Persistence of attenuated HIV-1 rev alleles in an epidemiologically linked cohort of long-term survivors infected with nef-deleted virus

Abstract

Background: The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with nef-deleted, attenuated strains of human immunodeficiency virus type 1 (HIV-1). Although the cohort members have experienced differing clinical courses and now comprise slow progressors (SP) as well as long-term nonprogressors (LTNP), longitudinal analysis of nef/long-terminal repeat (LTR) sequences demonstrated convergent nef/LTR sequence evolution in SBBC SP and LTNP. Thus, the in vivo pathogenicity of attenuated HIV-1 strains harboured by SBBC members is dictated by factors other than nef/LTR. Therefore, to determine whether defects in other viral genes contribute to attenuation of these HIV-1 strains, we characterized dominant HIV-1 rev alleles that persisted in 4 SBBC subjects; C18, C64, C98 and D36.

Results: The ability of Rev derived from D36 and C64 to bind the Rev responsive element (RRE) in RNA binding assays was reduced by approximately 90% compared to Rev derived from HIV-1NL4-3, C18 or C98. D36 Rev also had a 50-60% reduction in ability to express Rev-dependent reporter constructs in mammalian cells. In contrast, C64 Rev had only marginally decreased Rev function despite attenuated RRE binding. In D36 and C64, attenuated RRE binding was associated with rare amino acid changes at 3 highly conserved residues; Gln to Pro at position 74 immediately N-terminal to the Rev activation domain, and Val to Leu and Ser to Pro at positions 104 and 106 at the Rev C-terminus, respectively. In D36, reduced Rev function was mapped to an unusual 13 amino acid extension at the Rev C-terminus.

Conclusion: These findings provide new genetic and mechanistic insights important for Rev function, and suggest that Rev function, not Rev/RRE binding may be rate limiting for HIV-1 replication. In addition, attenuated rev alleles may contribute to viral attenuation and long-term survival of HIV-1 infection in a subset of SBBC members.

Figure 1

Amino acid sequences of persistent and dominant SBBC rev alleles. The HIV-1 Rev amino acid sequences shown represent those derived from the dominant and persistent rev alleles harboured by SBBC subjects C18, C64, C98 and D36. They are the consensus sequences of multiple independent Rev clones that persisted over a 4- to 6-year period in C64, C98 and D36, or which were dominant in a single blood sample obtained from C18 [see Additional file 1]. Amino acid alignments are compared to Rev from HIV-1_NL4-3. Dots indicate residues identical to HIV-1_NL4-3 Rev, and dashes indicate gaps. Boxed residues indicate amino acid substitutions which discriminate C18 and C98 Revs from C64 and D36 Revs. NLS; nuclear localization signal, RBD; RNA binding domain, NES; nuclear export signal.

Figure 2

Analysis of Rev/RRE binding. RNA binding assays were conducted with [³²P]-labelled RRE riboprobes and increasing concentrations of His-tagged Rev proteins, as described in Materials and Methods. Binding reactions containing increasing concentrations of His-tagged Matrix protein from HIV-1_NL4-3 were included as negative controls. Rev/RRE complexes were resolved by electrophoresis in 5% (wt/vol) native polyacrylamide gels and visualized by autoradiography (A). Bands were quantified by phosphorimager analysis, and the percentage of RNA binding was calculated by dividing the signal intensity of bands associated with Rev/RRE complexes by the signal intensity of all bands, and multiplying this number by 100 (B). The data shown are representative of three independent experiments. *p < 0.01, Student's t test.

Figure 3

Analysis of Rev protein expression and function in mammalian cells. Rev function was examined by co-transfection of CEM cells with pcDNA3.1-Rev plasmid and the Rev-dependent pDM128 CAT expression plasmid [31], as described in Materials and Methods. Cells co-transfected with pDM128 and pcDNA3.1 expressing HIV-1_NL4-3 Matrix protein or empty pcDNA3.1 vector were included as negative controls. Rev protein expression was determined by Western blotting with sheep anti-Rev polyclonal antisera (A). CAT activity in cell lysates was quantified and normalized to CAT activity in lysates of CEM cells co-transfected with pDM128 and NL4-3 Rev (B). Values shown are means of triplicate transfections. Error bars represent standard deviations. Results are representative of three independent experiments. *P < 0.01, Student's t test.