Abstract
HIV-1 Rev is an essential viral regulatory protein that facilitates the nuclear export of intron-containing viral mRNAs. It is organized into structured, functionally well-characterized motifs joined by less understood linker regions. Our recent competitive deep mutational scanning study confirmed many known constraints in Rev’s established motifs, but also identified positions of mutational plasticity, most notably in surrounding linker regions. Here, we probe the mutational limits of these linkers by testing the activities of multiple truncation and mass substitution mutations. We find that these regions possess previously unknown structural, functional or regulatory roles, not apparent from systematic point mutational approaches. Specifically, the N- and C-termini of Rev contribute to protein stability; mutations in a turn that connects the two main helices of Rev have different effects in different contexts; and a linker region which connects the second helix of Rev to its nuclear export sequence has structural requirements for function. Thus, Rev function extends beyond its characterized motifs, and is tuned by determinants within seemingly plastic portions of its sequence. Additionally, Rev’s ability to tolerate many of these massive truncations and substitutions illustrates the overall mutational and functional robustness inherent in this viral protein.
Highlights
Viral proteins maintain their function amidst many forces such as high viral polymerase error rates, immune pressures, and even competing selective pressures from overlapping reading frames and RNA structures
Rev recognizes a ~350nt structured RNA within the intron known as the Rev Response Element (RRE) using an arginine-rich motif (ARM) for binding, and forms an oligomeric assembly on the RRE using two hydrophobic oligomerization motifs or domains (ODs)[11,12]
This Rev-RRE complex recruits the host nuclear export factor Crm[1], using a nuclear export sequence (NES) in Rev, together with RanGTP to transport these RNAs to the cytoplasm and thereby circumvent splicing[5,6]
Summary
Viral proteins maintain their function amidst many forces such as high viral polymerase error rates, immune pressures, and even competing selective pressures from overlapping reading frames and RNA structures. We recently performed competitive deep mutational scanning (CDMS) in non-overlapped viral replication assays[10] These experiments allowed us to examine for the first time, the amino acid preferences/fitness in Rev at each position, when unconstrained by overlapping genes. Results from these experiments showed that, in general, the known structured/functional regions of Rev experience selective pressure for specific side chains while the linker regions between/flanking these structured domains appear to be relatively “plastic”, freely mutating between many different side chains with a variety of chemical properties (Fig. 1B,C). These experiments make evident Rev’s extraordinary genetic robustness, which is achieved via the use of malleable macromolecular interaction surfaces spaced by even more malleable linker regions that can more finely tune those interactions
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