V3 loop of the human immunodeficiency virus type 1 Env protein: interpreting sequence variability.

Two different states of human immunodeficiency virus type 1 are apparent in the asymptomatic and late stages of infection. Important determinants associated with these two states have been found within the V3 loop of the viral Env protein. In this study, two large data sets of published V3 sequences were analyzed to identify patterns of sequence variability that would correspond to these two states of the virus. We were especially interested in the pattern of basic amino acid substitutions, since the presence of basic amino acids in V3 has been shown to change virus tropism in cell culture. Four features of the sequence heterogeneity in V3 were observed: (i) approximately 70% of all nonconservative basic substitutions occur at four positions in V3, and V3 sequences with a basic substitution in at least one of these four positions contain approximately 95% of all nonconservative basic substitutions; (ii) substitution patterns within V3 are influenced by the identity of the amino acid at position 25; (iii) sequence polymorphisms account for a significant fraction of uncharged amino acid substitutions at several positions in V3, and sequence heterogeneity other than these polymorphisms is most significant at two positions near the tip of V3; and (iv) sequence heterogeneity in V3 (in addition to the basic amino acid substitutions) is approximately twofold greater in V3 sequences that contain basic amino acid substitutions. By using this sequence analysis, we were able to identify distinct groups of V3 sequences in infected patients that appear to correspond to these two virus states. The identification of these discrete sequence patterns in vivo demonstrates how the V3 sequence can be used as a genetic marker for studying the two states of human immunodeficiency virus type 1.     

Novel human endogenous sequences related to human immunodeficiency virus type 1.

Endogenous retrovirus-related sequences exist within the normal genomic DNA of all eukaryotes, and these endogenous sequences have been shown to be important to the nature and biology of related exogenous retroviruses and may also play a role in cellular functions. To date, no endogenous sequences related to human immunodeficiency virus type 1 (HIV-1) have been reported. Herein we describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee, and rhesus monkey DNAs from normal uninfected individuals. We also present the isolation and characterization of two of these endogenous HIV-1-related sequences, EHS-1 and EHS-2. With use of low-stringency Southern blot hybridization, complex banding patterns were detected in human DNA with 5' and 3' HIV-1-derived probes. When an HIV-1 env region probe was used, we detected a less complex, conserved banding pattern in human DNA as well as a related but distinct banding pattern in chimpanzee and rhesus monkey DNAs. EHS-1 and -2 were cloned from normal human genomic DNA libraries by using the env region probe. Clone EHS-1 shows sequence similarity with the domain of the envelope cellular protease cleavage site of HIV-1, while EHS-2 has sequence similarity to the overlapping reading frame for Rev and gp41. Stringent hybridization of EHS-1 back to primate genomic DNA indicates two distinct EHS-1 loci in normal human DNA, an identical band pattern in chimpanzee DNA, and a single locus in rhesus monkey DNA. Likewise, EHS-2 is present as a single highly conserved locus in all three species. An oligonucleotide derived from EHS-2 across a region of near identity to HIV-1 detects a complex banding pattern in all primates tested similar to that seen with the 3' HIV-1 probe. These data suggest that most of the HIV-1-related sequences identified in primate DNA share a common core of nucleic acid sequence found in both EHS-2 and rev and that some of these HIV-1-related sequences have additional larger regions of sequence similarity to HIV-1.     

V3-independent determinants of macrophage tropism in a primary human immunodeficiency virus type 1 isolate.

Human immunodeficiency virus type 1 isolates differ in their ability to productively infect macrophages, and several groups have mapped the genetic basis for macrophage tropism to regions of env that include the third hypervariable region (V3 loop). We recently described a primary isolate (89.6) which is highly macrophage tropic and yet differs from other macrophage-tropic strains studied in that it is cytopathic in T cells. Genetic mapping of macrophage tropism determinants in this virus was done by using chimeras generated with the prototypic non-macrophage-tropic strain HXB2. Replacement of a 2.7-kb env-containing region of HXB with corresponding sequences from 89.6 conferred the macrophage-tropic phenotype, but insertion of the 89.6 V3 loop along with V4/V5 sequences did not. Conversely, placement of HXB sequences that included V3 into 89.6 did not impair this strain's ability to replicate in macrophages. Sequence analysis of V3 shows that 89.6 differs markedly from previously described macrophage-tropic consensus sequences and that it is more similar to highly charged non-macrophage-tropic strains. This suggests either that macrophage tropism is defined by structural determinants resulting from complex interactions among multiple env regions rather than V3 sequence-specific requirements or that there are multiple mechanisms by which different strains may establish productive macrophage infection. In addition, because the HXB V3 loop supports productive macrophage infection in the background of 89.6, phenotypic characterization of V3 sequences should be considered specific to the viral context in which they are placed.     

cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging.

