A domain directly C-terminal to the major homology region of human immunodeficiency type 1 capsid protein plays a crucial role in directing both virus assembly and incorporation of Gag-Pol

We demonstrate here that a deletion of 14 amino acid residues directly C-terminal to the major homology region (MHR) of the HIV-1 capsid (CA) in Gag-Pol markedly affects the incorporation of Gag-Pol into virions. The 14-amino acid deletion also significantly impaired virus assembly. In agreement with previous reports, mutations at the very C-terminus of CA resulted in a remarkable reduction in virus production. However, HIV-1 Gag-Pol precursors containing the C-terminal CA mutation were still capable of being incorporated into virions at a level of about 50% that of the wild-type. These results suggest that the domain immediately C-terminal to the MHR is functionally involved in Gag-Gag and Gag/Gag-Pol interaction, and this supports the notion that Gag or Gag-Pol mutants blocked in assembly into virus particles can be rescued into virions provided they retain the domains that are able to interact with the Gag precursor. An HIV-1 Gag-Pol deletion mutant retaining a minimal sequence consisting of the MHR and the adjacent CA-SP1 was efficiently incorporated into virions. Analysis by immunofluorescence staining indicated that the subcellular localization patterns shown by the Gag-Pol mutants were not fully compatible with their efficiency in being incorporated into virions, suggesting that the ability of Gag-Pol mutants to be incorporated into virions largely depends on their interactions with the Gag precursor.     

A block in virus-like particle maturation following assembly of murine leukaemia virus in insect cells

Expression of the murine leukaemia virus (MLV) major Gag antigen p65(Gag) using the baculovirus expression system leads to efficient assembly and release of virus-like particles (VLP) representative of immature MLV. Expression of p180(Gag-Pol), facilitated normally in mammalian cells by readthrough of the p65(Gag) termination codon, also occurs efficiently in insect cells to provide a source of the MLV protease and a pattern of p65(Gag) processing similar to that observed in mammalian cells. VLP release from p180(Gag-Pol)-expressing cells however remains essentially immature with disproportionate levels of the uncleaved p65(Gag) precursor when compared to the intracellular Gag profile. Changing the p65(Gag) termination codon altered the level of p65(Gag) and p180(Gag-Pol) within expressing cells but did not alter the pattern of released VLP, which remained immature. Coexpression of p65(Gag) with a fixed readthrough p180(Gag-Pol) also led to only immature VLP release despite high intracellular protease levels. Our data suggest a mechanism that preferentially selects uncleaved p65(Gag) for the assembly of MLV in this heterologous expression system and implies that, in addition to their relative levels, active sorting of the correct p65(Gag) and p180(Gag-Pol) ratios may occur in producer cells.     

Naturally occurring amino acid polymorphisms in human immunodeficiency virus type 1 (HIV-1) Gag p7(NC) and the C-cleavage site impact Gag-Pol processing by HIV-1 protease

Human immunodeficiency virus type 1 (HIV-1) protease activity is targeted at nine cleavage sites comprising different amino acid sequences in the viral Gag-Pol polyprotein. Amino acid polymorphisms in protease and in regions of Gag, particularly p7(NC) and the C-cleavage site between p2 and p7(NC), occur in natural variants of HIV-1 within infected patients. Studies were designed to examine the role of natural polymorphisms in protease and to identify determinants in Gag that modulate protease processing activity. Closely related Gag-Pol regions from an HIV-1-infected mother and two children were evaluated for processing in an inducible expression system, for protease activity on cleavage-site analogues, and for impact on replication by recombinant viruses. Gag-Pol regions displayed one of three processing phenotypes based on the appearance of Gag intermediates and accumulation of mature p24(CA). Gag-Pol regions that were processed rapidly to produce p24(CA) resulted in high-level replication by recombinant viruses, while slow-processing Gag-Pol variants resulted in recombinant viruses that replicated with reduced kinetics in both T cell lines and peripheral blood mononuclear cells. Direct impact by Gag sequences on processing by protease was assessed by construction of chimeric Gag-Pol regions and by site-directed mutagenesis. Optimal protease activity occurred when Gag and Pol regions were derived from the same gag-pol allele. Heterologous Gag regions generally diminished rates and extent of protease processing. Natural polymorphisms in novel positions in p7(NC) and the C-cleavage site have a dominant effect on protease processing activity. Accumulation of Gag products after processing at the C site appears to delay subsequent cleavage and production of mature p24(CA).     

