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

Abstract

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.

FIG. 1.

Schematic of the Gag and Gag-Pol processing sites showing the 12 individual protease cleavage sites: 5 cleavage sites in Gag (p17/p24, p24/p2, p2/NC, p7/p1, and p1/p6^gag), 6 cleavage sites in Gag-Pol (NC/TFP, TFP/p6^pol, p6^pol/PR, PR/RT, RT/p66, and p66/IN), and a single site in Nef. The frequency of amino acid substitution at each of these cleavage sites is shown in Fig. 2 and Tables 3 and 4.

FIG.2.

Amino acid polymorphisms at Gag, Gag-Pol, and Nef cleavage sites. The letters refer to the amino acid substitutions; the numbers in parentheses refer to the number of times the substitution was observed. Each cleavage site sequence consists of the 5 amino acids upstream and the 5 amino acids downstream of the scissile bond, indicated by a shill. The labeling of amino acids is according to the convention of P1 to P5 going from the scissile bond toward the amino terminus and P1′ to P5′ going toward the carboxy terminus. Positively selected amino acids are marked with asterisks. Dots represent amino acids that are identical to those in the M MRCA.

FIG. 3.

Phylogenetic relationships of the South African Tygerberg virology (TV) cleavage site sequences relative to other subtypes in the group M data set. This representative maximum-likelihood tree is based on concatenation and analysis of the 12 protease site nucleotide sequences as a single segment of 360 bp. An indication of the degree of sequence dissimilarity is given by the distance from the central node. The percentage of bootstrap trees out of 1,000 replications supporting a particular phylogenetic group is shown alongside the node considered.