Molecular characterization of the HIV-1 gag nucleocapsid gene associated with vertical transmission

Abstract

Background: The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) plays a pivotal role in the viral lifecycle: including encapsulating the viral genome, aiding in strand transfer during reverse transcription, and packaging two copies of the viral genome into progeny virions. Another gag gene product, p6, plays an integral role in successful viral budding from the plasma membrane and inclusion of the accessory protein Vpr within newly budding virions. In this study, we have characterized the gag NC and p6 genes from six mother-infant pairs following vertical transmission by performing phylogenetic analysis and by analyzing the degree of genetic diversity, evolutionary dynamics, and conservation of functional domains.

Results: Phylogenetic analysis of 168 gag NC and p6 genes sequences revealed six separate subtrees that corresponded to each mother-infant pair, suggesting that epidemiologically linked individuals were closer to each other than epidemiologically unlinked individuals. A high frequency (92.8%) of intact open reading frames of NC and p6 with patient and pair specific sequence motifs were conserved in mother-infant pairs' sequences. Nucleotide and amino acid distances showed a lower degree of viral heterogeneity, and a low degree of estimates of genetic diversity was also found in NC and p6 sequences. The NC and p6 sequences from both mothers and infants were found to be under positive selection pressure. The two important functional motifs within NC, the zinc-finger motifs, were highly conserved in most of the sequences, as were the gag p6 Vpr binding, AIP1 and late binding domains. Several CTL recognition epitopes identified within the NC and p6 genes were found to be mostly conserved in 6 mother-infant pairs' sequences.

Conclusion: These data suggest that the gag NC and p6 open reading frames and functional domains were conserved in mother-infant pairs' sequences following vertical transmission, which confirms the critical role of these gene products in the viral lifecycle.

Figure 1

Phylogenetic analysis of 168 HIV-1 NC and p6 sequences from six mother-infant pairs; pairs B, C, D, E, F, and H. The neighbor-joining tree is based on the distance calculated between the nucleotide sequences from the six mother-infant pairs. Each terminal node represents one sequence. The values on the branches represent the occurrence of that branch over 1,000 bootstrap resamplings. Each pair formed a distinct subtree, and within each subtree the mother and infant sequences were generally separated into clusters, although some intermingling was observed. The formation of subtrees indicated that epidemiologically linked mother-infant pairs were closer to each other evolutionarily than to epidemiologically unlinked pairs, and that there was no PCR cross-contamination. The placement of the HIV-1 lab control strain NL4-3 indicates that no PCR contamination occurred.

Figure 2

Multiple sequence alignment of deduced amino acids of NC and p6 from mother-infant pairs B and C. Within the alignment, the top sequence is the NC consensus B (ConBNC) sequence to which the mother-infant pair sequences are compared. Each line of the alignment represents one clone sequence, and is identified by a clone number with M referring to mother and I referring to infants. The dots represent agreement with the consensus sequence, while substitutions are represented by a single letter amino acid code. Stop codons are shown as asterisks (*). The functional domains within the sequence are indicated above the alignment.

Figure 3

Multiple sequence alignment of deduced amino acids of NC and p6 from mother-infant pairs D and E. Within the alignment, the top sequence is the NC consensus B (ConBNC) sequence to which the mother-infant pair sequences are compared. Each line of the alignment represents one clone sequence, and is identified by a clone number with M referring to mother and I referring to infants. The dots represent agreement with the consensus sequence, while substitutions are represented by a single letter amino acid code. Stop codons are shown as asterisks (*). The functional domains within the sequence are indicated above the alignment.

Figure 4

Multiple sequence alignment of deduced amino acids of NC and p6 from mother-infant pairs F and H, including both infant H twins (I1H and I2H). Within the alignment, the top sequence is the NC consensus B (ConBNC) sequence to which the mother-infant pair sequences are compared. Each line of the alignment represents one clone sequence, and is identified by a clone number with M referring to mother and I referring to infants. The dots represent agreement with the consensus sequence, while substitutions are represented by a single letter amino acid code. Stop codons are shown as asterisks (*). The functional domains within the sequence are indicated above the alignment.