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We propose a new approach for calculating the three-dimensional (3D) structure of a protein from distance and dihedral angle constraints derived from experimental data. We suggest that such constraints can be obtained from experiments such as tritium planigraphy, chemical or enzymatic cleavage of the polypeptide chain, paramagnetic perturbation of nuclear magnetic resonance (NMR) spectra, measurement of hydrogen-exchange rates, mutational studies, mass spectrometry, and electron paramagnetic resonance. These can be supplemented with constraints from theoretical prediction of secondary structures and of buried/exposed residues. We report here distance geometry calculations to generate the structures of a test protein Staphylococcal nuclease (STN), and the HIV-1 rev protein (REV) of unknown structure. From the available 3D atomic coordinates of STN, we set up simulated data sets consisting of varying number and quality of constraints, and used our group's Self Correcting Distance Geometry (SECODG) program DIAMOD to generate structures. We could generate the correct tertiary fold from qualitative (approximate) as well as precise distance constraints. The root mean square deviations of backbone atoms from the native structure were in the range of 2.0 A to 8.3 A, depending on the number of constraints used. We could also generate the correct fold starting from a subset of atoms that are on the surface and those that are buried. When we used data sets containing a small fraction of incorrect distance constraints, the SECODG technique was able to detect and correct them. In the case of REV, we used a combination of constraints obtained from mutagenic data and structure predictions. DIAMOD generated helix-loop-helix models, which, after four self-correcting cycles, populated one family exclusively. The features of the energy-minimized model are consistent with the available data on REV-RNA interaction. Our method could thus be an attractive alternative for calculating protein 3D structures, especially in cases where the traditional methods of X-ray crystallography and multidimensional NMR spectroscopy have been unsuccessful.

Retroviruses exploit nuclear trafficking machinery at several distinct stages in their replication cycles. In this review, we will focus primarily on nucleocytoplasmic trafficking events that occur after the completion of reverse transcription and proviral integration. First, we will discuss nuclear export of unspliced viral RNA transcripts, which serves two essential roles: as the mRNA template for the translation of viral structural proteins and as the genome for encapsidation into virions. These full-length viral RNAs must overcome the cell’s quality control measures to leave the nucleus by co-opting host factors or encoding viral proteins to mediate nuclear export of unspliced viral RNAs. Next, we will summarize the most recent findings on the mechanisms of Gag nuclear trafficking and discuss potential roles for nuclear localization of Gag proteins in retrovirus replication.

The transcription of the HIV-1 provirus results in only one type of transcript—full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.

Background We observe the emergence of several promoter-variant viral strains in India during recent years. The variant viral promoters contain additional copies of transcription factor binding sites present in the viral modulatory region or enhancer, including RBEIII, LEF-1, Ap-1 and/or NF-κB. These sites are crucial for governing viral gene expression and latency. Here, we infer that one variant viral promoter R2N3-LTR containing two copies of RBF-2 binding sites (an RBEIII site duplication) and three copies of NF-κB motifs may demonstrate low levels of gene expression noise as compared to the canonical RN3-LTR or a different variant R2N4-LTR (a duplication of an RBEIII site and an NF-κB motif). To demonstrate this, we constructed a panel of sub-genomic viral vectors of promoter-variant LTRs co-expressing two reporter proteins (mScarlet and Gaussia luciferase) under the dual-control of Tat and Rev. We established stable pools of CEM.NKR-CCR5 cells (CEM-CCR5 RL reporter cells) and evaluated reporter gene expression under different conditions of cell activation. Results The R2N3-LTR established stringent latency that was highly resistant to reversal by potent cell activators such as TNF-α or PMA, or even to a cocktail of activators, compared to the canonical RN3- or the variant R2N4-LTR. The R2N3-LTR exhibited low-level basal gene expression in the absence of cell activation that enhanced marginally but significantly when activated. In the presence of Tat and Rev, trans-complemented in the form of an infectious virus, the R2N3-LTR demonstrated gene expression at levels comparable to the wild-type viral promoter. The R2N3-LTR is responsive to Tat and Rev factors derived from viral strains representing diverse genetic subtypes. Conclusion With extremely low-level transcriptional noise, the R2N3-LTR can serve as an excellent model to examine the establishment, maintenance, and reversal of HIV-1 latency. The R2N3-LTR would also be an ideal viral promoter to develop high-throughput screening assays to identify potent latency-reversing agents since the LTR is not affected by the usual background noise of the cell.

Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection. The HIV-1 RNA-binding protein rev facilitates nuclear export of viral RNA. Here, the authors use native mass spectrometry to study the interactions between rev-derived peptides and rev response elements of HIV-1 RNA, providing mechanistic insights into rev recognition and recruitment.

Human immunodeficiency virus-1 (HIV-1) is a retrovirus that integrates its reverse-transcribed genome as proviral DNA into the host genome to establish a successful infection. The viral genome integration requires safeguarding the subviral complexes, reverse transcription complex (RTC) and preintegration complex (PIC), in the cytosol from degradation, presumably effectively secured by the capsid surrounding these complexes. An intact capsid, however, is a large structure, which raises concerns about its translocation from cytoplasm to nucleus crossing the nuclear membrane, guarded by complex nuclear pore structures, which do not allow non-specific transport of large molecules. In addition, the generation of new virions requires the export of incompletely processed viral RNA from the nucleus to the cytoplasm, an event conventionally not permitted through mammalian nuclear membranes. HIV-1 has evolved multiple mechanisms involving redundant host pathways by liaison with the cell’s nucleocytoplasmic trafficking system, failure of which would lead to the collapse of the infection cycle. This review aims to assemble the current developments in temporal and spatial events governing nucleocytoplasmic transport of HIV-1 factors. Discoveries are anticipated to serve as the foundation for devising host-directed therapies involving selective abolishment of the critical interactomes between viral proteins and their host equivalents.

The HIV-1 Rev protein expressed in the early stage of virus replication is involved in the nuclear export of some forms of virus RNA. Naturally occurring polymorphisms in the Rev protein could influence its activity. The association between the genetic features of different virus variants and HIV infection pathogenesis has been discussed for many years. In this study, Rev diversity among HIV-1 group M clades was analyzed to note the signatures that could influence Rev activity and, subsequently, clinical characteristics. From the Los Alamos HIV Sequence Database, 4962 Rev sequences were downloaded and 26 clades in HIV-1 group M were analyzed for amino acid changes, conservation in consensus sequences, and the presence of clade-specific amino acid substitutions (CSSs) and the Wu–Kabat protein variability coefficient (WK). Subtypes G, CRF 02_AG, B, and A1 showed the largest amino acid changes and diversity. The mean conservation of the Rev protein was 80.8%. In consensus sequences, signatures that could influence Rev activity were detected. In 15 out of 26 consensus sequences, an insertion associated with the reduced export activity of the Rev protein, 95QSQGTET96, was identified. A total of 32 CSSs were found in 16 clades, wherein A6 had the 41Q substitution in the functionally significant region of Rev. The high values of WK coefficient in sites 51 and 82, located on the Rev interaction surface, indicate the susceptibility of these positions to evolutionary replacements. Thus, the noted signatures require further investigation.

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.

HIV-1 Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process Rev oligomerizes in association with a highly structured RNA motif, the Rev response element. Crystallographic studies of Rev have been hampered by the protein's tendency to aggregate, but Rev has now been found to form a stable soluble equimolar complex with a specifically engineered monoclonal Fab fragment. We have determined the structure of this complex at 3.2 Å resolution. It reveals a molecular dimer of Rev, bound on either side by a Fab, where the ordered portion of each Rev monomer (residues 9-65) contains two coplanar α-helices arranged in hairpin fashion. Subunits dimerize through overlapping of the hairpin prongs. Mating of hydrophobic patches on the outer surface of the dimer is likely to promote higher order interactions, suggesting a model for Rev oligomerization onto the viral RNA.

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions. HIV relies on the RRE RNA interaction with Rev protein for nuclear export of viral mRNAs. The structure of the high-affinity Rev binding site in RRE in two conformations suggests a mechanism for initial Rev binding and oligomerization onto RRE.