We have previously described a series of human immunodeficiency virus type 1-based vectors in which efficient RNA encapsidation appeared to correlate with the presence of a 1.1-kb env gene fragment encompassing the Rev-responsive element (RRE). In this report, we explore in detail the role of the RRE and flanking env sequences in vector expression and RNA encapsidation. The analysis of a new series of vectors containing deletions within the env fragment failed to identify a discrete packaging signal, although the loss of certain sequences reduced packaging efficiency three- to fourfold. Complete removal of the env fragment resulted in a 100-fold decrease in the vector transduction titer but did not abolish RNA encapsidation. We conclude that the RRE and 3' env sequences are not essential for human immunodeficiency virus type 1 vector encapsidation but may be important in vectors in which a heterologous gene has been placed adjacent to the 5' packaging signal, potentially disrupting its structure.     

A reliable phenotype predictor for human immunodeficiency virus type 1 subtype C based on envelope V3 sequences

In human immunodeficiency virus type 1 (HIV-1) subtype B infections, the emergence of viruses able to use CXCR4 as a coreceptor is well documented and associated with accelerated CD4 decline and disease progression. However, in HIV-1 subtype C infections, responsible for more than 50% of global infections, CXCR4 usage is less common, even in individuals with advanced disease. A reliable phenotype prediction method based on genetic sequence analysis could provide a rapid and less expensive approach to identify possible CXCR4 variants and thus increase our understanding of subtype C coreceptor usage. For subtype B V3 loop sequences, genotypic predictors have been developed based on position-specific scoring matrices (PSSM). In this study, we apply this methodology to a training set of 279 subtype C sequences of known phenotypes (228 non-syncytium-inducing [NSI] CCR5⁺ and 51 SI CXCR4⁺ sequences) to derive a C-PSSM predictor. Specificity and sensitivity distributions were estimated by combining data set bootstrapping with leave-one-out cross-validation, with random sampling of single sequences from individuals on each bootstrap iteration. The C-PSSM had an estimated specificity of 94% (confidence interval [CI], 92% to 96%) and a sensitivity of 75% (CI, 68% to 82%), which is significantly more sensitive than predictions based on other methods, including a commonly used method based on the presence of positively charged residues (sensitivity, 47.8%). A specificity of 83% and a sensitivity of 83% were achieved with a validation set of 24 SI and 47 NSI unique subtype C sequences. The C-PSSM performs as well on subtype C V3 loops as existing subtype B-specific methods do on subtype B V3 loops. We present bioinformatic evidence that particular sites may influence coreceptor usage differently, depending on the subtype.     

Use of random systematic mutagenesis to generate viable human rhinovirus 14 chimeras displaying human immunodeficiency virus type 1 V3 loop sequences.

Random systematic mutagenesis was used to generate a library of human rhinovirus 14 chimeras that each display a segment from the V3 loop of human immunodeficiency virus type 1. The sequence XXIGPGRAXX, where X could be any of the 20 amino acids, was inserted at the neutralizing immunogenic site II of human rhinovirus 14 between VP2 residues 159 and 160. Twenty-five unique chimeric viruses were isolated, and the identity of their randomized residues was determined. A nonrandom amino acid distribution that may reflect structural requirements for viability was observed at the randomized positions. Fifteen of 25 chimeras were neutralized by one or more of a panel of four anti-human immunodeficiency virus type 1 V3 loop antibody preparations, indicating that antigenicity had been successfully transplanted. Libraries of chimeric viruses produced by using the techniques described may be a source of vaccines and other immunotherapeutic reagents. The random systematic mutagenesis methodology described should be generally useful for the rapid transplantation of foreign sequences into viral coat and other proteins to produce libraries containing members with the desired properties.     

Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection.

Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.     

Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo.

The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function.     

Convergent evolution within the V3 loop domain of human immunodeficiency virus type 1 in association with disease progression.

