Highly Mutable Linker Regions Regulate HIV-1 Rev Function and Stability

Abstract

The HIV-1 protein Rev is an essential viral regulatory protein that facilitates the nuclear export of intron-containing viral mRNAs. Its sequence is organized into short, structured, functionally well-characterized motifs joined by less understood linker regions. We recently carried out a competitive deep mutational scanning study, which determined the relative fitness of every amino acid at every position of Rev in replicating viruses. This study confirmed many known constraints in Rev’s established interaction motifs, but also identified positions of mutational plasticity within these regions as well as in surrounding linker regions. Here, we probe the mutational limits of these linkers by designing and 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 nuclear export assays and viral replication assays; and a linker region which connects the second helix of Rev to its nuclear export sequence has structural requirements for function. Thus, we find that Rev function extends beyond its characterized motifs, and is in fact further tuned by determinants within seemingly plastic portions of its sequence. At the same time, Rev’s ability to tolerate many of these massive truncations and substitutions illustrates the overall mutational and functional robustness inherent in this viral protein.Author Summary (non-technical summary)HIV-1 Rev is an essential viral protein that controls a critical step in the HIV life cycle. It is responsible for transporting viral mRNA messages from the nucleus to the cytoplasm where they can contribute to the formation new virus particles. In order to understand how different regions of the Rev protein sequence are involved in its function, we introduced truncations and mass substitution mutations in the protein sequence and tested their effect on protein function. Through this study, we not only confirmed previous work highlighting known functionally important regions in Rev, but also found that a large portion of Rev, with little known functional roles influence Rev function and stability. We also show that although protein sequence is critical to its function, Rev can tolerate large variations to its sequence without disrupting its function significantly.

Functional and Structural Segregation of Overlapping Helices in HIV-1

“…To confirm that our selection data reflect Rev functional requirements, we assayed individual alanine mutants at the nine NES residues in a p24 Rev-dependent RRE reporter assay (Smith et al, 1990). Mutation of any of the leucines (positions 75, 78, 81, and 83) or proline at position 76 resulted in loss of function (Figure 4A; high p24 = active Rev), consistent with previous data of Crm1 binding residues (Figure 1C) (Fornerod et al, 1997;Güttler et al, 2010;Booth, Cheng and Frankel, 2014;Jayaraman et al, 2019).…”

“…The present study shows that residues can functionally segregate even when both overlapping segments are structured. We probed residues in the Env gp41 LLP-2 that overlap with the Rev NES and compared the limits to which this domain can tolerate mutation to the already wellestablished mutational limits of Rev (Jayaraman et al, 2019). Interestingly, we observed that critical residues in both proteins are located on one face of a helix such that they was not certified by peer review) is the author/funder.…”

“…This arrangement is a consequence of the genetic code where the wobble bases of functionally critical codons in one reading frame can be chosen to accommodate critical residues in the alternative frame, while the first two codon bases overlap with more mutation-tolerant residues located on a non-binding surface of the overlapped protein (Figure 7). Specifically, the important hydrophobic residues of the Rev NES are oriented on one surface towards the Crm1 binding groove, with consecutive prolines altering the helical register compared to other Crm1 cargoes (Booth, Cheng and Frankel, 2014;Jayaraman et al, 2019). The non-binding face of the NES helix is highly mutable and capable of sampling nearly every amino acid with roughly equal fitness.…”

HIV Rev-isited

“…Recent mutagenesis research suggests that the CTD may help stabilize Rev and prevent aggregation [173]. Conversely, an increase in HIV-1 fitness is observed when stop codons are included in the CTD, suggesting it may play an inhibitory role [174]. More work must be carried out to resolve the molecular function of the CTD.…”

Binding stoichiometry and structural model of the HIV-1 Rev/Importin β complex

“…75-83) which is recognized in the nucleus by CRM1 in complex with RanGTP. Residues C-terminal to the NES enhance protein stability 77,78 , become partly ordered when Rev forms filaments 67 , and have been predicted to interact with the NTD 79 and to regulate accessibility of the NES 80 . The NTD includes an a-helical hairpin (res.…”