Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Behrens, Ryan T.; Aligeti, Mounavya; Pocock, Ginger M.; Higgins, Christina A.; Sherer, Nathan M.

doi:10.1128/jvi.02107-16

Cited by 41 publications

(38 citation statements)

References 117 publications

(89 reference statements)

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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: 52%

“…Intriguingly, the CDMS data suggest a fitness advantage to C-terminal truncations and thus a possible inhibitory role (Figure 5A, B). A recent study proposed that the Rev NES can be masked in the cytoplasm, controlling Rev trafficking in and out of the nucleus [29]. We speculate that removing C-terminal residues might decrease the masking and thereby promote export of viral RNAs to provide a fitness advantage.…”

Section: Discussionmentioning

confidence: 90%

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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: 52%

Section: Discussionmentioning

confidence: 90%

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

Jayaraman

Fernandes

Yang

et al. 2018

Preprint

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“…Because the nucleotide mutations in RRE resulted in amino acid changes in Env (Fig. 1A), the reporter HIV-1 defective for Env expression was pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) to allow endocytosis-mediated viral entry (11). We found that HIV-1 bearing mutant RREs responded to trans-complemented Rev similarly to WT HIV-1, with comparable infectivity ( Fig.…”

Section: Resultsmentioning

confidence: 87%

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

Chen

et al. 2019

J Virol

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HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity. IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

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“…7). Besides, the nuclear export activities of chicken anemia virus VP1, IAV NS2 and human immunode ciency virus type 1 Rev are also modulated via CRM1-mediated pathway [47][48][49]. In this study, we found that EBV BLLF2 could shuttle between the cytoplasm and nucleus.…”

Section: Discussionmentioning

confidence: 61%

Nuclear Import of Epstein-Barr virus BLLF2 is Mediated by an Importin β1-Dependent Mechanism

Guo

Deng

et al. 2020

Preprint

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Background: Epstein-Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins that require to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. A nuclear membrane phosphoprotein encoded by EBV BLLF2, is believed to associate with viral DNA packaging and primary egress across the nuclear membrane. Results: Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assays and western blot were performed to explore the nuclear import mechanism of BLLF2. As results, BLLF2 was shown to be a nucleocytoplasmic shuttling protein, which was mediated neither by chromosomal region maintenance 1 (CRM1)- nor transporter associated with antigen processing (TAP)-dependent pathway. Yet, two functional nuclear localization signals (NLSs) of BLLF2, NLS1 (16KRQALETVPHPQNRGR31) and NLS2 (48PPVAKRRR58), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proved to transport into the nucleus via Ran-dependent and importin β1-dependent pathway. Conclusions: This mechanism may contribute to a more extensive insight of the assembly and synthesis of EB virions in the nucleus, thus affording a new direction for the treatment of viruses.

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Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Cited by 41 publications

References 117 publications

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

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

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

Nuclear Import of Epstein-Barr virus BLLF2 is Mediated by an Importin β1-Dependent Mechanism

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