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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.

The HIV-1 protein Rev controls a critical step in viral replication by mediating the nuclear export of unspliced and singly-spliced viral mRNAs. Multiple Rev subunits assemble on the Rev Response Element (RRE), a structured region present in these RNAs, and direct their export through the Crm1 pathway. Rev-RRE assembly occurs via several Rev oligomerization and RNA-binding steps, but how these steps are coordinated to form an export–competent complex is unclear. Here, we report the first crystal structure of a Rev dimer-RRE complex, revealing a dramatic rearrangement of the Rev-dimer upon RRE binding through re-packing of its hydrophobic protein–protein interface. Rev-RNA recognition relies on sequence-specific contacts at the well-characterized IIB site and local RNA architecture at the second site. The structure supports a model in which the RRE utilizes the inherent plasticity of Rev subunit interfaces to guide the formation of a functional complex. To be able to multiply, viruses have to first infect a host cell and then hijack the host's molecular machinery to make viral proteins. One stage of this process takes place in the nucleus of the host cell and involves the viral DNA being transcribed to make RNA molecules. These RNA molecules must then be exported from the nucleus to the cytoplasm, where the viral proteins are made. In the case of HIV-1, a protein called Rev has an important role in the export process. The Rev protein, which is supplied by the virus, binds to a region on the viral RNA molecules called the Rev Response Element. The Rev protein then binds to a group of host proteins called the Crm1 export complex to send the viral RNA molecules to the cytoplasm. Jayaraman et al. now provide the first in-depth 3D structure of two Rev molecules bound to a fragment of the Rev Response Element. The Rev molecules change shape when they bind to the element, and specific regions of the element were found to be important for this. The experiments suggest that the Rev Response Element directs the positioning of the Rev proteins on itself to match the shape needed to bind to Crm1 export complex. In parallel work from the same laboratory, Booth et al. have produced a 3D structure of the whole complex. Both structures shed new light on how the HIV-1 virus is able to multiply in its host, which may aid future efforts to develop new treatments for the disease.

Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant selective force on a particular nucleotide can be attributed to just one gene. While this arrangement has been observed in regions in which one gene is structured and the other is disordered, we sought to explore how overlapping genes balance constraints when both protein products are structured over the same sequence. We use a combination of sequence analysis, functional assays, and selection experiments to examine an overlapped region in HIV-1 that encodes helical regions in both Env and Rev. We find that functional segregation occurs even in this overlap, with each protein spacing its functional residues in a manner that allows a mutable non-binding face of one helix to encode important functional residues on a charged face in the other helix. Additionally, our experiments reveal novel and critical functional residues in Env and have implications for the therapeutic targeting of HIV-1.

The HIV-1 Tat protein hijacks P-TEFb kinase to activate paused RNA polymerase II (RNAP II) at the viral promoter. Tat binds additional host factors, but it is unclear how they regulate RNAP II elongation. Here, we identify the cytoplasmic ubiquitin ligase UBE2O as critical for Tat transcriptional activity. Tat hijacks UBE2O to ubiquitinate the P-TEFb kinase inhibitor HEXIM1 of the 7SK snRNP, a fraction of which also resides in the cytoplasm bound to P-TEFb. HEXIM1 ubiquitination sequesters it in the cytoplasm and releases P-TEFb from the inhibitory 7SK complex. Free P-TEFb then becomes enriched in chromatin, a process that is also stimulated by treating cells with a CDK9 inhibitor. Finally, we demonstrate that UBE2O is critical for P-TEFb recruitment to the HIV-1 promoter. Together, the data support a unique model of elongation control where non-degradative ubiquitination of nuclear and cytoplasmic 7SK snRNP pools increases P-TEFb levels for transcriptional activation.

RNA is a crucial structural component of many ribonucleoprotein (RNP) complexes, including the ribosome, spliceosome, and signal recognition particle, but the role of RNA in guiding complex formation is only beginning to be explored. In the case of HIV, viral replication requires assembly of an RNP composed of the Rev protein homooligomer and the Rev response element (RRE) RNA to mediate nuclear export of unspliced viral mRNAs. Assembly of the functional Rev-RRE complex proceeds by cooperative oligomerization of Rev on the RRE scaffold and utilizes both protein-protein and protein-RNA interactions to organize complexes with high specificity. The structures of the Rev protein and a peptide-RNA complex are known, but the complete RNP is not, making it unclear to what extent RNA defines the composition and architecture of Rev-RNA complexes. Here we show that the RRE controls the oligomeric state and solubility of Rev and guides its assembly into discrete Rev-RNA complexes. SAXS and EM data were used to derive a structural model of a Rev dimer bound to an essential RRE hairpin and to visualize the complete Rev-RRE RNP, demonstrating that RRE binding drives assembly of Rev homooligomers into asymmetric particles, reminiscent of the role of RNA in organizing more complex RNP machines, such as the ribosome, composed of many different protein subunits. Thus, the RRE is not simply a passive scaffold onto which proteins bind but instead actively defines the protein composition and organization of the RNP.