Characterization of human immunodeficiency virus type 1 Pr160 gag-pol mutants with truncations downstream of the protease domain

We have constructed a series of HIV-1 Gag-pol mutants by progressive deletion of the pol sequence downstream of the viral protease (PR) domain. Effects of the truncation mutations on virus particle production and Gag particle processing were analyzed. Analysis indicated that removal of the integrase (IN) domain had no major effect on the efficiency of particle processing, but resulted in a marked reduction in virus particle budding. Deletion of both the IN and RNase H domains, however, restored the production of virus particles to wild-type level. The proteolytic processing of virus particle was significantly impaired when the p51RT domain was truncated. All of the truncated Gag-pol proteins could be incorporated into virus particles and demonstrated an immunofluorescence staining pattern similar to that of the wild type (wt). Our data are consistent with the proposal that signals for directing the Gag-pol transport and particle incorporation are determined by its N-terminal Gag domain. Truncated Gag-pol retaining an intact p51RT was able to complement a PR-defective mutant to produce infectious pseudotyped virions, with a virus titer 20-70% of that of wt. Pseudotyped virions produced by the Gag-pol lacking an intact p51RT were noninfectious or poorly infectious. This suggests that an intact p51RT domain is required for the Gag-pol to mediate production of mature infectious virus particles in trans.     

Moloney murine leukemia virus protease expressed in bacteria is enzymatically active

Summary.  Replication of Moloney murine leukemia virus requires a readthrough translation mechanism to generate the Gag-Pol polyprotein. One of the final products of this polyprotein is the protease (PR), which is required to generate the mature virion proteins. The assembly of Gag and Gag-Pol polyproteins into a virion followed by activation of the viral protease is necessary to produce a mature, infectious particle. These events are believed to occur near the cell membrane just prior to the budding of the virion. We report here the autoproteolytic activity of the viral PR when a Gag-PR fusion protein is expressed in E. coli. Efficient cleavage at the p12/CA, CA/NC and NC/PR junctions was observed. Thus the Moloney murine leukemia virus PR is capable of cleaving its substrates in the absence of specific host factors. Accepted September 20, 1997 Received April 10, 1997     

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types.     

The role of lysine residue at amino acid position 165 of human immunodeficiency virus type 1 CRF01_AE Gag in reducing viral drug susceptibility to protease inhibitors

Recombinant human immunodeficiency virus type 1 (HIV-1) containing a CRF01_AE Gag, AE-Gag62, was significantly less susceptible to protease inhibitors (PIs) than the subtype B reference strain, NL4-3; therefore, the mechanism of how AE-Gag62 reduced viral drug susceptibility to PIs was studied in this report. The results showed that the lysine residue at amino acid position 165 (K165) of AE-Gag62 played a role in reducing the drug susceptibility of the recombinant virus to PIs. In addition, K165 potentially appears more frequently in CRF01_AE viruses than in the viruses of other major HIV-1 subtypes. Although K165 had no effect on the extent of recombinant protease-mediated in vitro Gag cleavage, it enhanced the incorporation of the Gag-Pol precursor protein, p160, into virions. Taken together, these results suggest that K165 of CRF01_AE Gag affects the regulation of virion assembly or maturation, and reduces viral drug susceptibility to PIs.     

