Conservation of functional domains and limited heterogeneity of HIV-1 reverse transcriptase gene following vertical transmission

Abstract

Background: The reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the life cycle of the virus by converting the single stranded RNA genome into double stranded DNA that integrates into the host chromosome. In addition, RT is also responsible for the generation of mutations throughout the viral genome, including in its own sequences and is thus responsible for the generation of quasi-species in HIV-1-infected individuals. We therefore characterized the molecular properties of RT, including the conservation of functional motifs, degree of genetic diversity, and evolutionary dynamics from five mother-infant pairs following vertical transmission.

Results: The RT open reading frame was maintained with a frequency of 87.2% in five mother-infant pairs' sequences following vertical transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in mother-infant pairs' sequences. Both mothers and infants RT sequences were under positive selection pressure, as determined by the ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 132 mother-infant RT sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked mother-infant pairs. The functional domains of RT which are responsible for reverse transcription, DNA polymerization and RNase H activity were mostly conserved in the RT sequences analyzed in this study. Specifically, the active sites and domains required for primer binding, template binding, primer and template positioning and nucleotide recruitment were conserved in all mother-infant pairs' sequences.

Conclusion: The maintenance of an intact RT open reading frame, conservation of functional domains for RT activity, preservation of several amino acid motifs in epidemiologically linked mother-infant pairs, and a low degree of genetic variability following vertical transmission is consistent with an indispensable role of RT in HIV-1 replication in infected mother-infant pairs.

Figure 1

Phylogenetic analysis of HIV-1 RT of 132 RT sequences from five mother-infant pairs, including B, C, D, F and H. The neighbor-joining tree is based on the distance calculated between the nucleotide sequences from the five mother-infant pairs. Each terminal node represents one RT gene sequence. The numbers on the branch points indicate the percent occurrence of branches over 1,000 bootstrap resamplings of the data set. The sequences from each mother formed distinct clusters and are well discriminated and in confined subtrees, indicating that the variants from the same mother-infant pair are closer to each other than to other sequences and that there was no PCR cross-contamination. These data were strongly supported by the high bootstrap values indicated on the branch points.

Figure 2

Multiple sequence alignment of deduced amino acids of HIV-1 reverse transcriptase (RT) gene from mother-infant pair B involved in vertical transmission. In the alignment, the top sequence is the consensus RT sequence of subtype or clade B (CON B) to which mother-infant pair-B RT sequences are aligned. In mother-infant pair B sequences, each line refers to a clone identified by a clone number with M referring to mothers and I referring to infants. The structural elements of RT are indicated above the alignment. Dots represent amino acid agreement with CON-B and substitutions are shown by single letter codes for the changed amino acid. Stop codons are shown as x and dashes represent gaps or truncated protein. Relevant amino acid motifs and domains essential for RT activity are shown by spanning arrowheads indicated above the alignment.

Figure 3

Multiple sequence alignment of deduced amino acids of HIV-1 reverse transcriptase (RT) gene from mother-infant pair C in reference to consensus subtype B (CON B) RT sequence. In the alignment, the top sequence is CON B RT sequence and the bottom sequences are mother-infant pair C sequences (M refers to mother sequences and I to sequences). The number of clones sequenced is represented with clone numbers. The structural elements of RT are indicated above the alignment. Dots represent amino acid agreement with CON-B and substitutions are shown by single letter codes for the changed amino acid. Stop codons are shown as x and dashes represent gaps or truncated protein. Spanning arrowheads indicated above the alignment shows relevant amino acid motifs and domains essential for RT function.

Figure 4

Multiple sequence alignment of deduced amino acids of HIV-1 reverse transcriptase (RT) gene from mother-infant pair D. The patient sequences are aligned in reference to consensus RT sequence of HIV-1 subtype or clade B (CON B) at the top. In the mother-infant pair sequences, each line refers to a clone identified by a clone number with M referring to mother and I to infants. The structural elements of RT are indicated above the alignment. Dots represent amino acid agreement with CON-B and substitutions are shown by single letter codes for the changed amino acid. Stop codons are shown as x and dashes represent gaps or truncated protein. Relevant amino acid motifs and domains essential for RT activity are shown by spanning arrowheads indicated above the alignment.

Figure 5

Multiple sequence alignment of deduced amino acids of HIV-1 reverse transcriptase gene from mother-infant pair F. In the alignment, the top sequence (CON B) is the consensus subtype B RT sequence and the bottom sequences are from mother-infant pair F sequences (M stands for mother sequences and I for infant sequences and the number of clones for mother and infant are indicated by clone number). The structural elements of RT are indicated above the alignment. Dots represent amino acid agreement with CON-B and substitutions are shown by single letter codes for the changed amino acid. Stop codons are shown as x and dashes represent gaps or truncated protein. Relevant amino acid motifs and domains essential for RT functions are shown by spanning arrowheads indicated above the alignment.

Figure 6

Multiple sequence alignment of deduced amino acids of HIV-1 reverse transcriptase (RT) gene from mother H, who had given birth to infected twins, H1 and H2 (alignment shown in Figure 7). In the mother H sequences, each line refers to a clone identified by a clone number with M referring to mother. The mother sequences are aligned in reference to consensus RT sequence of HIV-1 subtype or clade B (CON B) shown at the top. The structural elements of RT are indicated above the alignment. Dots represent amino acid agreement with CON-B and substitutions are shown by single letter codes for the changed amino acid. Stop codons are shown as x and dashes represent gaps or truncated protein. Spanning arrowheads indicated above the alignment shows relevant amino acid motifs and domains required for RT activity.

Figure 7

Multiple sequence alignment of deduced amino acids of HIV-1 reverse transcriptase gene (RT) from infected twin infants, H1 and H2 of mother H (alignment shown in Figure 6). In the alignment, the top sequence is the consensus subtype B RT sequence (CON B) and the bottom sequences are of infants H1 and H2 represented by I and clone numbers. Dots represent amino acid agreement with CON-B and substitutions are shown by single letter codes for the changed amino acid. Stop codons are shown as x and dashes represent gaps or truncated protein. Relevant amino acid motifs and domains essential for RT activity are shown by spanning arrowheads indicated above the alignment.