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

<p>Abstract</p> <p>Background</p> <p>The 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.</p> <p>Results</p> <p>We 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.</p> <p>Conclusions</p> <p>Similar 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 <it>bona fide </it>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.</p

Receptor use by pathogenic arenaviruses

The arenavirus family contains several important human pathogens including Lassa fever virus (LASV), lymphocytic choriomeningitis virus (LCMV) and the New World clade B viruses Junin (JUNV) and Machupo (MACV). Previously, α-dystroglycan (α-DG) was identified as a receptor recognized by LASV and certain strains of LCMV. However, other studies have suggested that α-DG is probably not used by the clade B viruses, and the receptor(s) for these pathogens is currently unknown. Using pseudotyped retroviral vectors displaying arenavirus glycoproteins (GPs), we are able to explore the role played by the GP in viral entry in the absence of other viral proteins. By examining the ability of the vectors to transduce DG knockout murine embryonic stem (ES) cells, we have confirmed that LASV has an absolute requirement for α-DG in these cells. However, the LCMV GP can still direct substantial entry into murine ES cells in the absence of α-DG, even when the GP from the clone 13 variant is used that has previously been reported to be highly dependent on α-DG for entry. We also found that neither LASV or LCMV pseudotyped vectors were able to transduce human or murine lymphocytes, presumably due to the glycosylation state of α-DG in these cells. In contrast, the JUNV and MACV GPs displayed broad tropism on human, murine and avian cell types, including lymphocytes, and showed no requirement for α-DG in murine ES cells. These findings highlight the importance of molecules other than α-DG for arenavirus entry. An alternate receptor is present on murine ES cells that can be used by LCMV but not by LASV, and which is not available on human or murine lymphocytes, while a distinct and widely expressed receptor(s) is used by the clade B viruses.Facultad de Ciencias Exacta

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 (<it>trans</it>-Golgi network). Sequestration of tetherin in this compartment was independent of tetherin's normal endocytosis trafficking pathway.</p> <p>Conclusions</p> <p>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.</p

Receptor use by pathogenic arenaviruses

The arenavirus family contains several important human pathogens including Lassa fever virus (LASV), lymphocytic choriomeningitis virus (LCMV) and the New World clade B viruses Junin (JUNV) and Machupo (MACV). Previously, α-dystroglycan (α-DG) was identified as a receptor recognized by LASV and certain strains of LCMV. However, other studies have suggested that α-DG is probably not used by the clade B viruses, and the receptor(s) for these pathogens is currently unknown. Using pseudotyped retroviral vectors displaying arenavirus glycoproteins (GPs), we are able to explore the role played by the GP in viral entry in the absence of other viral proteins. By examining the ability of the vectors to transduce DG knockout murine embryonic stem (ES) cells, we have confirmed that LASV has an absolute requirement for α-DG in these cells. However, the LCMV GP can still direct substantial entry into murine ES cells in the absence of α-DG, even when the GP from the clone 13 variant is used that has previously been reported to be highly dependent on α-DG for entry. We also found that neither LASV or LCMV pseudotyped vectors were able to transduce human or murine lymphocytes, presumably due to the glycosylation state of α-DG in these cells. In contrast, the JUNV and MACV GPs displayed broad tropism on human, murine and avian cell types, including lymphocytes, and showed no requirement for α-DG in murine ES cells. These findings highlight the importance of molecules other than α-DG for arenavirus entry. An alternate receptor is present on murine ES cells that can be used by LCMV but not by LASV, and which is not available on human or murine lymphocytes, while a distinct and widely expressed receptor(s) is used by the clade B viruses.Facultad de Ciencias Exacta

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

AbstractCorrection A confocal image in Figure 3B of Hauser et al. 2010, Retrovirology 2010, 7:51 ), showing TGN staining of Vphu-HcRed expressing cells (middle row), was incorrect. This image has now been replaced with the correct image

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▿

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

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

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