Infection of XC cells by MLVs and Ebola virus is endosome-dependent but acidification-independent

Inhibitors of endosome acidification or cathepsin proteases attenuated infections mediated by envelope proteins of xenotropic murine leukemia virus-related virus (XMRV) and Ebola virus, as well as ecotropic, amphotropic, polytropic, and xenotropic murine leukemia viruses (MLVs), indicating that infections by these viruses occur through acidic endosomes and require cathepsin proteases in the susceptible cells such as TE671 cells. However, as previously shown, the endosome acidification inhibitors did not inhibit these viral infections in XC cells. It is generally accepted that the ecotropic MLV infection in XC cells occurs at the plasma membrane. Because cathepsin proteases are activated by low pH in acidic endosomes, the acidification inhibitors may inhibit the viral infections by suppressing cathepsin protease activation. The acidification inhibitors attenuated the activities of cathepsin proteases B and L in TE671 cells, but not in XC cells. Processing of cathepsin protease L was suppressed by the acidification inhibitor in NIH3T3 cells, but again not in XC cells. These results indicate that cathepsin proteases are activated without endosome acidification in XC cells. Treatment with an endocytosis inhibitor or knockdown of dynamin 2 expression by siRNAs suppressed MLV infections in all examined cells including XC cells. Furthermore, endosomal cathepsin proteases were required for these viral infections in XC cells as other susceptible cells. These results suggest that infections of XC cells by the MLVs and Ebola virus occur through endosomes and pH-independent cathepsin activation induces pH-independent infection in XC cells.     

Isolation of an RD-114-Like Oncornavirus from a Cat Cell Line

A clone of cells derived from a continuous line of cat cells (CCC) spontaneously produced an RNA C-type virus (CCC virus) which did not have the group-specific antigen of the standard strains of feline leukemia viruses but did have that of the RD-114 virus. Single-hit infection of a virus yielding CCC cell with only the feline leukemia virus pseudotype of murine sarcoma virus [MSV(FeLV)] resulted in the release of a pseudotype of MSV coated with the CCC virus envelope. Host range, transmission of virus, helper functions, interference properties, and specific neutralization showed that the CCC and the RD-114 isolates as well as their respective MSV pseudotypes are closely similar if not identical. Parental, virus-negative cells frozen before the existence of RD-114 were chemically induced to yield CCC-like virus de novo. Infection of susceptible human cells with the chemically induced virus resulted in interference with the CCC virus pseudotype of MSV but not with the FeLV pseudotype of MSV.     

Primate gammaretroviruses require an ancillary factor not required for murine gammaretroviruses to infect BHK cells

BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice.     

Glycosylation-dependent inactivation of the ecotropic murine leukemia virus receptor.

The ecotropic murine leukemia virus (E-MuLV) receptor expressed on Mus dunni tail fibroblast (MDTF) cells is a receptor for all E-MuLVs with the notable of Moloney murine leukemia virus (Mo-MuLV). Substitution of isoleucine for valine at position 214 in the third extracellular region (the putative E-MuLV binding site) of the MDTF receptor molecule allows this molecule to function as a Mo-MuLV receptor (M.V. Eiden, K. Farrell, J. Warsowe, L. A. Mahan, and C. A. Wilson, J. Virol. 67:4056-4061, 1993). We have now determined that treating MDTF cells with tunicamycin, an inhibitor of N-linked glycosylation, also renders them susceptible to Mo-MuLV infection. Two potential N-linked glycosylation sites are present in the third extracellular regions of both the NIH 3T3 and MDTF ecotropic receptors. The glycosylation site at position 229 of the MDTF receptor cDNA was eliminated by substituting a threonine codon for the asparagine codon. Mo-MuLV-resistant human HOS cells, expressing this form of the receptor, are susceptible to Mo-MuLV infection. Thus, our studies suggest that without a glycan moiety at position 229, the valine residue at 214 is no longer restrictive for Mo-MuLV infection. BHK-21 and CHO K1 hamster cells also express glycosylation-inactivated forms of the ecotropic receptor. Sequence analysis of these receptors together with our analysis of MDTF receptor function suggests that a single asparagine-linked glycosylation site is responsible for glycosylation inactivation of these receptors.     

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.     

Reevaluation of host ranges of feline leukemia virus subgroups

We reevaluated the host ranges of feline leukemia virus (FeLV) subgroups A, B and C using pseudotype assays based on recombinant NB-tropic murine leukemia virus, which is not usually blocked after viral entry in mammalian cells. Pseudotype viruses of FeLV-B and -C infected a variety of cell lines from many mammalian species. Unexpectedly, FeLV-A pseudotype viruses of two independent isolates from the UK and US also infected a variety of non-feline cell lines including cells from humans, rabbits, pigs and minks. Moreover, both isolates of FeLV-A productively infected human embryonic kidney 293 and mink Mv-1-Lu cells. We conclude that FeLV-A is not strictly ecotropic.     

