Novel Vpx virus-like particles to improve cytarabine treatment response against acute myeloid leukemia.

Knowledge of the molecular pathogenesis of acute myeloid leukemia has advanced in recent years. Despite novel treatment options, acute myeloid leukemia remains a survival challenge for elderly patients. We have recently shown that the triphosphohydrolase SAMHD1 is one of the factors determining resistance to Ara-C treatment. Here, we designed and tested novel and simpler virus-like particles incorporating the lentiviral protein Vpx to efficiently and transiently degrade SAMHD1 and increase the efficacy of Ara-C treatment. The addition of minute amounts of lentiviral Rev protein during production enhanced the generation of virus-like particles. In addition, we found that our 2nd generation of virus-like particles efficiently targeted and degraded SAMHD1 in AML cell lines with high levels of SAMHD1, thereby increasing Ara-CTP levels and response to Ara-C treatment. Primary AML blasts were generally less responsive to VLP treatment. In summary, we have been able to generate novel and simpler virus-like particles that can efficiently deliver Vpx to target cells.

Spatial resolution of HIV-1 post-entry steps in resting CD4 T cells.

Resting CD4 T cells resist productive HIV-1 infection. The HIV-2/simian immunodeficiency virus protein viral accessory protein X (Vpx) renders these cells permissive to infection, presumably by alleviating blocks at cytoplasmic reverse transcription and subsequent nuclear import of reverse-transcription/pre-integration complexes (RTC/PICs). Here, spatial analyses using quantitative virus imaging techniques reveal that HIV-1 capsids containing RTC/PICs are readily imported into the nucleus, recruit the host dependency factor CPSF6, and translocate to nuclear speckles in resting CD4 T cells. Reverse transcription, however, remains incomplete, impeding proviral integration and viral gene expression. Vpx or pharmacological inhibition of the deoxynucleotide triphosphohydrolase (dNTPase) activity of the restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) increases levels of nuclear reverse-transcribed cDNA and facilitates HIV-1 integration. Nuclear import and intranuclear transport of viral complexes therefore do not pose important blocks to HIV-1 in resting CD4 T cells, and the limitation to reverse transcription by SAMHD1's dNTPase activity constitutes the main pre-integration block to infection.

Interaction Between Vpx and SAMHD1, Vital for SAMHD1 Inhibition.

It was confirmed that the sterile alpha motif and HD domain 1 (SAMHD1) limits human immunodeficiency virus type 1 (HIV-1) replication. In contrast, viral protein x (Vpx) in HIV-2 and some simian immunodeficiency viruses can counteract this effect. The possible interaction between SAMHD1 and Vpx was suggested by previous studies; however, there are no data to confirm this interaction. Therefore, this study aimed to study the interaction between two proteins and the properties of Vpx protein for the first time using bioinformatic tools. Vpx and SAMHD1 sequences were obtained from the National Center for Biotechnology Information GenBank. Several software were used to define Vpx properties and the interaction between Vpx and different SAMHD1 isoforms. Our findings indicated the difference in interaction sites among different Vpx. However, in all Vpx proteins, this region is from amino acids 4 to 90. In addition, two regions (26-31 and 134-139) and two amino acids 425 and 429 in SAMHD1 are vital in the possible interaction. In addition, our analysis determined the physicochemical and immunological properties of the Vpx. Considering all factors, this study could confirm that Vpx interacts with SAMHD1, which could inhibit SAMHD1. Moreover, our findings can pave the way for future studies to express and purify Vpx in the laboratory and study this protein in vitro.

Enhanced infection efficiency and cytotoxicity mediated by vpx-containing lentivirus in chimeric antigen receptor macrophage (CAR-M).

