Novel Arenavirus Entry Inhibitors Discovered by Using a Minigenome Rescue System for High-Throughput Drug Screening.

UNLABELLED: Certain members of the Arenaviridae family are category A agents capable of causing severe hemorrhagic fevers in humans. Specific antiviral treatments do not exist, and the only commonly used drug, ribavirin, has limited efficacy and can cause severe side effects. The discovery and development of new antivirals are inhibited by the biohazardous nature of the viruses, making them a relatively poorly understood group of human pathogens. We therefore adapted a reverse-genetics minigenome (MG) rescue system based on Junin virus, the causative agent of Argentine hemorrhagic fever, for high-throughput screening (HTS). The MG rescue system recapitulates all stages of the virus life cycle and enables screening of small-molecule libraries under biosafety containment level 2 (BSL2) conditions. The HTS resulted in the identification of four candidate compounds with potent activity against a broad panel of arenaviruses, three of which were completely novel. The target for all 4 compounds was the stage of viral entry, which positions the compounds as potentially important leads for future development. IMPORTANCE: The arenavirus family includes several members that are highly pathogenic, causing acute viral hemorrhagic fevers with high mortality rates. No specific effective treatments exist, and although a vaccine is available for Junin virus, the causative agent of Argentine hemorrhagic fever, it is licensed for use only in areas where Argentine hemorrhagic fever is endemic. For these reasons, it is important to identify specific compounds that could be developed as antivirals against these deadly viruses.

Determinants in HIV-2 Env and tetherin required for functional interaction.

BACKGROUND: The interferon-inducible factor BST-2/tetherin blocks the release of nascent virions from the surface of infected cells for certain enveloped virus families. The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein. We sought to further understand the features of the Env protein and tetherin that are important for this interaction, and to evaluate the selective pressure on HIV-2 to maintain such an activity. RESULTS: By examining Env mutants with changes in the ectodomain of the protein (virus ROD14) or the cytoplasmic tail (substitution Y707A) that render the proteins unable to counteract tetherin, we determined that an interaction between Env and tetherin is important for this activity. Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env. The replication of virus carrying the ROD14 substitutions was significantly slower than the matched wild-type virus, but it acquired second-site mutations during passaging in the cytoplasmic tail of Env which restored the ability of the protein to both bind to and counteract tetherin. CONCLUSIONS: These results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.

Pre-clinical modeling of CCR5 knockout in human hematopoietic stem cells by zinc finger nucleases using humanized mice.

Genetic strategies to block expression of CCR5, the major co-receptor of human immunodeficiency virus type 1 (HIV-1), are being developed as anti-HIV therapies. For example, human hematopoietic stem/precursor cells (HSPC) can be modified by the transient expression of CCR5-targeted zinc finger nucleases (ZFNs) to generate CCR5-negative cells, which could then give rise to HIV-resistant mature CD4(+) T cells following transplantation into patients. The safety and anti-HIV effects of such treatments can be evaluated by transplanting ZFN-treated HSPC into immunodeficient mice, where the extent of human cell engraftment, lineage differentiation and anti-HIV activity arising from the engineered HSPC can be examined. In this way, humanized mice are providing a powerful small animal model for pre-clinical studies of novel anti-HIV therapies.

Anti-tetherin activities of HIV-1 Vpu and Ebola virus glycoprotein do not involve removal of tetherin from lipid rafts.

BST-2/tetherin is an interferon-inducible host restriction factor that blocks the release of newly formed enveloped viruses. It is enriched in lipid raft membrane microdomains, which are also the sites of assembly of several enveloped viruses. Viral anti-tetherin factors, such as the HIV-1 Vpu protein, typically act by removing tetherin from the cell surface. In contrast, the Ebola virus glycoprotein (GP) is unusual in that it blocks tetherin restriction without apparently altering its cell surface localization. We explored the possibility that GP acts to exclude tetherin from the specific sites of virus assembly without overtly removing it from the cell surface and that lipid raft exclusion is the mechanism involved. However, we found that neither GP nor Vpu had any effect on tetherin's distribution within lipid raft domains. Furthermore, GP did not prevent the colocalization of tetherin and budding viral particles. Contrary to previous reports, we also found no evidence that GP is itself a raft protein. Together, our data indicate that the exclusion of tetherin from lipid rafts is not the mechanism used by either HIV-1 Vpu or Ebola virus GP to counteract tetherin restriction.

