag gene is expressed, its cleavage and processing are incomplete. Furthermore, analysis of intermediate cleavage products provided information regarding the order of sequences coding for the individual viral proteins within the avian type C viral gag gene.     

Presentation Overrides Specificity: Probing the Plasticity of Alphaviral Proteolytic Activity through Mutational Analysis

Semliki Forest virus (genus Alphavirus) is an important model for studying regulated nonstructural (ns) polyprotein processing. In this study, we evaluated the strictness of the previously outlined cleavage rules, accounting for the timing and outcome of each of three cleavages within the ns polyprotein P1234, and assessed the significance of residues P6 to P4 within the cleavage sites using an alanine scanning approach. The processing of the 1/2 and 3/4 sites was most strongly affected following changes in residues P5 and P4, respectively. However, none of the mutations had a detectable effect on the processing of the 2/3 site. An analysis of recombinant viruses bearing combinations of mutations in cleavage sites revealed tolerance toward the cooccurrence of native and mutated cleavage sites within the same polyprotein, suggesting a remarkable plasticity of the protease recognition pocket. Even in a virus in which all of the cleavage sequences were replaced with alanines in the P6, P5, and P4 positions, the processing pattern was largely preserved, without leading to reversion of cleavage site mutations. Instead, the emergence of second-site mutations was identified, among which Q706R/L in nsP2 was confirmed to be associated with the recognition of the P4 position within the modified cleavage sites. Our results imply that the spatial arrangement of the viral replication complex inherently contributes to scissile-site presentation for the protease, alleviating stringent sequence recognition requirements yet ensuring the precision and the correct order of processing events. Obtaining a proper understanding of the consequences of cleavage site manipulations may provide new tools for taming alphaviruses.     

Aminopeptidase substrate preference affects HIV epitope presentation and predicts immune escape patterns in HIV-infected individuals

Viruses evade immune detection partly through immune-associated mutations. Analyses of HIV sequences derived from infected individuals have identified numerous examples of HLA-associated mutations within or adjacent to T cell epitopes, but the potential impact of most mutations on epitope production and presentation remains unclear. The multistep breakdown of proteins into epitopes includes trimming of N-extended peptides into epitopes by aminopeptidases before loading onto MHC class I molecules. Definition of sequence signatures that modulate epitope production would lead to a better understanding of factors driving viral evolution and immune escape at the population level. In this study, we identified cytosolic aminopeptidases cleavage preferences in primary cells and its impact on HIV Ag degradation into epitopes in primary human cell extracts by mass spectrometry and on epitope presentation to CTL. We observed a hierarchy of preferred amino acid cleavage by cytosolic aminopeptidases. We demonstrated that flanking mutations producing more or less cleavable motifs can increase or decrease epitope production and presentation by up to 14-fold. We found that the efficiency of epitope production correlates with cleavability of flanking residues. These in vitro findings were supported by in vivo population-level analyses of clinically derived viral sequences from 1134 antiretroviral-naive HIV-infected individuals: HLA-associated mutations immune pressures drove the selection of residues that are less cleavable by aminopeptidases predominantly at N-flanking sites, leading to reduced epitope production and immune recognition. These results underscore an important and widespread role of Ag processing mutations in HIV immune escape and identify molecular mechanisms underlying impaired epitope presentation.     

Mutagenesis of the yellow fever virus NS2B/3 cleavage site: determinants of cleavage site specificity and effects on polyprotein processing and viral replication.

The determinants of cleavage site specificity of the yellow fever virus (YF) NS3 proteinase for its 2B/3 cleavage site have been studied by using site-directed mutagenesis. Mutations at residues within the GARR decreases S sequence were tested for effects on cis cleavage of an NS2B-3(181) polyprotein during cell-free translation. At the P1 position, only the conservative substitution R-->K exhibited significant levels of cleavage. Conservative and nonconservative substitutions were tolerated at the P1' and P2 positions, resulting in intermediate levels of cleavage. Substitutions at the P3 and P4 positions had no effects on cleavage efficiency in the cell-free assay. Processing at other dibasic sites was studied by using transient expression of a sig2A-5(356) polyprotein. Cleavage at the 2B/3 site was not required for processing at downstream sites. However, increased accumulation of high-molecular-weight viral polyproteins was generally observed for mutations which reduced cleavage efficiency at the 2B/3 site. Several mutations were also tested for their effects on viral replication. Virus was not recovered from substitutions which blocked or substantially reduced cleavage in the cell-free assay, suggesting that efficient cleavage at the 2B/3 site is required for flavivirus replication.     

