Rev-Rev Response Element Activity Selection Bias at the Human Immunodeficiency Virus Transmission Bottleneck

Abstract

Background: Sexual transmission of human immunodeficiency virus (HIV) is inefficient and results in selection of viral variants based on incompletely understood factors. Functional variation in the Rev-Rev response element (RRE) regulatory axis of HIV affect replication kinetics and relative expression of viral proteins. We explored whether differences in this axis among viral isolates affect transmission fitness.

Methods: HIV sequences were identified from nine female-to-male transmission pairs. Using a rapid flow cytometric assay, we analyzed Rev-RRE functional activity of primary isolates.

Results: Rev-RRE activity was significantly lower in recipient viruses compared with corresponding donor viruses. In most transmission events, recipient virus Rev-RRE activity clustered at the extreme low end of the range of donor virus activity.

Conclusions: These data indicate selection pressure on the Rev-RRE axis during female-to-male sexual transmission. Variation in Rev-RRE activity may permit viral adaptation to different fitness landscapes and could play an important role in HIV pathogenesis.

Keywords: HIV; HIV Rev; viral pathogenesis; viral transmission.

Figure 1.

Phylogenetic trees for individual transmission pairs. A phylogenetic tree was generated using the neighbor-joining method for 401 single-genome human immunodeficiency virus (HIV) sequences. The sequences of 4 hundred primary isolates associated with 18 individuals in 9 linked female-to-male HIV transmission pairs were obtained from GenBank. The laboratory strain NL4-3 was included in tree generation as an outgroup but was excluded from the figure display for clarity. Portions of the tree corresponding to the individual transmission pairs, A–I, are displayed separately. Tip symbols differentiate sequences from donors and recipients, as well as genomes containing Rev–Rev response element pairs that were selected or not selected for inclusion in functional assays. Horizontal bars represent nucleotide substitutions per site. See also Supplementary Figures 1 and 2.

Figure 2.

Proportion of viral variants represented in functional activity assays. A subset of the unique Rev–Rev response element (RRE) cognate pairs from primary isolates were included in the functional activity assays. For each individual, the proportion of the sequenced viral variants from that individual's plasma containing a Rev–RRE sequence included in the functional assays is shown. Individuals are indicated by the transmission pair, A through I, and the position within each transmission pair, either the donor or recipient partner.

Figure 3.

Rev–Rev response element (RRE) functional activity of viral variants from donors and recipients. The relative functional activity of selected Rev-RRE pairs from primary isolates in both donors and recipients is shown. Each human immunodeficiency virus transmission pair is displayed, from A through I, with Rev-RRE cognate pairs from donor viral sequences on the left of each box and Rev-RRE cognate pairs from recipient viral sequences on the right. Each bubble is a unique Rev-RRE pair in the indicated individual's quasispecies. The position of the bubble on the y-axis represents the relative level of Rev-RRE functional activity for that pair. The area of each bubble is scaled to the relative prevalence of the Rev-RRE pair sequence within the individual's sequenced quasispecies. The weighted average of variant Rev-RRE activity for each individual quasispecies is indicated by the dashed horizontal line. Rev-RRE activity distributions of the donor and the recipient variants were compared for each transmission pair by means of independent-samples Mann-Whitney U test, and the P value for this comparison is shown in each plot. Activity units are multiples of the Rev-RRE activity of the laboratory strain NL4-3.

Figure 4.

Contributions of Rev and the Rev–response element (RRE) to cognate pair functional activity. The relative functional activity of Rev-RRE cognate pairs from primary isolates, as well as the activity of the component Rev with NL4-3 RRE and the component RRE with NL4-3 Rev is shown. Relative activity is shown as multiples of the activity of the NL4-3 cognate pair without units, such that 1 corresponds to the NL4-3 Rev/NL4-3 RRE pair. Observations from technical replicates (n = 5 or 6) are shown as dots. Bars represent the mean values of individual observations, and error bars represent standard deviation. Statistical comparisons were performed using 1-way analysis of variant with adjustment for multiple comparisons using the Dunnett T3 method. Primary isolate 1 was obtained from donor I (accession no. KR820312), isolate 2 from recipient B (KR820385), isolate 3 from donor G (KY112428), isolate 4 from donor C (KY112346), and isolate 5 from recipient A (JX973051). Cognate pair activity values replicate values shown in Figure 3. Abbreviation: NS, not significant. *P < .05; **P < .01; ***P < .001.