Effects of mutations in an HIV-1 gag gene containing a 107-codon tandem repeat in the matrix region on assembly and processing of the protein product

It has been demonstrated previously that a human immunodeficiency virus (HIV) type 1 Gag mutant (MA2) with a tandem repeat of 107-matrix codons in the matrix domain could direct virus particle assembly and budding [Wang et al. (2000c): J Med Virol 61:423-432]. Since the regions involved functionally in HIV Gag assembly and transport have been mapped to the matrix domain, it was interesting to test the effects of the duplicated matrix-coding sequence on Gag assembly, transport, and virus processing of some assembly-defective HIV matrix mutants. In this study, a number of HIV matrix mutations were introduced into either the proximal or distal copy of the duplicated matrix-coding sequence. Assembly, release, processing, and subcellular localization of the Gag mutants were analyzed by transient expression in 293T cells. The result indicates that the budding defect of HIV matrix mutants could be moderately or significantly reversed when the additional 107-matrix codons were present; however, these matrix double mutations affected significantly the virus particle processing. Mislocalized matrix mutants could also be redistributed to a certain degree in the presence of the duplicated matrix copy. Although the subcellular distribution patterns of the matrix mutants did not correlate completely with the budding efficiency, the data suggest that the budding defect caused by the matrix mutations could be masked to some extent by the duplicated matrix coding sequence.     

A novel real-time PCR assay to determine relative replication capacity for HIV-1 protease variants and/or reverse transcriptase variants

The emergence of drug-resistant viruses is a major issue in the treatment of HIV-1 infections. Quite often these drug-resistant viruses have a reduced replication capacity. A novel assay was developed to study the impact of mutations selected during therapy on viral replication capacity. Two HIV-1 HXB2 reference clones were constructed for this assay based on viral competition experiments, which are identical except for the presence of two silent nucleotide changes in p24 in one of the two clones. Within these two reference clones, three different contiguous deletions were constructed: (I) the C-terminus of Gag and protease, (II) the N-terminus of RT and (III) the C-terminus of Gag and protease together with the N-terminus of RT. Using these reference clones, recombinant viruses were created and viral competition experiments were performed. The proportion of each virus during the competition experiments was determined with a real-time PCR assay based on the two silent nucleotide changes in p24 in one of the two reference clones. With this novel assay it was possible to detect accurately differences in replication capacity due to mutations in the C-terminus of Gag and protease and/or the N-terminus of RT.     

Functional domains in the feline immunodeficiency virus nucleocapsid protein

Retroviral nucleocapsid (NC) proteins are small Gag-derived products containing one or two zinc finger motifs that mediate genomic RNA packaging into virions. In this study, we addressed the role of the feline immunodeficiency virus (FIV) NC protein in the late stages of virus replication by analyzing the assembly phenotype of FIV NC mutant viruses and the RNA binding activity of a panel of recombinant FIV NC mutant proteins. Substitution of serine for the first cysteine residue in the NC proximal zinc finger was sufficient to impair both virion assembly and genomic RNA binding. A similar defective phenotype with respect to particle formation and RNA binding was observed when the basic residues Lys28 and Lys29 in the region connecting both zinc fingers were replaced by alanine. In contrast, mutation of the first cysteine residue in the distal zinc finger had no effect on virion production and allowed substantial RNA binding activity of the mutant NC protein. Moreover, this NC mutant virus exhibited wild-type replication kinetics in the feline MYA-1 T-cell line. Interestingly, amino acid substitutions disrupting the highly conserved PSAP and LLDL motifs present in the C-terminus of the FIV NC abrogated virion formation without affecting the NC RNA binding activity. Our results indicate that the proximal zinc finger of the FIV NC is more important for virion production and genomic RNA binding than the distal motif. In addition, this study suggests that assembly domains in the FIV NC C-terminus may be functionally equivalent to those present in the p6 domain of the Gag polyprotein of primate lentiviruses.     

Role of a conserved sequence in the maturation and function of the NDV HN glycoprotein

The hemagglutinin-neuraminidase (HN) proteins of viruses in the Paramyxouirus genus have a short conserved sequence, G(A, S)EGR(I, L, V). The role of this sequence in the intracellular processing and function of the Newcastle disease virus HN protein was explored by site directed mutagenesis. Mutations in this region fall into two categories. One set of mutants (G398A, E400D, R402K, and a deletion removing amino acids 400–403) was defective in folding. These mutant proteins formed little or no mature, disulfide linked oligomer. They had few or no antigenic sites found on the mature protein and they were transported to the cell surface poorly or not at all. The second class of mutants (A399G, G401A, G401L) was minimally affected in folding and intracellular transport. When normalized to surface expression, this group of mutant proteins had wild type levels of attachment activity, neuraminidase activity, and fusion promotion activity. Thus mutations in this region directly affect intracellular processing but not the biological activities of the protein. This sequence may, therefore, be conserved in the HN proteins of Paramyxoviruses because it is critical to the folding of the molecule.     

