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.

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.

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.

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

BACKGROUND: 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. RESULTS: 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 beta 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. CONCLUSIONS: Similar to the mechanism whereby Vpu targets BST-2/tetherin for degradation, these findings implicate beta-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.

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.

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, alpha-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.

Receptor use by the Whitewater Arroyo virus glycoprotein.

Whitewater Arroyo virus (WWAV) is a North American New World arenavirus, first isolated from rats in New Mexico in 1993, and tentatively associated with three human fatalities in California in 1999-2000. However, it remains unclear whether WWAV was the cause of these, or any other, human infections. One important characteristic of viruses that influences pathogenic potential is the choice of cellular receptor and the corresponding tropism of the virus. In the arenaviruses, these properties are determined largely by the viral glycoprotein (GP). We have previously noted for the New World clade B arenaviruses, which include four severe human pathogens, that the ability to cause human disease correlates with the ability of the GP to use the human transferrin receptor 1 (hTfR1) to enter cells. In addition, pseudotyped retroviral vectors displaying the GPs from pathogenic clade B viruses transduced a range of cell lines in vitro that were distinct from those that could be transduced by non-pathogenic clade B viruses. WWAV was initially classified as a New World clade A virus, based on sequence analysis of its nucleoprotein gene. However, more extensive analyses have revealed that WWAV and the other North American arenaviruses are probably recombinant clade A/B viruses, and that the WWAV GP is more closely related to the clade B GPs. Based on this finding, we sought to understand more about the possible pathogenic potential of WWAV by determining whether its clade B-like GP exhibited the characteristics of a pathogenic or non-pathogenic clade B virus. Our studies found that WWAV GP did not use hTfR1 for entry, and that its overall in vitro tropism was most similar to the GPs from the non-pathogenic clade B viruses. Although many viral factors in addition to GP receptor use and tropism determine whether a virus is able to cause disease in humans, our analysis of the WWAV GP does not support the idea that WWAV is a human pathogen.

Differences in tropism and pH dependence for glycoproteins from the Clade B1 arenaviruses: implications for receptor usage and pathogenicity.

The Clade B lineage of the New World arenaviruses contains four viruses capable of causing severe hemorrhagic fevers in humans. Within this group, the B1 sub-lineage contains the pathogenic viruses Junin (JUNV) and Machupo (MACV), as well as the non-pathogenic Tacaribe virus (TCRV). In order to elucidate differences that may determine pathogenicity, we studied the entry pathways directed by the glycoproteins (GPs) from these related B1 viruses, using pseudotyped retroviral vectors and GP1 immunoadhesin constructs. Our data revealed variations in the efficiency with which different cell types could be transduced by B1 vectors, and this correlated with the ability of the immunoadhesins to bind to those cells. Interestingly, the tropism directed by the TCRV GP proved to be distinct from that of JUNV and MACV, in particular on lymphocyte cell lines. In addition, the GPs showed variations in their sensitivity to an inhibitor of endosome acidification, with the TCRV GP again being the outlier. Together these data suggest that more than one entry pathway can be used by these closely related viruses and that the ability to cause human disease may be highly dependent on receptor usage.

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, alpha-dystroglycan (alpha-DG) was identified as a receptor recognized by LASV and certain strains of LCMV. However, other studies have suggested that alpha-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 alpha-DG in these cells. However, the LCMV GP can still direct substantial entry into murine ES cells in the absence of alpha-DG, even when the GP from the clone 13 variant is used that has previously been reported to be highly dependent on alpha-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 alpha-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 alpha-DG in murine ES cells. These findings highlight the importance of molecules other than alpha-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.