Genetically modified macrophage infusion has been proven to be a novel treatment for cancer. One of the most important processes in macrophage-based therapy is the efficient transfer of genes. HIV-1-derived lentiviruses were widely used as delivery vectors in chimeric antigen receptor T and NK cell construction. While macrophages are relatively refractory to this lentiviral vector transduction as a result of the myeloid-specific restriction factor SAMHD1, which inhibited the virion cycle through exhausting the dNTPs pool and degradating RNAs. An efficient macrophage transduction strategy has been developed via packaging the HIV-2 accessory protein Vpx into the virion. Vpx counteracts SAMHD1 through CRL4 (DCAF1) E3 ubiquitin ligase mediated SAMHD1 degradation, yet the influence by the introduction of Vpx on macrophage has not been fully evaluated. Here, we constructed the chimeric lentiviral vector HIV-1-Vpx and systematically analyzed the infection efficiency of this vector in time-dependent manner. Our results showed that the simplified chimeric virus exhibited dramatically enhanced infection in human macrophages compared to normal lentivirus. Moreover, transcriptome sequencing was performed to evaluate the cellular status after chimeric virus infection. The sequencing results indicated that Vpx introduction promoted macrophage remodeling towards a proinflammatory phenotype, without affecting classic M1/M2 cell surface markers. Our results suggest that the Vpx-containing lentivirus could be used as an ideal tool for the generation of genetically engineered macrophages with high gene transfer efficiency and poised proinflammatory gene sets, especially for solid tumor treatment.

Vpx requires active cellular dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages.

HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.

Purified recombinant lentiviral Vpx proteins maintain their SAMHD1 degradation efficiency in resting CD4+ T cells.

Different protein purification methods exist. Yet, they need to be adapted for specific downstream applications to maintain functional integrity of the recombinant proteins. This study established a purification protocol for lentiviral Vpx (viral protein X) and test its ability to degrade sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) ex vivo in resting CD4+ T cells. For this purpose, we cloned a novel eukaryotic expression plasmid for Vpx including C-terminal 10x His- and HA-tags and confirmed that those tags did not alter the ability to degrade SAMHD1. We optimized purification conditions for Vpx produced in HEK293T cells with CHAPS as detergent and Co-NTA resins yielding the highest solubility and protein amounts. Size exclusion chromatography (SEC) further enhanced the purity of recombinant Vpx proteins. Importantly, nucleofection of resting CD4+ T cells demonstrated that purified recombinant Vpx protein efficiently degraded SAMHD1 in a proteasome-dependent manner. In conclusion, this protocol is suitable for functional downstream applications of recombinant Vpx and might be transferrable to other recombinant proteins with similar functions/properties as lentiviral Vpx.

Effect and significance of viral proteins regulating activity of retroelement long interspersed element 1 / 中国生物制品学杂志

@#Abstract：Long interspersed element⁃1（LINE⁃1）is the only known active and autonomously transposable retroelement in human cells，which is related to autoimmune diseases and plays important roles in activating and regulating the antiviral innate immunity of cells，especially the level of interferon（IFN）. This paper reviews the mechanisms of several non ⁃ structural proteins from human immunodeficiency virus（HIV），hepatitis B virus（HBV）and other viruses participating in the regulation of LINE ⁃ 1 activity. These mechanisms not only ensure the normal expression of viral genome，but also participate in the cellular innate immunity regulation，the inhibition of which may provide new strategies to develop treatments of diseases caused by viruses.

Viral Particle-Mediated SAMHD1 Depletion Sensitizes Refractory Glioblastoma to DNA-Damaging Therapeutics by Impairing Homologous Recombination.

The current standard-of-care treatment for glioblastoma includes DNA damaging agents, γ-irradiation (IR) and temozolomide (TMZ). These treatments fail frequently and there is limited alternative strategy. Therefore, identifying a new therapeutic target is urgently needed to develop a strategy that improves the efficacy of the existing treatments. Here, we report that tumor samples from GBM patients express a high level of SAMHD1, emphasizing SAMHD1's importance. The depletion of SAMHD1 using virus-like particles containing Vpx, VLP(+Vpx), sensitized two independent GBM cell lines (LN-229 and U-87) to veliparib, a well-established PARP inhibitor, and slowed cell growth in a dose-dependent manner. In the mouse GBM xenograft model, Vpx-mediated SAMHD1 depletion reduced tumor growth and SAMHD1 knockout (KO) improved survival. In combination with IR or TMZ, SAMHD1 KO and exposure to 50% growth inhibitory dose (gID50) of VLP(+Vpx) displayed a synergistic effect, resulting in impaired HR, and improved LN-229 cells' sensitivity to TMZ and IR. In conclusion, our finding demonstrates that SAMHD1 promotes GBM resistance to treatment, and it is a plausible therapeutic target to improve the efficacy of TMZ and IR in GBM. Furthermore, we show that Vpx could be a potential therapeutic tool that can be utilized to deplete SAMHD1 in GBM.

