The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4) includes the regulatory PSTAIRE helix

BACKGROUND: The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK) CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21(CIP )inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. RESULTS: To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity. CONCLUSION: Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane

Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2-arrest

AbstractThe HIV-1 accessory protein Vpr induces G2 cell cycle arrest and apoptosis. Previous studies indicate that the induction of G2-arrest requires the localization of Vpr to the nuclear envelope. Here we show that treatment of Vpr-expressing HeLa cells with the caspase 3 inhibitor Z-DEVD-fmk induced accumulation of Vpr at the nuclear lamina, while other proteins or structures of the nuclear envelope were not influenced. Furthermore, Z-DEVD-fmk enhances the Vpr-mediated G2-arrest that even occurred in HIV-1NL4–3-infected T-cells. Mutation of Pro-35, which is important for the integrity of helix-α1 in Vpr, completely abrogated the Z-DEVD-fmk-mediated accumulation of Vpr at the nuclear lamina and the enhancement of G2-arrest. As expected, inhibition of caspase 3 reduced the induction of apoptosis by Vpr. Taken together, we could show that besides its role in Vpr-mediated apoptosis induction caspase 3 influences the localization of Vpr at the nuclear envelope and thereby augments the Vpr-induced G2-arrest

Inhibitors of Deubiquitinating Enzymes Block HIV-1 Replication and Augment the Presentation of Gag-Derived MHC-I Epitopes

In recent years it has been well established that two major constituent parts of the ubiquitin proteasome system (UPS)—the proteasome holoenzymes and a number of ubiquitin ligases—play a crucial role, not only in virus replication but also in the regulation of the immunogenicity of human immunodeficiency virus type 1 (HIV-1). However, the role in HIV-1 replication of the third major component, the deubiquitinating enzymes (DUBs), has remained largely unknown. In this study, we show that the DUB-inhibitors (DIs) P22077 and PR-619, specific for the DUBs USP7 and USP47, impair Gag processing and thereby reduce the infectivity of released virions without affecting viral protease activity. Furthermore, the replication capacity of X4- and R5-tropic HIV-1NL4-3 in human lymphatic tissue is decreased upon treatment with these inhibitors without affecting cell viability. Most strikingly, combinatory treatment with DIs and proteasome inhibitors synergistically blocks virus replication at concentrations where mono-treatment was ineffective, indicating that DIs can boost the therapeutic effect of proteasome inhibitors. In addition, P22077 and PR-619 increase the polyubiquitination of Gag and thus its entry into the UPS and the major histocompatibility complex (MHC)-I pathway. In summary, our data point towards a model in which specific inhibitors of DUBs not only interfere with virus spread but also increase the immune recognition of HIV-1 expressing cells

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane

Propagation of SARS-CoV-2 in a Closed Cell Culture Device: Potential GMP Compatible Production Platform for Live-Attenuated Vaccine Candidates under BSL-3 Conditions?

Live-attenuated SARS-CoV-2 vaccines present themselves as a promising approach for the induction of broad mucosal immunity. However, for initial safety assessment in clinical trials, virus production requires conditions meeting Good Manufacturing Practice (GMP) standards while maintaining biosafety level 3 (BSL-3) requirements. Since facilities providing the necessary complex ventilation systems to meet both requirements are rare, we here describe a possibility to reproducibly propagate SARS-CoV-2 in the automated, closed cell culture device CliniMACS Prodigy® in a common BSL-3 laboratory. In this proof-of-concept study, we observed an approximately 300-fold amplification of SARS-CoV-2 under serum-free conditions with high lot-to-lot consistency in the infectious titers obtained. With the possibility to increase production capacity to up to 3000 doses per run, this study outlines a potential fast-track approach for the production of live-attenuated vaccine candidates based on highly pathogenic viruses under GMP-like conditions that may contribute to pandemic preparedness

Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs against SARS-CoV-2

Currently, human infections with the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are accelerating the ongoing spread of the pandemic. Several innovative types of vaccines have already been developed, whereas effective options of antiviral treatments still await a scientific implementation. The development of novel anti-SARS-CoV-2 drug candidates demands skillful strategies and analysis systems. Promising results have been achieved with first generation direct-acting antivirals targeting the viral polymerase RdRp or the protease 3CLpro. Such recently approved or investigational drugs like remdesivir and GC376 represent a basis for further development and optimization. Here, we establish a multi-readout assay (MRA) system that enables the antiviral assessment and mechanistic characterization of novel test compounds, drug repurposing and combination treatments. Our SARS-CoV-2-specific MRA combines the quantitative measurement of several parameters of virus infection, such as the intracellular production of proteins and genomes, enzymatic activities and virion release, as well as the use of reporter systems. In this regard, the antiviral efficacy of remdesivir and GC376 has been investigated in human Caco-2 cells. The readouts included the use of spike- and double-strand RNA-specific monoclonal antibodies for in-cell fluorescence imaging, a newly generated recombinant SARS-CoV-2 reporter virus d6YFP, the novel 3CLpro-based FRET CFP::YFP and the previously reported FlipGFP reporter assays, as well as viral genome-specific RT-qPCR. The data produced by our MRA confirm the high antiviral potency of these two drugs in vitro. Combined, this MRA approach may be applied for broader analyses of SARS-CoV-2-specific antivirals, including compound screenings and the characterization of selected drug candidates

Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner

<div><p>There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.</p></div

The 51 aa EIAV p9 protein and HIV-2 or SIV p6 are not degraded in S10.

<p><b>(A)</b> 400 ng of HIV-2 or SIV <i>s</i>p6, or 100 ng of HIV-1 <i>s</i>p6, or EIAV <i>s</i>p9 were incubated with 5 μg S10 extract for the indicated times at 37°C, and remaining <i>s</i>p6 or <i>s</i>p9 was detected by Western blot. <b>(B)</b> Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. <b>(C)</b> 10 ng <i>s</i>p6BY were incubated with 5 μg S10 and increasing concentrations of HIV-1, HIV-2 or SIV <i>s</i>p6 or EIAV <i>s</i>p9 for 30 min at 37°C. <i>s</i>p6BY was detected by measurement of fluorescence excitation. <b>(D)</b> Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. <b>(E)</b> Virions produced in HEK293T cells transfected with expression plasmids pNLgp2/Udel-1 (HIV-2) or pSIV3+ (SIV) were isolated, lysed with 0.5% Triton X-100 and incubated with 5 μg S10 extract or 10 ng rIDE for 30 min at 37°C. <b>(F)</b> Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. <b>(G)</b> Sequence alignment of p6 peptides from HIV-1, HIV-2, SIV and the EIAV p9 peptide. The sequence of HIV-2 p6 originates from the isolate ROD10, SIV p6 from SIVmac239, and EIAV p9 from the isolate EIAV_Wyoming [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174254#pone.0174254.ref029" target="_blank">29</a>].</p