BackgroundBispecific antibodies (BsAbs) targeting PD-1 and VEGF and bispecific T cell engagers (BiTEs) have the potential to transform cancer treatment, however only a subset of patients obtain deep and durable responses. BiTEs show potent anti-tumor activity by redirecting T cells to tumor cells expressing the targeted antigen while PD-1/VEGF BsAbs aim to address TME-related resistance by combining immune checkpoint blockade with angiogenesis inhibition to enhance T cell function. Despite these advancements, combining these immunotherapies with complementary strategies is critical to unlock their full potential.Interleukin-2 (IL-2) promotes the proliferation and effector function of T cells. High-dose IL-2 monotherapy can induce complete responses in cancer patients, but its clinical application is severely limited by acute vascular toxicities, notably capillary leak syndrome and severe hypotension via the systemic activation of lymphocytes and natural killer (NK) cells. IL-2 activates lymphocytes and NK cells through the intermediate-affinity dimeric IL-2 receptor (IL-2Rβγ, composed of CD122/CD132). In contrast, certain lymphocytes such as antigen-activated T-cells exhibit increased sensitivity to IL-2 due to their expression of the high-affinity trimeric IL-2 receptor (IL-2Rαβγ, composed of CD25/CD122/CD132). To avoid systemic lymphocyte and NK cell activation, but maintain anti-tumor efficacy, we have developed a novel pegylated, α/β-IL-2 agonist (STK-012) engineered for preferential binding to the IL-2Rabg receptor, which is highly upregulated on antigen-activated T-cells.Methods and ResultsWe show that the murine surrogate of STK-012, mSTK-012, is effective in various syngeneic solid tumor models without inducing acute vascular toxicities. mSTK-012 led to enhanced expansion of tumor antigen-specific CD25+PD-1+CD8+ T cells both systemically and within the tumor microenvironment, resulting in complete responses and durable tumor immune memory with mSTK-012 monotherapy.Furthermore, mouse tumor models resistant to PD1/VEGF BsAbs or different targeting BiTEs were developed. We demonstrate that mSTK-012, in combination with either of these modalities, further enhanced intratumoral T cell proliferation, infiltration, and activity, leading to enhanced thereby rescuing anti-tumor efficacy. Also, combination treatment of mSTK-012 with either PD1/VEGF BsAbs or BiTEs did not result in acute vascular toxicities.ConclusionsCurrently, STK-012 is in Phase I trials for first line Non-small cell lung cancer (NSCLC) patients (NCT05098132) in combination with pembrolizumab and chemotherapy. These findings highlight the potential of STK-012 to overcome key limitations of the next wave immunotherapies by selectively expanding and activating antigen-specific T cells while avoiding typical IL-2 systemic toxicities.

ERM (Ezrin-Radixin-Moesin) proteins are regulated membrane-cytoskeletal crosslinkers. Through their regulated cross linking, they control various cellular activities that require change in cell morphology, maintenance of cell polarity and cell adhesion and other trafficking events. They are organized into an N-terminal FERM domain (∼300 residues) that binds to plasma membrane proteins, a C-terminal C-ERMAD domain (∼100 residues) that binds to f-actin filaments and a central coiled coil region (∼150 residues). In the dormant state, the FERM and C-ERMAD domains are bound to each other thus masking their binding surfaces for other targets. Unmasking is achieved by activation signals like phosphorylation and phosphoinositide binding which weaken the interaction between the domains. The crystal structure of a non-covalent complex of Moesin FERM/C-ERMAD (N/C complex) revealed a globular FERM domain and a non-globular C-ERMAD that binds to the FERM domain through five distinct structural elements---a beta strand and four alpha helices. Since this interface is the target of regulatory signals, we have characterized the macroscopic energetics and microscopic residue-specific stabilities of Ezrin C-ERMAD at the Ezrin N/C interface using isothermal titration calorimetry (ITC) and hydrogen-deuterium exchange (HX) NMR spectroscopy. pH dependent HX studies were carried out in order to determine kinetic/thermodynamic properties of every residue. This work has led to the identification of the most stable regions of C-ERMAD and the main anchors through which it binds on the FERM surface. ITC studies on buffer dependence of the system have identified a potential small molecule that might promote the dormant form of the complex. We also investigated the activation mechanisms of the two main regulatory events in weakening the N/C binding. Phosphorylation at T567 of C-ERMAD was studied using a T→E phosphorylation mimicking mutant and the role of PIP2 in weakening the N/C interaction was studied using IP3, the soluble head-group of PIP2. Results of both studies indicate an overall weakening of the most stable elements of C-ERMAD and that the effects of these regulatory events are propagated across the interface.

Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant selective force on a particular nucleotide can be attributed to just one gene. While this arrangement has been observed in regions in which one gene is structured and the other is disordered, we sought to explore how overlapping genes balance constraints when both protein products are structured over the same sequence. We use a combination of sequence analysis, functional assays and selection experiments to examine an overlapped region in HIV-1 that encodes helical regions in both Env and Rev. We find that functional segregation occurs even in this overlap, with each protein spacing its functional residues in a manner that allows a mutable non-binding face of one helix to encode important functional residues on a charged face in the other helix. Additionally, our experiments reveal novel and critical functional residues in Env and have implications for the therapeutic targeting of HIV-1. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE179046 * https://github.com/jferna10/EnvPaper/