HIV-2/SIV Vpx antagonises NF-κB activation by targeting p65.

BACKGROUND: The NF-κB family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF-κB transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF-κB and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins targeting the NF-κB pathway to mitigate the anti-viral effects of NF-κB-dependent host immunity. RESULTS: In this study we have demonstrated using a variety of assays, in a number of different cell types including primary cells, that plasmid-encoded or virus-delivered simian immunodeficiency virus (SIV) accessory protein Vpx is a broad antagonist of NF-κB signalling active against diverse innate NF-κB agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF-κB transcription factor p65, but not NF-κB family members p50 or p100, preventing nuclear translocation of p65. We found that broad antagonism of NF-κB activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity. CONCLUSIONS: We have discovered a novel mechanism by which lentiviruses antagonise NF-κB activation by targeting p65. These findings extend our knowledge of how lentiviruses manipulate universal regulators of immunity to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists. Importantly our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of NF-κB.

Modular Lentiviral Vectors for Highly Efficient Transgene Expression in Resting Immune Cells.

Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4+ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4+ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.

SAMHD1 and Viral Ways around It.

The SAM and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase that plays a crucial role for a variety of different cellular functions. Besides balancing intracellular dNTP concentrations, facilitating DNA damage repair, and dampening excessive immune responses, SAMHD1 has been shown to act as a major restriction factor against various virus species. In addition to its well-described activity against retroviruses such as HIV-1, SAMHD1 has been identified to reduce the infectivity of different DNA viruses such as the herpesviruses CMV and EBV, the poxvirus VACV, or the hepadnavirus HBV. While some viruses are efficiently restricted by SAMHD1, others have developed evasion mechanisms that antagonize the antiviral activity of SAMHD1. Within this review, we summarize the different cellular functions of SAMHD1 and highlight the countermeasures viruses have evolved to neutralize the restriction factor SAMHD1.

Role of Intracellular Distribution of Feline and Bovine SAMHD1 Proteins in Lentiviral Restriction.

Human SAMHD1 (hSAM) restricts lentiviruses at the reverse transcription step through its dNTP triphosphohydrolase (dNTPase) activity. Besides humans, several mammalian species such as cats and cows that carry their own lentiviruses also express SAMHD1. However, the intracellular distribution of feline and bovine SAMHD1 (fSAM and bSAM) and its significance in their lentiviral restriction function is not known. Here, we demonstrated that fSAM and bSAM were both predominantly localized to the nucleus and nuclear localization signal (11KRPR14)-deleted fSAM and bSAM relocalized to the cytoplasm. Both cytoplasmic fSAM and bSAM retained the antiviral function against different lentiviruses and cytoplasmic fSAM could restrict Vpx-encoding SIV and HIV-2 more efficiently than its wild-type (WT) protein as cytoplasmic hSAM. Further investigation revealed that cytoplasmic fSAM was resistant to Vpx-induced degradation like cytoplasmic hSAM, while cytoplasmic bSAM was not, but they all demonstrated the same in vitro dNTPase activity and all could interact with Vpx as their WT proteins, indicating that cytoplasmic hSAM and fSAM can suppress more SIV and HIV-2 by being less sensitive to Vpx-mediated degradation. Our results suggested that fSAM- and bSAM-mediated lentiviral restriction does not require their nuclear localization and that fSAM shares more common features with hSAM. These findings may provide insights for the establishment of alternative animal models to study SAMHD1 in vivo.

Enhanced enzyme kinetics of reverse transcriptase variants cloned from animals infected with SIVmac239 lacking viral protein X.

