Contributions of Individual Domains to Function of the HIV-1 Rev Response Element

O’Carroll, Ina P.; Thappeta, Yashna; Li, Fan; Ramirez-Valdez, Edric A.; Smith, Sean G.; Wang, Yun‐Xing; Rein, Alan

doi:10.1128/jvi.00746-17

Cited by 10 publications

(7 citation statements)

References 27 publications

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“…Unexpectedly, none of the truncated Rev proteins could be detected by western blot, despite some (such as Rev ∆1-10) being functional in both reporter and replication assays (Figure 4A, B). Previous reports have shown that Rev function is robust even at very low expression levels [29, 30]. Indeed, we observed that serial dilutions of Rev plasmid demonstrated high reporter activity at even very low levels and that activity was actually reduced at higher plasmid levels (Figure S2).…”

Section: Resultssupporting

confidence: 53%

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

Jayaraman

Fernandes

Yang

et al. 2018

Preprint

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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.

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Section: Resultssupporting

confidence: 53%

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

Jayaraman

Fernandes

Yang

et al. 2018

Preprint

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“…4A,B ). Previous reports have shown that Rev function is robust even at very low expression levels 36 , 37 . Indeed, we observed that serial dilutions of Rev plasmid demonstrated high reporter activity at even very low levels and that activity was actually reduced at higher plasmid levels (Fig.…”

Section: Resultsmentioning

confidence: 93%

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

Jayaraman

Fernandes

Yang

et al. 2019

Sci Rep

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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.

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“…S3). Notably, whereas no sites beyond IIB and IA have been conclusively identified, indeed, as judged by quantitative functional reporter assays much of the RRE can be deleted as long as the two sites are properly opposed to one another (O’Carroll et al, 2017), it has repeatedly been found that Rev assembly on the RRE is dependent on protein-protein interactions (Daugherty et al, 2008; Pond et al, 2009). Our model indicates that: (a) Rev initially contacts the RRE at only two points, and then assembles through Rev-Rev interactions rather than “along” the RNA, (b) all the C-terminal domains are arrayed in a well-separated manner about the RRE to optimally engage Crm1 (Rausch and Le Grice, 2015), and (c) the ARMs of the two monomers involved in the C-C interaction are oriented outwards ready to engage the distal Region 3 of the RNA proposed to fold back at the checkpoint (Bai et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for nuclear export

Watts

Eren

Zhuang

et al. 2018

Journal of Structural Biology

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HIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occurs is not known. The Rev assembly domain has a helical-hairpin structure which associates through three (A-A, B-B and C-C) interfaces. The RRE has the topology of an upper-case letter A, with the two known Rev binding sites mapping onto the legs of the A. We have determined a crystal structure for the Rev assembly domain at 2.25 Å resolution, without resort to either mutations or chaperones. It shows that B-B dimers adopt an arrangement reversed relative to that previously reported, and join through a C-C interface to form tetramers. The new subunit arrangement shows how four Rev molecules can assemble on the two sites on the RRE to form the specificity checkpoint, and how further copies add through A-A interactions. Residues at the C-C interface, specifically the Pro31-Trp45 axis, are a potential target for intervention.

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Contributions of Individual Domains to Function of the HIV-1 Rev Response Element

Cited by 10 publications

References 27 publications

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

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

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

A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for nuclear export

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