Substitutions in the glycoprotein (GP) of the Candid#1 vaccine strain of Junin virus increase dependence on human transferrin receptor 1 for entry and destabilize the metastable conformation of GP.

Candid#1 (Cd1) is an attenuated vaccine strain of Junin virus, the causative agent of Argentine hemorrhagic fever. Although several substitutions are present in Cd1, their importance for attenuation has not been established. We functionally characterized the substitutions present in the Cd1 glycoprotein (GP) and identified F427I in the transmembrane domain of the GP2 subunit as reducing infectivity in a reconstituted viral system. We further showed that this phenotype derives from the destabilization of the GP metastable conformation. Lastly, we identified an increased dependence of Cd1 GP on human transferrin receptor type 1 (hTfR-1) for entry, which may affect the tropism of the attenuated strain in vivo.

Lack of adaptation to human tetherin in HIV-1 group O and P.

BACKGROUND: HIV-1 viruses are categorized into four distinct groups: M, N, O and P. Despite the same genomic organization, only the group M viruses are responsible for the world-wide pandemic of AIDS, suggesting better adaptation to human hosts. Previously, it has been reported that the group M Vpu protein is capable of both down-modulating CD4 and counteracting BST-2/tetherin restriction, while the group O Vpu cannot antagonize tetherin. This led us to investigate if group O, and the related group P viruses, possess functional anti-tetherin activities in Vpu or another viral protein, and to further map the residues required for group M Vpu to counteract human tetherin. RESULTS: We found a lack of activity against human tetherin for both the Vpu and Nef proteins from group O and P viruses. Furthermore, we found no evidence of anti-human tetherin activity in a fully infectious group O proviral clone, ruling out the possibility of an alternative anti-tetherin factor in this virus. Interestingly, an activity against primate tetherins was retained in the Nef proteins from both a group O and a group P virus. By making chimeras between a functional group M and non-functional group O Vpu protein, we were able to map the first 18 amino acids of group M Vpu as playing an essential role in the ability of the protein to antagonize human tetherin. We further demonstrated the importance of residue alanine-18 for the group M Vpu activity. This residue lies on a diagonal face of conserved alanines in the TM domain of the protein, and is necessary for specific Vpu-tetherin interactions. CONCLUSIONS: The absence of human specific anti-tetherin activities in HIV-1 group O and P suggests a failure of these viruses to adapt to human hosts, which may have limited their spread.

Ebola virus glycoprotein counteracts BST-2/Tetherin restriction in a sequence-independent manner that does not require tetherin surface removal.

BST-2/tetherin is an interferon-inducible protein that restricts the release of enveloped viruses from the surface of infected cells by physically linking viral and cellular membranes. It is present at both the cell surface and in a perinuclear region, and viral anti-tetherin factors including HIV-1 Vpu and HIV-2 Env have been shown to decrease the cell surface population. To map the domains of human tetherin necessary for both virus restriction and sensitivity to viral anti-tetherin factors, we constructed a series of tetherin derivatives and assayed their activity. We found that the cytoplasmic tail (CT) and transmembrane (TM) domains of tetherin alone produced its characteristic cellular distribution, while the ectodomain of the protein, which includes a glycosylphosphatidylinositol (GPI) anchor, was sufficient to restrict virus release when presented by the CT/TM regions of a different type II membrane protein. To counteract tetherin restriction and remove it from the cell surface, HIV-1 Vpu required the specific sequence present in the TM domain of human tetherin. In contrast, the HIV-2 Env required only the ectodomain of the protein and was sensitive to a point mutation in this region. Strikingly, the anti-tetherin factor, Ebola virus GP, was able to overcome restriction conferred by both tetherin and a series of functional tetherin derivatives, including a wholly artificial tetherin molecule. Moreover, GP overcame restriction without significantly removing tetherin from the cell surface. These findings suggest that Ebola virus GP uses a novel mechanism to circumvent tetherin restriction.