Phylogenetic analysis was used to study in vivo genetic variation of the V3 region of human immunodeficiency virus type 1 in relation to disease progression in six infants with vertically acquired human immunodeficiency virus type 1 infection. Nucleotide sequences from each infant formed a monophyletic group with similar average branch lengths separating the sets of sequences. In contrast to the star-shaped phylogeny characteristic of interinfant viral evolution, the shape of the phylogeny formed by sequences from the infants who developed AIDS tended to be linear. A computer program, DISTRATE, was written to analyze changes in DNA distance values over time. For the six infants, the rate of divergence from the initial variant was inversely correlated with CD4 cell counts averaged over the first 11 to 15 months of life (r = -0.87, P = 0.024). To uncover evolutionary relationships that might be dictated by protein structure and function, tree-building methods were applied to inferred amino acid sequences. Trees constructed from the full-length protein fragment (92 amino acids) showed that viruses from each infant formed a monophyletic group. Unexpectedly, V3 loop protein sequences (35 amino acids) that were found at later time points from the two infants who developed AIDS clustered together. Furthermore, these sequences uniquely shared amino acids that have been shown to confer a T-cell line tropic phenotype. The evolutionary pattern suggests that viruses from these infants with AIDS acquired similar and possibly more virulent phenotypes.     

Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus.

To identify RNA and protein sequences involved in packaging of human immunodeficiency virus type 1 (HIV-1), various mutations were introduced into the viral genome. Portions of the human immunodeficiency virus type 1 genome between the first splice donor site and the gag initiation codon were deleted to investigate the RNA packaging site (psi). Point mutations that alter cysteine residues in one or both zinc finger motifs of p7, a cleavage product of the gag precursor, were created to study the role of the gag zinc fingers in packaging. The psi site mutants and the gag mutants exhibited similar phenotypes. Cells transfected with the mutant genomes, while expressing normal levels of human immunodeficiency virus type 1 RNA and proteins, produced viral particles that were normal in protein content but lacked detectable viral RNA. These mutant virions were unable to productively infect cells. The combination of human immunodeficiency virus type 1 packaging mutations should minimize fortuitous assembly of infectious virus and may provide a means to produce noninfectious particles for candidate vaccines.     

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.     

Disulfide bond formation in the human immunodeficiency virus type 1 Nef protein.

Substitution of alanine for cysteine residues of the human immunodeficiency virus type 1 LAI (BRU) and ELI Nef proteins was used to determine pairing of the cysteine residues present in each protein. The results show that under nonreducing conditions, alternative pairing of the cysteines occurs. The preferred pairing of cysteine residues of the LAI and ELI proteins differs. In the experimental system used, viruses carrying the ELI nef allele are found to express Nef proteins which accelerate virus replication. Mutation in critical cysteine residues of the protein reduce the rate of virus replication. In the same system, viruses harboring the LAI nef allele fail to replicate. These observations raise the possibility that differences in the observed biological activity of nef alleles may be attributed, at least in part, to differences in the secondary structure of the proteins.     

Transcellular transactivation by the human immunodeficiency virus type 1 tat protein.

The human immunodeficiency virus type 1 (HIV-1) transactivator (tat) protein produced in one cell activated HIV-1 promoter-directed gene expression in a second cell, provided the cells were in direct contact with one another. This observation suggests that the tat protein produced in HIV-1-infected cells has a physiological effect on neighboring cells.     

Binding of human immunodeficiency virus type 1 nucleocapsid protein to psi-RNA-SL3

The interaction of the nucleocapsid protein NCp7, from the pNL4-3 isolate of HIV-1, with psi-RNA-SL3, with the sequence 5'-GGACUAGCGGAGGCUAGUCC, was studied using non-denaturing gel electrophoresis. Two kinds of experiments were performed, using buffered solutions of radiolabeled RNA and unlabeled protein. In the 'dilution' experiments, the total RNA concentration, RT, was varied for a series of solutions, but kept equal to the total protein concentration, PT, In the 'titration' experiments, solutions having RT constant but with varying PT were analyzed. The solutions were electrophoresed and the autoradiographic spot intensities, proportional to the amounts of the different species present, were measured. The intensities were fit to a number of equilibrium models, differing in species stoichiometries, by finding the best values of the binding constants. It was shown that NCp7 protein and SL3 RNA combine to form at least two complexes. When PT is below approximately 10 microM, a complex that contains two RNAs and one protein forms. Increasing PT to approximately 100 microM causes the 2:1 complex to oligomerize, forming a species having eight RNAs and four proteins. For the dilution experiments, run at 5 degrees C at an ionic strength of 31 mM, we found K1 for the 2:1 complex is approximately 10(11) M(-2) and K2 for the 8:4 complex is approximately 10(16) M(-3). The titration experiments returned K1 approximately 10(7) M(-2) (poorly determined) and K2 approximately 10(19) M(-3). The analysis was complicated by the loss of RNA at higher protein concentrations, due to formation of an insoluble species containing both RNA and protein, which does not enter the gel. Correcting for this changes the calculated values of equilibrium constants, but not the molecularities determined by our analysis. The observation that a small complex can oligomerize to form a larger species is consistent with the fact that NCp7 organizes and condenses the genome in the virus particle.     

Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140

The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015-3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.     

Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines

The ability to elicit an immune response to a spectrum of human immunodeficiency virus type 1 (HIV-1) gene products from divergent strains is a desirable feature of an AIDS vaccine. In this study, we examined combinations of plasmids expressing multiple HIV-1 genes from different clades for their ability to elicit humoral and cellular immune responses in mice. Immunization with a modified Env, gp145DeltaCFI, in combination with a Gag-Pol-Nef fusion protein plasmid elicited similar CD4(+) and CD8(+) cellular responses to immunization with either vector alone. Further, when mice were immunized with a mixture of Env from three clades, A, B, and C, together with Gag-Pol-Nef, the overall potency and balance of CD4(+)- and CD8(+)-T-cell responses to all viral antigens were similar, with only minor differences noted. In addition, plasmid mixtures elicited antibody responses comparable to those from individual inoculations. These findings suggest that a multigene and multiclade vaccine, including components from A, B, and C Env and Gag-Pol-Nef, can broaden antiviral immune responses without immune interference. Such combinations of immunogens may help to address concerns about viral genetic diversity for a prospective HIV-1 vaccine.     

Variability in the human immunodeficiency virus type 1 gp120 Env protein linked to phenotype-associated changes in the V3 loop

Isolates of human immunodeficiency virus type 1 (HIV-1) are classified according to the chemokine receptor (coreceptor) used in conjunction with CD4 to target and enter cells: viruses using CCR5 and CXCR4 are classified as R5 and X4, respectively. The major determinant of entry-related HIV-1 phenotypes is known to reside in the third variable region of gp120 (V3). It is clear, however, that positions outside of V3 play some role in influencing phenotype, although marked context dependence and extensive variability among HIV-1 isolates have made the identification of these positions difficult. We used the presence of previously described substitutions in V3 to classify a large set of HIV-1 subtype B gp120 sequences available in public databases as X4-like or R5-like. Using these classifications, we searched for positions outside of V3 where either amino acid composition or variability differed significantly among sequences of different inferred phenotypes. Our approach took the epidemiological relationships among sequences into account. A cluster of positions linked to changes in V3 was identified between amino acids 190 and 204 of gp120, immediately C-terminal of V2; changes at position 440 in C4 were also linked to inferred phenotype. Structural data place these positions at the coreceptor-binding face of gp120 in a surface-exposed location. We also noted a significant increase in net positive charge in a highly variable region of V2. This study both confirms previous observations and predicts specific positions that contribute to a functional relationship between V3, V2, and C4.     

Functional domains of the capsid protein of human immunodeficiency virus type 1.

A series of deletions was introduced into the CA domain of the human immunodeficiency virus type 1 Gag polyprotein to examine its role in virus particle and core formation. The mutations resulted in two phenotypes, indicating the existence of two functionally distinct regions within the CA domain. Deletions within a conserved stretch of 20 amino acids referred to as the major homology region (MHR) and deletions C terminal to this region blocked virus replication and significantly reduced the ability to form viral particles. Deletions N terminal to the MHR also prevented virus replication, but the mutants retained the ability to assemble and release viral particles with the same efficiency as the wild-type virus. The mutant particles contained circular rather than cone-shaped cores, and while they were of a density similar to that of wild-type particles, they were more heterogeneous in size. These results indicate that CA domain sequences N terminal to the MHR are essential for the morphogenesis of the mature cone-shaped core.     

Subcellular localization of the Vif protein of human immunodeficiency virus type 1.

The Vif (viral infectivity factor) protein of human immunodeficiency virus type 1 (HIV-1) has been shown to dramatically enhance the infectivity of HIV-1 virus particles during virus production. The subcellular localization of Vif was examined to elucidate cellular pathways which may be important for Vif function. Indirect immunofluorescence staining of Vif demonstrated a diffuse cytoplasmic distribution and showed that most Vif was not associated with the Golgi complex, a proposed site of localization (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). Subcellular fractionation of transfected COS cells and HIV-1-infected Jurkat and CEM cells demonstrated that Vif is a cytoplasmic protein which exists in both a soluble cytosolic form and membrane-associated form. The membrane-associated form of Vif is a peripheral membrane protein which is tightly associated with the cytoplasmic side of cellular membranes. The C terminus of Vif was required for the stable association of Vif with membranes. The C terminus was also essential for Vif function, suggesting that the association of Vif with membranes is likely to be important for its biological activity. The highly conserved regions at residues 103 to 115 and 142 to 150 were important for Vif function but did not affect membrane association, indicating that these regions are likely to be important for other, as-yet-unknown functions.