HIV-1 Rev-RRE functional activity in primary isolates is highly dependent on minimal context-dependent changes in Rev

Abstract

During HIV infection, intron-containing viral mRNAs are exported from the cell nucleus to the cytoplasm to complete the replication cycle. Cellular restrictions on the export of incompletely spliced transcripts are overcome by a viral protein, Rev, and an RNA structure found in all unspliced and incompletely spliced viral mRNAs, the Rev Response Element (RRE). Primary HIV isolates display substantial variation in the sequence and functional activity of Rev proteins. We analyzed Rev from two primary isolates with disparate activity that resulted in differences in in vitro fitness of replication-competent viral constructs. The results showed that amino acid differences within the oligomerization domain, but not the arginine-rich motif or the nuclear export signal, determined the level of Rev activity. Two specific amino acid substitutions were sufficient to alter the low-activity Rev to a high-activity phenotype. Other mutations in Rev sequences had unpredictable effects on activity that differed between the two Rev backbones. The sensitivity of Rev function level to small sequence changes likely permits modulation of Rev-RRE activity during HIV infection, which may play a role in pathogenesis. The functional consequences of Rev mutations differed between primary isolates, highlighting the challenge of generalizing studies of Rev conducted using laboratory HIV strains.

Rev-Rev Response Element Activity Selection Bias at the HIV Transmission Bottleneck

“…First, the Rev-RRE axis impacts viral replication capacity. We previously demonstrated with replication-competent HIV constructs that Rev activity is positively correlated with replication kinetics (27). Second, Rev-RRE activity alters the relative expression of viral proteins encoded by completely spliced mRNA species (i.e.…”

“…The relative functional activity of the selected Rev-RRE cognate pairs was determined using a lentiviral vector based assay as previously described (see Methods) (27,34). As shown in Figure 3, there was an almost 9-fold difference in functional activity between the most-active and least-active Rev-RRE cognate pairs.…”

Structural and computational studies of HIV-1 RNA

“…The associated structural transitions were identified within the three-way junction of the stem-loop 2 of the RRE RNA which was known to be responsible for initial Rev binding [ 148 ]. In another study [ 149 ], novel amino acid substitutions were identified in the oligomerization domain of the Rev protein which affected the Rev-RRE functional activity. The substitutions of two amino acids at the positions 14 and 19 were shown to sufficiently alter the activity of the Rev protein [ 149 ].…”

“…In another study [ 149 ], novel amino acid substitutions were identified in the oligomerization domain of the Rev protein which affected the Rev-RRE functional activity. The substitutions of two amino acids at the positions 14 and 19 were shown to sufficiently alter the activity of the Rev protein [ 149 ].…”

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

“…HIV primary isolates exhibit sequence differences in both rev and the RRE, and this in turn results in substantial functional activity variation in the Rev-RRE axis between variants [ 24 , 25 ]. Small sequence differences in Rev, the RRE, or both can yield significant differences in Rev-RRE axis activity [ 26 , 27 ]. As the Rev-RRE interaction is necessary for the nuclear export and translation of intron-containing but not fully spliced viral mRNAs, differences in the level of Rev-RRE activity affect not only viral replication kinetics but also the relative expression of many viral proteins [ 28 ].…”