HIV-1 Vpu and HIV-2 Env counteract BST-2/tetherin by sequestration in a perinuclear compartment.

BACKGROUND: In the absence of the Vpu protein, newly formed HIV-1 particles can remain attached to the surface of human cells due to the action of an interferon-inducible cellular restriction factor, BST-2/tetherin. Tetherin also restricts the release of other enveloped viral particles and is counteracted by a several viral anti-tetherin factors including the HIV-2 Env, SIV Nef and KSHV K5 proteins. RESULTS: We observed that a fraction of tetherin is located at the surface of restricting cells, and that co-expression of both HIV-1 Vpu and HIV-2 Env reduced this population. In addition, Vpu, but not the HIV-2 Env, reduced total cellular levels of tetherin. An additional effect observed for both Vpu and the HIV-2 Env was to redirect tetherin to an intracellular perinuclear compartment that overlapped with markers for the TGN (trans-Golgi network). Sequestration of tetherin in this compartment was independent of tetherin's normal endocytosis trafficking pathway. CONCLUSIONS: Both HIV-1 Vpu and HIV-2 Env redirect tetherin away from the cell surface and sequester the protein in a perinuclear compartment, which likely blocks the action of this cellular restriction factor. Vpu also promotes the degradation of tetherin, suggesting that it uses more than one mechanism to counteract tetherin restriction.

Anti-tetherin activities in Vpu-expressing primate lentiviruses.

BACKGROUND: The anti-viral activity of the cellular restriction factor, BST-2/tetherin, was first observed as an ability to block the release of Vpu-minus HIV-1 from the surface of infected cells. However, tetherin restriction is also counteracted by primate lentiviruses that do not express a Vpu protein, where anti-tetherin functions are provided by either the Env protein (HIV-2, SIVtan) or the Nef protein (SIVsm/mac and SIVagm). Within the primate lentiviruses, Vpu is also present in the genomes of SIVcpz and certain SIVsyk viruses. We asked whether, in these viruses, anti-tetherin activity was always a property of Vpu, or if it had selectively evolved in HIV-1 to perform this function. RESULTS: We found that despite the close relatedness of HIV-1 and SIVcpz, the chimpanzee viruses use Nef instead of Vpu to counteract tetherin. Furthermore, SIVcpz Nef proteins had activity against chimpanzee but not human tetherin. This specificity mapped to a short sequence that is present in the cytoplasmic tail of primate but not human tetherins, and this also accounts for the specificity of SIVsm/mac Nef for primate but not human tetherins. In contrast, Vpu proteins from four diverse members of the SIVsyk lineage all displayed an anti-tetherin activity that was active against macaque tetherin. Interestingly, Vpu from a SIVgsn isolate was also found to have activity against human tetherin. CONCLUSIONS: Primate lentiviruses show a high degree of flexibility in their use of anti-tetherin factors, indicating a strong selective pressure to counteract tetherin restriction. The identification of an activity against human tetherin in SIVgsn Vpu suggests that the presence of Vpu in the ancestral SIVmus/mon/gsn virus believed to have contributed the 3' half of the HIV-1 genome may have played a role in the evolution of viruses that could counteract human tetherin and infect humans.

Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of sufficient Vif to allow efficient HIV-1 replication in cells expressing relatively high levels of A3G.

Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.

Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune-deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome-editing technologies in HSPCs.