Improved Coreceptor Usage Prediction and Genotypic Monitoring of R5-to-X4 Transition by Motif Analysis of Human Immunodeficiency Virus Type 1 env V3 Loop Sequences

Early studies of the biological properties of human immunodeficiency virus type 1 (HIV-1) found that virus isolates could be placed into as few as two phenotypic categories (defined in vitro as either non-syncytium-inducing [NSI] or syncytium-inducing [SI]) in certain CD4+ T-cell lines. These phenotypes were often found to be associated with differences in growth properties and cytopathicity on peripheral blood mononuclear cells (PBMC) (1, 14, 46) and in cellular host range (3, 48). Ultimately, the difference between the NSI and SI phenotypes was shown to be due largely to the differential use of chemokine receptors as coreceptors for viral entry: NSI viruses predominantly use CCR5, while SI viruses can use CCR5 and CXCR4 or CXCR4 exclusively (2, 29, 31, 52, 54). Results determined on the basis of SI phenotype and/or coreceptor usage typing showed that although HIV-1 present at primary infections used the CCR5 coreceptor (R5 virus) ∼90% of the time (63, 67, 68), a substantial proportion of individuals eventually developed virus that used the CXCR4 coreceptor (X4 virus). These X4/SI viruses are associated with accelerated CD4 decline and more rapid progression of HIV-1 disease (8, 28, 33, 43, 47). Little is known about the mechanisms by which these viruses come to predominate among the HIV-1 strains present in an infected person. For example, it is not known whether X4 emergence is a primary pathogenic event or is secondary to some other event, i.e., whether the virus itself causes accelerated disease progression or whether another event causes the acceleration and perhaps also leads to X4 outgrowth. Another important unanswered question is whether X4 viruses arise multiple times during the course of disease and, if so, why they do not become dominant whenever they emerge. There is also uncertainty about the frequency with which phenotype transition occurs. Phenotypic studies suggest that 50 to 60% of progressing subjects acquire X4/SI virus (26, 57, 58), but the results of a detailed longitudinal genotypic study have indicated the occurrence, sometimes transient, of at least one of four X4-associated mutations in nine of nine individuals (50). Coreceptor usage of a particular virus is established by functional assays (growth on MT2 cells [28] or infection of indicator cell lines [64]). These assays are limited, however, in that results are generally reported only as positive or negative and provide no insight into the sequence of mutations responsible for the phenotype switch—information which may further clarify the role of X4 viruses in pathogenesis, as we discuss below. Certain mutations, particularly in the V3 loop of env (5-7, 15, 16, 22, 23, 51), are strongly associated with syncytium induction and CXCR4 usage; in particular, basic amino acids at V3 positions 11 and 25 (amino acid coordinates 306 and 322 [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"K03455","term_id":"1906382","term_text":"K03455"}}K03455] in standard reference HXB2) very frequently distinguish primary X4 from R5 viruses (9, 15, 16, 21, 42, 66), with positions 24 and 27 implicated in some cases (10, 37). However, while evolutionary studies of the R5-X4 transition have been undertaken (30, 38, 61, 62), the actual mutational pathway or pathways by which R5 viruses that establish infections in vivo evolve into X4 viruses (i.e., the specific evolutionary sequence of mutations required) has been largely unexplored. It is not clear whether the appearance of basic amino acids at sites 11 and 25 is sufficient or even necessary in most cases in vivo to lead to the outgrowth of X4 virus or whether, instead, a more gradual process of mutation accumulation takes place. If mutant accumulation does occur in a more gradual process, this may provide an opportunity for both early detection and arrest of X4 development. Viruses that can use both coreceptors (R5X4 viruses) are known to arise around the time of R5-to-X4 transition (2, 8, 38, 61), but their evolutionary role in that transition is not certain. The answers to questions of in vivo evolution cannot be approached by phenotypic assays alone, and analyses based on the appearance of positively charged mutations at V3 sites 11 and 25 have led to incomplete and sometimes ambiguous conclusions regarding the transition process (35). The V3 loop is highly variable within and between individuals, and bioinformatic approaches suggest that many changes not yet examined virologically are likely to influence coreceptor usage (21, 24, 42). To gain a broader understanding of the mutations that contribute to X4 phenotype and of the temporal sequence in which they occur, we used position-specific scoring matrices (PSSM) (19, 20) to analyze V3 sequences. PSSM are used to detect nonrandom distributions of amino acids at adjacent sites associated with empirically determined groupings of sequences. They are frequently used to search DNA or protein sequences for particular motifs, e.g., transcriptional regulatory sites (55), coiled-coil domains (32), major histocompatibility complex class I binding sites (41), and others. A PSSM uses background genetic variation as a baseline comparison, or “null model,” to facilitate comparison of the residues of a sequence fragment to those of a group of aligned sequences known to have the desired property. The comparison leads to a score that can be interpreted as indicating the likelihood that the sequence fragment has the property of interest. In our study, the empirical groupings consist of V3 loop sequences associated with X4 (or SI) virus and R5 (or NSI) viruses. Using the PSSM as described below, a sequence can be assigned a score: the higher the score, the more closely the sequence resembles those of known X4 viruses. We used the PSSM score for two purposes. First, we developed a PSSM-based phenotype predictor usable for all V3 sequences. We explored the statistical properties of this predictor and showed that it outperforms simple methods that categorize sequences on the basis of the presence of basic amino acids at sites 11 or 25. We validated the predictor with two sets of V3 sequences from phenotyped viruses different from those used to produce the PSSM matrix. Second, we showed that the score can serve as a measure of the transition from R5 to X4 phenotype. Since the PSSM score can act as a continuous indicator of X4 evolution, we used it to identify common temporal patterns among 11 serially sampled individuals. By scoring reconstructed ancestors of the sampled virus for each subject, we demonstrated that the progression from low-scoring (R5-like) to high-scoring (X4-like) viruses was generally gradual but that the loss of putative X4 virus at the later stages of infection can occur in two different ways.