HIV Type 1 (HIV-1) and simian immunodeficiency virus (SIV) display differential replication kinetics in macrophages. This is because high expression levels of the active host deoxynucleotide triphosphohydrolase sterile α motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) deplete intracellular dNTPs, which restrict HIV-1 reverse transcription, and result in a restrictive infection in this myeloid cell type. Some SIVs overcome SAMHD1 restriction using viral protein X (Vpx), a viral accessory protein that induces proteasomal degradation of SAMHD1, increasing cellular dNTP concentrations and enabling efficient proviral DNA synthesis. We previously reported that SAMHD1-noncounteracting lentiviruses may have evolved to harbor RT proteins that efficiently polymerize DNA, even at low dNTP concentrations, to circumvent SAMHD1 restriction. Here we investigated whether RTs from SIVmac239 virus lacking a Vpx protein evolve during in vivo infection to more efficiently synthesize DNA at the low dNTP concentrations found in macrophages. Sequence analysis of RTs cloned from Vpx (+) and Vpx (-) SIVmac239-infected animals revealed that Vpx (-) RTs contained more extensive mutations than Vpx (+) RTs. Although the amino acid substitutions were dispersed indiscriminately across the protein, steady-state and pre-steady-state analysis demonstrated that selected SIVmac239 Vpx (-) RTs are characterized by higher catalytic efficiency and incorporation efficiency values than RTs cloned from SIVmac239 Vpx (+) infections. Overall, this study supports the possibility that the loss of Vpx may generate in vivo SIVmac239 RT variants that can counteract the limited availability of dNTP substrate in macrophages.

Lentiviral-Vector-Based Dendritic Cell Vaccine Synergizes with Checkpoint Blockade to Clear Chronic Viral Infection.

Dendritic cell vaccines are a promising strategy for the treatment of cancer and infectious diseases but have met with mixed success. We report on a lentiviral vector-based dendritic cell vaccine strategy that generates a cluster of differentiation 8 (CD8) T cell response that is much stronger than that achieved by standard peptide-pulsing approaches. The strategy was tested in the mouse lymphocytic choriomeningitis virus (LCMV) model. Bone marrow-derived dendritic cells from SAMHD1 knockout mice were transduced with a lentiviral vector expressing the GP33 major-histocompatibility-complex (MHC)-class-I-restricted peptide epitope and CD40 ligand (CD40L) and injected into wild-type mice. The mice were highly protected against acute and chronic variant CL-13 LCMVs, resulting in a 100-fold greater decrease than that achieved with peptide epitope-pulsed dendritic cells. Inclusion of an MHC-class-II-restricted epitope in the lentiviral vector further increased the CD8 T cell response and resulted in antigen-specific CD8 T cells that exhibited a phenotype associated with functional cytotoxic T cells. The vaccination synergized with checkpoint blockade to reduce the viral load of mice chronically infected with CL-13 to an undetectable level. The strategy improves upon current dendritic cell vaccine strategies; is applicable to the treatment of disease, including AIDS and cancer; and supports the utility of Vpx-containing vectors.

The stability of HIV-2 Vpx and Vpr proteins is regulated by the presence or absence of zinc-binding sites and poly-proline motifs with distinct roles.

The Vpx and Vpr proteins of human immunodeficiency virus type 2 (HIV-2) are important for virus replication. Although these proteins are homologous, Vpx is expressed at much higher levels than Vpr. Previous studies demonstrated that this difference results from the presence of an HHCC zinc-binding site in Vpx that is absent in Vpr. Vpx has another unique region, a poly-proline motif (PPM) of seven consecutive prolines at the C-terminus. Using PPM point mutants of Vpx, this study demonstrated that these seven consecutive prolines are critical for suppressing proteasome degradation of Vpx in the absence of Gag. Both the PPM and the zinc-binding site stabilize Vpx but do so via different mechanisms. PPM and zinc-binding site mutants overexpressed in Escherichia coli aggregated readily, indicating that these motifs normally prevent exposure of the hydrophobic region outside the structure. Furthermore, introduction of the zinc-binding site and the PPM into Vpr increased the level of Vpr expression so that it was as high as that of Vpx. Intriguingly, HIV-2 has evolved to express Vpx at high levels and Vpr at low levels based on the presence and absence of these two motifs with distinct roles.

The C-terminal domain of feline and bovine SAMHD1 proteins has a crucial role in lentiviral restriction.

SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.

Viral protein X reduces the incorporation of mutagenic noncanonical rNTPs during lentivirus reverse transcription in macrophages.

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain- and HD domain-containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase-mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.

Efficient pre-catalytic conformational change of reverse transcriptases from SAMHD1 non-counteracting primate lentiviruses during dNTP incorporation.