Signal peptide peptidase cleavage of GB virus B core protein is required for productive infection in vivo

Chronic infection by hepatitis C virus (HCV) is a leading cause of liver disease for which better therapies are urgently needed. Because a clearer understanding of the viral life cycle may suggest novel anti-viral approaches, we studied the role of host signal peptide peptidase (SPP) in viral infection. This intramembrane protease cleaves within a C-terminal signal sequence in the viral core protein, but the molecular determinants of cleavage and whether it is required for infection in vivo are unknown. To answer these questions, we studied SPP processing in GB virus B (GBV-B) infection. GBV-B is the closest phylogenetic relative of HCV and offers an accurate surrogate model for HCV infection. We demonstrate that SPP also processes GBV-B core protein and that a serine residue in the hydrophobic region of the signal sequence (present also in HCV) is critical for efficient SPP cleavage. The small size of the serine side chain combined with its ability to form intra- and interhelical hydrogen bonds likely contributes to recognition of the signal sequence as a substrate for SPP. By introducing mutations with differing effects on SPP processing into an infectious GBV-B molecular clone, we demonstrate that SPP processing of the core protein is required for productive infection in primates. These results broaden our understanding of the mechanism and requirements for SPP cleavage and reveal a functional role in vivo for intramembrane proteolysis in host-pathogen interactions. Moreover, they identify SPP as a potential therapeutic target for reducing the impact of HCV infection.     

Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.     

Human immunodeficiency virus type 1 gag-protease fusion proteins are enzymatically active.

We have introduced mutations into the region of the genome of human immunodeficiency virus type 1 (HIV-1) that encodes the cleavage sites between the viral protease (PR) and the adjacent upstream region of the polyprotein precursor. Segments containing these mutations were introduced into plasmids, and the retroviral proteins were expressed in Escherichia coli. The mutations prevented cleavage between the PR and the adjacent polypeptide; however, other PR cleavage sites in the polyprotein were cleaved normally, showing that the release of free PR is not a prerequisite for the appropriate processing of HIV-1 precursors.     

Processing of the intracellular form of the west Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study.

According to the existing model of flavivirus polyprotein processing, one of the cleavages in the amino-terminal part of the flavivirus polyprotein by host cell signalases results in formation of prM (precursor to one of the structural proteins, M) and the membrane-bound intracellular form of the viral capsid protein (Cint) retaining the prM signal sequence at its carboxy terminus. This hydrophobic anchor is subsequently removed by the viral protease, resulting in formation of the mature viral capsid protein found in virions (Cvir). We have prepared in vitro expression cassettes coding for both forms of the capsid protein, for the prM protein, for the C-prM precursor, and for the viral protease components of West Nile flavivirus and characterized their translation products. Using Cint and Cvir translation products as molecular markers, we have observed processing of the intracellular form of the West Nile capsid protein by the viral protease in vitro both upon cotranslation of the C-prM precursor and the viral protease-encoding cassette and by incubation of C-prM translation products with a detergent-solubilized extract of cells infected with a recombinant vaccinia virus expressing the active viral protease. The cleavage of Cint by the viral protease at the predicted dibasic site was verified by introduction of point mutations into the cleavage site and an adjacent region. These studies provide the first direct demonstration of processing of the intracellular form of the flavivirus capsid protein by the viral protease.     

Cleavage site mutations in the encephalomyocarditis virus P3 region lethally abrogate the normal processing cascade.

Site-specific mutations within the proteinase 3C-dependent P3 region cleavage sequences of encephalomyocarditis virus have been constructed. The mutations altered the normal QG cleavage site dipeptide pairs of the 2C/3A, 3A/3B, 3B/3C, and 3C/3D junctions into QV, QC, QF, QY, and RG sequences. When translated in vitro in the context of full-length viral polyproteins, all mutations blocked endogenous 3C-mediated processing at their engineered sites and produced stable forms of the expected viral P3 precursors that were also resistant to cleavage by exogenously added recombinant 3C. Relative to wild-type viral sequences, each mutant form of P3 had a somewhat different ability to mediate overall polyprotein processing. Mutations at the 2C/3A, 3A/3B, and 3B/3C sites, for example, were generally less impaired than 3C/3D mutations, when the cleavage reactions were quantitated with cotranslated L-P1-2A precursors. A notable exception was mutant 3B3C(QG-->RG), which proved far less active than sibling mutants 3B3C(QG-->QF) and 3B3C(QG-->QV), a finding that possibly implicates this segment in the proper folding of an active 3C. When transfected into HeLa cells, all mutant sequences were lethal, presumably because of the reduced L-P1-2A processing levels or reduced RNA synthesis capacity. However, when specifically tested for the latter activity, all mutations except those at the 3C/3D cleavage site were indeed able to initiate and perpetuate viral RNA replication in transfected cells, albeit to RNA accumulation levels lower than those produced by wild-type sequences. The transfection effects could be mimicked with cell-free synthesized proteins, in that translation samples containing locked 3CD polymerase precursors were catalytically inactive in poly(A)-oligo(U)-dependent assays, while all other mutant processing samples initiated detectable RNA synthesis. Surprisingly, not only did the 3B/3C mutant sequences prove capable of directing RNA synthesis, but the viral RNA thus synthesized could be immunolabeled and precipitated with 3C-specific monoclonal antibody reagents, indicating an unexpected covalent attachment of the proteinase to the RNA product whenever this cleavage site was blocked.     

