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Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes 1 . These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend both on the strength of the contacts and on the inherent propensities to form binding-competent conformational states 2 , 3 . Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions 4 , 5 . However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities affect cellular activity. Here we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state. Systematic alteration of HIV-1 TAR RNA and quantitative determination of its propensity to bind to the Tat protein establish a key role role for a rare and short-lived RNA state in Tat-dependent transactivation in cells.

HIV Tat binding to the exterior of Aβ fibrils induces lateral aggregation and formation of fibers with increased adhesion, rigidity and mechanical resistance, thus potentially accounting for their higher neurotoxicity. Deposition of amyloid-β plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-β peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aβ fibrils increases β-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aβ aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-β–Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.

Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using \"nunchuck\" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay (\"negative Gaussian\") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.

Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell’s RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat·P-TEFb complex containing HIV-1 Tat, human Cdk9 (also known as CDK9), and human cyclin T1 (also known as CCNT1). Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that would specifically inhibit the Tat·P-TEFb complex and block HIV replication. Tat complex structure The Tat protein of the human immunodeficiency virus (HIV) has been a target for structural studies for many years, with a view to possible therapeutic intervention. Tat is active early in an infection, hijacking the host's positive transcription elongation factor, P-TEFb. The latter, unusually for a host protein, is therefore also a potential drug target. Tahirov et al . report the first crystal structure for HIV Tat in complex with P-TEFb. The 2.1-Å crystal structure shows that, although the interaction interface between Tat and P-TEFb is large, Tat binding changes the structure of P-TEFb. This points to possible opportunities for developing inhibitors that block only the form of P-TEFb that is used by the virus. Here the 2.1 Å crystal structure of human immunodeficiency virus (HIV) Tat protein complexed with the positive transcription elongation factor P-TEFb is reported. This shows that Tat binding changes the structure of P-TEFb, which may suggest opportunities for developing inhibitors that block only the form of P-TEFb used by the virus.

RNA–protein interactions lie at the basis of numerous regulatory and functional cellular biological processes, including transcriptional control, RNA processing, nuclear export, and viral replication. Despite their fundamental biological significance, direct structural investigation of RNA–protein complexes in live human cells remains an unresolved problem due to resolution limits in spatial information, delivery of molecules, and real-time monitoring under native conditions. Existing studies rely on pre-existing in vitro complexes added to cells and therefore overlook important aspects of endogenous binding and localization. Here, we report the first in-cell nuclear magnetic resonance (NMR) study of the de novo formation of an RNA–protein complex in living human cells. By using a model system involving the HIV-1 Tat protein and its high-affinity RNA aptamer, we expressed Tat endogenously in HeLa cells and introduced the aptamer by electroporation. Direct observation was made of native complex formation within the intracellular milieu. In-cell NMR spectra exhibited characteristic chemical shift perturbations and nuclear Overhauser effect (NOE) signatures indicative of specific RNA–protein binding under physiological conditions. Comparison directly with in vitro spectra confirmed structural integrity and binding specificity in the intracellular environment. Remarkably, we obtained a partial NOE-based assignment of the RNA upon complexation with Tat in living cells—an unprecedented step towards cellular structural biology. Complementary confocal microscopy validated nuclear co-localization, enabling functionally relevant interaction. This work shows the first direct, real-time evidence for native RNA–protein complex assembly in human cells. It provides a new paradigm for probing RNA-mediated regulatory events in vivo and expands the horizon of therapeutic RNA design.

Due to an unexpected cell-penetrating property, zinc-finger nucleases (ZFNs) can be delivered to several mammalian cell types as proteins. Dose-dependent disruption of an endogenous gene was achieved with reduced activity at known off-target sites. Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.

In patients infected with the human immunodeficiency virus (HIV), the HIV-Tat protein may be continually produced despite adequate antiretroviral therapy. As the HIV-infected population is aging, it is becoming increasingly important to understand how HIV-Tat may interact with proteins such as amyloid β and Tau which accumulate in the aging brain and eventually result in Alzheimer’s disease. In this review, we examine the in vivo data from HIV-infected patients and animal models and the in vitro experiments that show how protein complexes between HIV-Tat and amyloid β occur through novel protein-protein interactions and how HIV-Tat may influence the pathways for amyloid β production, degradation, phagocytosis, and transport. HIV-Tat may also induce Tau phosphorylation through a cascade of cellular processes that lead to the formation of neurofibrillary tangles, another hallmark of Alzheimer’s disease. We also identify gaps in knowledge and future directions for research. Available evidence suggests that HIV-Tat may accelerate Alzheimer-like pathology in patients with HIV infection which cannot be impacted by current antiretroviral therapy.