Unlike HIV-1, HIV-2 and some SIV strains replicate at high dNTP concentrations even in macrophages due to their accessory proteins, Vpx or Vpr, that target SAMHD1 dNTPase for proteasomal degradation. We previously reported that HIV-1 reverse transcriptase (RT) efficiently synthesizes DNA even at low dNTP concentrations because HIV-1 RT displays faster pre-steady state kpol values than SAMHD1 counteracting lentiviral RTs. Here, since the kpol step consists of two sequential sub-steps post dNTP binding, conformational change and chemistry, we investigated which of the two sub-steps RTs from SAMHD1 non-counteracting viruses accelerate in order to complete reverse transcription in the limited dNTP pools found in macrophages. Our study demonstrates that RTs of SAMHD1 non-counteracting lentiviruses have a faster conformational change rate during dNTP incorporation, supporting that these lentiviruses may have evolved to harbor RTs that can efficiently execute the conformational change step in order to circumvent SAMHD1 restriction and dNTP depletion in macrophages.

Interference with SAMHD1 Restores Late Gene Expression of Modified Vaccinia Virus Ankara in Human Dendritic Cells and Abrogates Type I Interferon Expression.

Attenuated poxviruses like modified vaccinia virus Ankara (MVA) are promising vectors for vaccines against infectious diseases and cancer. However, host innate immune responses interfere with the viral life cycle and also influence the immunogenicity of vaccine vectors. Sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a phosphohydrolase and reduces cellular deoxynucleoside triphosphate (dNTP) concentrations, which impairs poxviral DNA replication in human dendritic cells (DCs). Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) encode an accessory protein called viral protein X (Vpx) that promotes proteasomal degradation of SAMHD1, leading to a rapid increase in cellular dNTP concentrations. To study the function of SAMHD1 during MVA infection of human DCs, the SIV vpx gene was introduced into the MVA genome (resulting in recombinant MVA-vpx). Infection of human DCs with MVA-vpx led to SAMHD1 protein degradation and enabled MVA-vpx to replicate its DNA genome and to express genes controlled by late promoters. Late gene expression by MVA-vpx might improve its vaccine vector properties; however, type I interferon expression was unexpectedly blocked by Vpx-expressing MVA. MVA-vpx can be used as a tool to study poxvirus-host interactions and vector safety.IMPORTANCE SAMHD1 is a phosphohydrolase and reduces cellular dNTP concentrations, which impairs poxviral DNA replication. The simian SIV accessory protein Vpx promotes degradation of SAMHD1, leading to increased cellular dNTP concentrations. Vpx addition enables poxviral DNA replication in human dendritic cells (DCs), as well as the expression of viral late proteins, which is normally blocked. SAMHD1 function during modified vaccinia virus Ankara (MVA) infection of human DCs was studied with recombinant MVA-vpx expressing Vpx. Infection of human DCs with MVA-vpx decreased SAMHD1 protein amounts, enabling MVA DNA replication and expression of late viral genes. Unexpectedly, type I interferon expression was blocked after MVA-vpx infection. MVA-vpx might be a good tool to study SAMHD1 depletion during poxviral infections and to provide insights into poxvirus-host interactions.

Promiscuous Targeting of Cellular Proteins by Vpr Drives Systems-Level Proteomic Remodeling in HIV-1 Infection.

HIV-1 encodes four "accessory proteins" (Vif, Vpr, Vpu, and Nef), dispensable for viral replication in vitro but essential for viral pathogenesis in vivo. Well characterized cellular targets have been associated with Vif, Vpu, and Nef, which counteract host restriction and promote viral replication. Conversely, although several substrates of Vpr have been described, their biological significance remains unclear. Here, we use complementary unbiased mass spectrometry-based approaches to demonstrate that Vpr is both necessary and sufficient for the DCAF1/DDB1/CUL4 E3 ubiquitin ligase-mediated degradation of at least 38 cellular proteins, causing systems-level changes to the cellular proteome. We therefore propose that promiscuous targeting of multiple host factors underpins complex Vpr-dependent cellular phenotypes and validate this in the case of G2/M cell cycle arrest. Our model explains how Vpr modulates so many cell biological processes and why the functional consequences of previously described Vpr targets, identified and studied in isolation, have proved elusive.