Jill Oldenburg

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

BackgroundIn 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.ResultsWe 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 tetherins normal endocytosis trafficking pathway.ConclusionsBoth 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.

Ebola Virus Glycoprotein Counteracts BST-2/Tetherin Restriction in a Sequence-Independent Manner That Does Not Require Tetherin Surface Removal

ABSTRACT 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.

New World Clade B Arenaviruses Can Use Transferrin Receptor 1 (TfR1)-Dependent and -Independent Entry Pathways, and Glycoproteins from Human Pathogenic Strains Are Associated with the Use of TfR1

ABSTRACT Arenaviruses are rodent-borne viruses, with five members of the family capable of causing severe hemorrhagic fevers if transmitted to humans. To date, two distinct cellular receptors have been identified that are used by different pathogenic viruses, α-dystroglycan by Lassa fever virus and transferrin receptor 1 (TfR1) by certain New World clade B viruses. Our previous studies have suggested that other, as-yet-unknown receptors are involved in arenavirus entry. In the present study, we examined the use of TfR1 by the glycoproteins (GPs) from a panel of New World clade B arenaviruses comprising three pathogenic and two nonpathogenic strains. Interestingly, we found that TfR1 was only used by the GPs from the pathogenic viruses, with entry of the nonpathogenic strains being TfR1 independent. The pathogenic GPs could also direct entry into cells by TfR1-independent pathways, albeit less efficiently. A comparison of the abilities of TfR1 orthologs from different species to support arenavirus entry found that the human and feline receptors were able to enhance entry of the pathogenic strains, but that neither the murine or canine forms were functional. Since the ability to use TfR1 is a characteristic feature of the human pathogens, this interaction may represent an important target in the treatment of New World hemorrhagic fevers. In addition, the ability to use TfR1 may be a useful tool to predict the likelihood that any existing or newly discovered viruses in this family could infect humans.

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

ABSTRACT 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.

Tissue-specific restriction of cyclophilin A-independent HIV-1- and SIV-derived lentiviral vectors

The host factor α isoform of the tripartite motif 5 (TRIM5α) restricts human immunodeficiency virus type 1 (HIV-1) infection in certain non-human primate species. Restriction of HIV-1 is enhanced by binding of the viral capsid to cyclophilin A (CypA) in target cells, although CypA is not absolutely required for restriction in rhesus macaque cells. Simian immunodeficiency virus (SIV) is not restricted by rhesus macaque TRIM5α and its capsid does not bind to CypA. Here, the effect of lentiviral CypA dependence on restriction in different tissues was examined by engineering an HIV-1 capsid quadruple mutant (V86P/H87Q/I91V/M96I) lentiviral vector (HIVquad) that is CypA-independent. Whereas HIV-1 was restricted in rhesus macaque and owl monkey epithelial cells, infection with the HIVquad vector was efficient at high viral concentrations. In contrast, HIVquad was largely restricted in primary rhesus macaque CD34+ cells. Human epithelial and primary CD34+ cells were permissive for HIV-1, HIVquad and SIV, whereas transduction of human T cells by HIVquad or SIV was impaired. The restrictive human cells did not express increased levels of TRIM5α, and restriction was not relieved by abolishing CypA, consistent with HIVquad and SIV being CypA-independent. Pseudotyping of lentiviral vectors with the gibbon ape leukemia virus envelope altered their sensitivity to perturbations of the virus–CypA interaction compared to pseudotyping with vesicular stomatitis virus glycoproteins, suggesting that the viral entry pathway modulates restriction. Together, these studies reveal that an HIV-1 capsid quadruple mutant can partially overcome lentiviral restriction in non-human primate epithelial cells, but not in hematopoietic cells. Similarly, human cells vary in their permissiveness for CypA-independent lentiviruses, and suggest the presence of tissue-specific factor(s) that can inhibit lentiviral transduction independently of viral interaction with TRIM5α and CypA.

Vpu-dependent block to incorporation of GaLV Env into lentiviral vectors

BackgroundThe gibbon ape leukemia virus (GaLV) Env protein mediates entry into a wide range of human cells and is frequently used to pseudotype retroviral vectors. However, an incompatibility exists between GaLV Env and lentiviral vectors that results in decreased steady-state levels of the mature GaLV Env in cells and prevents its incorporation into lentiviral vector particles.ResultsWe identified the HIV-1 Vpu protein as the major cause of the depletion in GaLV Env levels that occurs when lentiviral vector components are present. This activity of Vpu targeted the mature (cleaved) form of the GaLV Env that exists within or beyond the trans-Golgi. The activity required two conserved phospho-serines in the cytoplasmic tail of Vpu that are known to recruit β TrCP, a substrate adaptor for an SCF E3 ubiquitin ligase complex, and could be blocked by mutation of lysine 618 in the GaLV Env tail. Moreover, the Vpu-mediated decrease of GaLV Env levels was inhibited by the lysosomal inhibitor, bafilomycin A1. Interestingly, this activity of Vpu was only observed in the presence of other lentiviral vector components.ConclusionsSimilar to the mechanism whereby Vpu targets BST-2/tetherin for degradation, these findings implicate β-TrCP-mediated ubiquitination and the endo-lysosomal pathway in the degradation of the GaLV Env by lentiviral vector components. Possibly, the cytoplasmic tail of the GaLV Env contains features that mimic bona fide targets of Vpu, important to HIV-1 replication. Furthermore, the lack of effect of Vpu on GaLV Env in the absence of other HIV-1 proteins, suggests that a more complex interaction may exist between Vpu and its target proteins, with the additional involvement of one or more component(s) of the HIV-1 replication machinery.

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

ABSTRACT Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.

Zinc Finger Nuclease Editing of Hematopoietic Stem Cells as an Anti-HIV Therapy

Hematopoietic stem cell (HSC) transplanted humanized mice are a valuable small animal model for preclinical testing of HSC-based gene therapies, since the engraftment and subsequent differentiation of the cells in the mice allows a rigorous assessment of whether the genetic manipulation in any way impacts HSC function. In addition, since the HSC give rise to human CD4+ T cells, the mice can support an HIV-1 infection. This means that the mice are particularly suited to the evaluation of anti-HIV gene therapies, where the actual target human cell and gene therapy reagents can be evaluated in a system that supports infection by the authentic human virus. In this chapter we review the role played by humanized mouse models in the preclinical development of a promising anti-HIV approach based on disruption of the human CCR5 gene in human HSC, using zinc finger nuclease editing.