HIV-1 Tat is an indispensible transactivator for HIV gene transcription and replication. It has been shown to exit cells as a free protein and enter neighboring cells or interact with surface receptors of neighboring cells to regulate gene expression and cell function. In this study, we report, for the first time, exosome-associated Tat release and uptake. Using a HIV-1 LTR-driven luciferase reporter-based cell assay and Western blotting or in combination with exosome inhibitor, OptiPrep gradient fractionation, and exosome depletion, we demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, Tat-transfected U373.MG and 293T, and HIV-infected MT4. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant-negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key regulatory step in human immunodeficiency virus-1 (HIV-1) transcription and thus in the reversal of HIV latency. By binding to the nascent transactivating response region (TAR) RNA, HIV-1 Tat recruits the human super elongation complex (SEC) to the promoter and releases paused Pol II. Structural studies of TAR interactions have been largely focused on interactions between the TAR bulge and the arginine-rich motif (ARM) of Tat. Here, the crystal structure of the TAR loop in complex with Tat and the SEC core was determined at a 3.5-Å resolution. The bound TAR loop is stabilized by cross-loop hydrogen bonds. It makes structure-specific contacts with the side chains of the Cyclin T1 Tat-TAR recognition motif (TRM) and the zinc-coordinating loop of Tat. The TAR loop phosphate backbone forms electrostatic and VDW interactions with positively charged side chains of the CycT1 TRM. Mutational analysis showed that these interactions contribute importantly to binding affinity. The Tat ARM was present in the crystallized construct; however, it was not visualized in the electron density, and the TAR bulge was not formed in the RNA construct used in crystallization. Binding assays showed that TAR bulge-Tat ARM interactions contribute less to TAR binding affinity than TAR loop interactions with the CycT1 TRM and Tat core. Thus, the TAR loop evolved to make high-affinity interactions with the TRM while Tat has three roles: scaffolding and stabilizing the TRM, making specific interactions through its zinc-coordinating loop, and making electrostatic interactions through its ARM.

Among various delivery systems for vaccine and drug delivery, cell-penetrating peptides (CPPs) have been known as a potent delivery system because of their capability to penetrate cell membranes and deliver some types of cargoes into cells. Several CPPs were found in the proteome of viruses such as Tat originated from human immunodeficiency virus-1 (HIV-1), and VP22 derived from herpes simplex virus-1 (HSV-1). In the current study, a wide-range of CPPs was identified in the proteome of SARS-CoV-2, a new member of coronaviruses family, using in silico analyses. These CPPs may play a main role for high penetration of virus into cells and infection of host. At first, we submitted the proteome of SARS-CoV-2 to CellPPD web server that resulted in a huge number of CPPs with ten residues in length. Afterward, we submitted the predicted CPPs to C2Pred web server for evaluation of the probability of each peptide. Then, the uptake efficiency of each peptide was investigated using CPPred-RF and MLCPP web servers. Next, the physicochemical properties of the predicted CPPs including net charge, theoretical isoelectric point (pI), amphipathicity, molecular weight, and water solubility were calculated using protparam and pepcalc tools. In addition, the probability of membrane binding potential and cellular localization of each CPP were estimated by Boman index using APD3 web server, D factor, and TMHMM web server. On the other hand, the immunogenicity, toxicity, allergenicity, hemolytic potency, and half-life of CPPs were predicted using various web servers. Finally, the tertiary structure and the helical wheel projection of some CPPs were predicted by PEP-FOLD3 and Heliquest web servers, respectively. These CPPs were divided into: a) CPP containing tumor homing motif (RGD) and/or tumor penetrating motif (RXXR); b) CPP with the highest Boman index; c) CPP with high half-life (~100 hour) in mammalian cells, and d) CPP with +5.00 net charge. Based on the results, we found a large number of novel CPPs with various features. Some of these CPPs possess tumor-specific motifs which can be evaluated in cancer therapy. Furthermore, the novel and potent CPPs derived from SARS-CoV-2 may be used alone or conjugated to some sequences such as nuclear localization sequence (NLS) for vaccine and drug delivery.