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.     

Analysis of retroviral protease cleavage sites reveals two types of cleavage sites and the structural requirements of the P1 amino acid

Retroviruses encode a protease which cleaves the viral Gag and Gag/Pol protein precursors into mature products. To understand the target sequence specificity of the viral protease, the amino acid sequences from 46 known processing sites from 10 diverse retroviruses were compared. Sequence preference was evident in positions P4 through P3' when compared to flanking sequences. Approximately 80% of all cleavage site sequences could be grouped into two classes based on the sequence composition flanking the scissile bond. The sequences at the amino-terminal cleavage site of the major capsid protein of Gag is always a member of one of the two classes while the carboxyl-terminal cleavage site is of the other class, suggesting a biological role for the two classes. Known processing site sequences proved useful in a motif searching strategy to identify processing sites in retroviral protein sequences, particularly in Gag. In all known cleavage sites, the P1 amino acid is hydrophobic and unbranched at the beta-carbon. The sequence requirements of the P1 position were tested by site-directed mutagenesis of the P1 Phe codon in an HIV-1 Pol cleavage site. Mutations were tested for protease-mediated cleavage of the Pol precursor expressed in Escherichia coli.     

Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication.

To study the role of specific regions of the yellow fever virus NS2B protein in proteolytic processing and association with the NS3 proteinase domain, a series of mutations were created in the hydrophobic regions and in a central conserved hydrophilic region proposed as a domain important for NS2B function. The effects of these mutations on cis cleavage at the 2B/3 cleavage site and on processing at other consensus cleavage sites for the NS3 proteinase in the nonstructural region were then characterized by cell-free translation and transient expression in BHK cells. Association between NS2B and the NS3 proteinase domain and the effects of mutations on complex formation were investigated by nondenaturing immunoprecipitation of these proteins expressed in infected cells, by cell-free translation, or by recombinant vaccinia viruses. Mutations within the hydrophobic regions had subtle effects on proteolytic processing, whereas mutations within the conserved domain dramatically reduced cleavage efficiency or abolished all cleavages. The conserved domain of NS2B is also implicated in formation of an NS2B-NS3 complex on the basis of the ability of mutations in this region to eliminate both association of these two proteins and trans-cleavage activity. In addition, mutations which either eliminated proteolytic processing or had no apparent effect on processing were found to abolish recovery of infectious virus following RNA transfection. These results suggest that the conserved region of NS2B is a domain essential for the function of the NS3 proteinase. Hydrophobic regions of NS2B whose structural integrity may not be essential for proteolytic processing may have additional functions during viral replication.     

Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage.

Retroviral integration requires cis-acting sequences at the termini of linear double-stranded viral DNA and a product of the retroviral pol gene, the integrase protein (IN). IN is required and sufficient for generation of recessed 3' termini of the viral DNA (the first step in proviral integration) and for integration of the recessed DNA species in vitro. Human immunodeficiency virus type 1 (HIV-1) IN, expressed in Escherichia coli, was purified to near homogeneity. The substrate sequence requirements for specific cleavage and integration of retroviral DNA were studied in a physical assay, using purified IN and short duplex oligonucleotides that correspond to the termini of HIV DNA. A few point mutations around the IN cleavage site substantially reduced cleavage; most other mutations did not have a drastic effect, suggesting that the sequence requirements are limited. The terminal 15 bp of the retroviral DNA were demonstrated to be sufficient for recognition by IN. Efficient specific cutting of the retroviral DNA by IN required that the cleavage site, the phosphodiester bond at the 3' side of a conserved CA-3' dinucleotide, be located two nucleotides away from the end of the viral DNA; however, low-efficiency cutting was observed when the cleavage site was located one, three, four, or five nucleotides away from the terminus of the double-stranded viral DNA. Increased cleavage by IN was detected when the nucleotides 3' of the CA-3' dinucleotide were present as single-stranded DNA. IN was found to have a strong preference for promoting integration into double-stranded rather than single-stranded DNA.