Contribution of the Gag-Pol transframe domain p6* and its coding sequence to morphogenesis and replication of human immunodeficiency virus type 1

The human immunodeficiency virus type-1 (HIV-1) transframe domain p6* is located between the nucleocapsid protein (NC) and the protease (PR) within the Gag-Pol precursor. This flexible, 68-amino-acid HIV-1 p6* domain has been suggested to negatively interfere with HIV PR activity in vitro proposing a contribution of either the C-terminal p6* tetrapeptide, internal cryptic PR cleavage sites, or a zymogen-related mechanism to a regulated PR activation. To assess these hypotheses in the viral context, a series of recombinant HX10-based provirus constructs has been established with clustered amino acid substitutions throughout the entire p6* coding sequence. Comparative analysis of the mutant proviral clones in different cell culture systems revealed that mutations within the well-conserved amino-terminal p6* region modified the Gag/Gag-Pol ratio and thus resulted in the release of viruses with impaired infectivity. Clustered amino acid substitutions destroying (i) the predicted cryptic PR cleavage sites or (ii) homologies to the pepsinogen propeptide did not influence viral replication in cell culture, whereas substitutions of the carboxyl-terminal p6* residues 62 to 68 altering proper release of the mature PR from the Gag-Pol precursor drastically reduced viral infectivity. Thus, the critical contribution of p6* and overlapping cis-acting sequence elements to timely regulated virus maturation and infectivity is closely linked to precise ribosomal frameshifting and proper N-terminal release of the viral PR from the Gag-Pol precursor, clearly disproving the hypothesis that sequence motifs in the central part of p6* modulate PR activation and viral infectivity.     

The virion-associated Gag-Pol is decreased in chimeric Moloney murine leukemia viruses in which the readthrough region is replaced by the frameshift region of the human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 translational frameshift event to synthesize the precursor of its enzymes, Gag-Pol, when ribosomes from the infected cells translate the full-length viral messenger RNA. Translation of the same RNA according to conventional translational rules produces Gag, the precursor of the structural proteins of the virus. The efficiency of the frameshift controls the ratio of Gag-Pol to Gag, which is critical for viral infectivity. The Moloney murine leukemia virus (MoMuLV) uses a different strategy, the programmed readthrough of a stop codon, to synthesize Gag-Pol. In this study, we investigated whether different forms of the HIV-1 frameshift region can functionally replace the readthrough signal in MoMuLV. Chimeric proviral DNAs were obtained by inserting into the MoMuLV genome the HIV-1 frameshift region encompassing the slippery sequence where the frameshift occurs, followed by the frameshift stimulatory signal. The inserted signal was either a simple stem-loop, previously considered as the stimulatory signal, or a longer bulged helix, now shown to be the complete stimulatory signal, or a mutated version of the complete signal with a three-nucleotide deletion. Although the three chimeric viruses can propagate essentially as the wild-type virus in NIH 3T3 cells, single-round infectivity assays revealed that the infectivity of the chimeric virions is about three to fivefold lower than that of the wild-type virions, depending upon the nature of the frameshift signal. It was also observed that the Gag-Pol to Gag ratio was decreased about two to threefold in chimeric virions. Comparison of the readthrough efficiency of MoMuLV to the HIV-1 frameshift efficiency, by monitoring the expression of a luciferase reporter in cultured cells, revealed that the frameshift efficiencies were only 30-60% of the readthrough efficiency. Altogether, these observations indicate that replacement of the readthrough region of MoMuLV with the frameshift region of HIV-1 results in virions that are replication competent, although less infectious than wild-type MoMuLV. This type of chimera could provide an interesting tool for in vivo studies of novel drugs targeted against the HIV-1 frameshift event.