Functional Murine Leukemia Virus Vectors Pseudotyped with the Visna Virus Envelope Show Expanded Visna Virus Cell Tropism

Pseudotype virus vectors serve as a powerful tool for the study of virus receptor usage and entry. We describe the development of murine leukemia virus (MuLV) particles pseudotyped with the visna virus envelope glycoprotein and encoding a green fluorescent protein reporter as a tool to study the expression of the visna virus receptor. Functional MuLV/visna virus pseudotypes were obtained when the cytoplasmic tail of the visna virus envelope TM protein was truncated to 3, 7, or 11 amino acids in length. MuLV/visna virus particles were used to transduce a panel of cell types from various organisms, including sheep, goat, human, hamster, mouse, monkey, and quail. The majority of the cells examined were susceptible to MuLV/visna pseudotype viruses, supporting the notion that the visna virus cellular receptor is a widely expressed protein found in many species. Of 16 different cell types tested, only mouse embryo fibroblast NIH 3T3 cells, hamster ovary CHO cells, and the human promonocyte cell line U937 cells were not susceptible to transduction by the pseudotyped virus. The production of functional MuLV/visna virus pseudotypes has provided a sensitive, biologically relevant system to study visna virus cell entry and envelope-receptor interactions.     

A transient three-plasmid expression system for the production of hepatocytes targeting retroviral vectors

Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses （MLVs）, but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses （MoMLV）-based retroviral vectors to transduce hepatocytes. A chimeric envelope （Env） expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa ＋1 at the Nterminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 104 infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.     

Heparin binds to murine leukemia virus and inhibits Env-independent attachment and infection

Certain glycosaminoglycans (GAGs), including heparin, inhibit infection by murine leukemia virus (MLV). We now show that this is due to inhibition of virus attachment independent of the interaction between viral envelope proteins (Env) and their cellular receptors. Heparin blocked the binding of both Env-deficient and amphotropic MLV (MLV-A) particles to NIH 3T3 fibroblasts, CHO cells which lack the amphotropic retroviral receptor Pit-2, and CHO cells transfected with Pit-2 (CHO-Pit-2). Heparin also inhibited the transduction of NIH 3T3 cells by MLV-A over a similar concentration range. This effect was observed within 15 min of exposure to retrovirus. Preloading target cells with heparin had no effect on transduction and both MLV-A and Env-deficient retrovirus bound efficiently to heparin-coated agarose beads, suggesting that heparin interacts with the virus rather than the target cell. This requires both a strong negative charge and a specific structure since GAGs with different charge and carbohydrate composition inhibited virus infection variably. The specificity of GAG-virus interaction also depends on the producer cells, since virus packaged by murine GP+EnvAM12 cells was 1,000-fold more sensitive to inhibition by chondroitin sulfate A than was virus packaged by human FLYA13 packaging cells. No evidence for an interaction between MLV and cell surface proteoglycans was found, however, since the attachment of MLV-A and envelope-defective virus to proteoglycan-deficient CHOpgsA-745 cells was similar to that seen with both wild-type and CHO-Pit-2 cells. Although the molecular mechanism is unclear, this study presents evidence that Env receptor-independent attachment is an important step in MLV infection.     

Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.

We have constructed hybrid retrovirus packaging cell lines that express the gibbon ape leukemia virus env and the Moloney murine leukemia virus gag-pol proteins. These cells were used to produce a retrovirus vector at over 10(6) CFU/ml, with a host range that included rat, hamster, bovine, cat, dog, monkey, and human cells. The gag-pol and env expression plasmids were separately transfected to reduce the potential for helper virus production, which was not observed. The NIH 3T3 mouse cells from which the packaging lines were made are not infectable by gibbon ape leukemia virus; thus, the generation and spread of possible recombinant viruses in the packaging cells is greatly reduced. These simian virus-based packaging cells extend the host range of currently available murine and avian packaging cells and should be useful for efficient gene transfer into higher mammals.     

Host cell cathepsins potentiate Moloney murine leukemia virus infection

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B-/- cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.     

Role of a third extracellular domain of an ecotropic receptor in Moloney murine leukemia virus infection

The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.     

N-Linked glycosylation is required for XC cell-specific syncytium formation by the R peptide-containing envelope protein of ecotropic murine leukemia viruses

The XC cell line undergoes extensive syncytium formation after infection with ecotropic murine leukemia viruses (MLVs) and is frequently used to titrate these viruses. This cell line is unique in its response to the ecotropic MLV envelope protein (Env) in that it undergoes syncytium formation with cells expressing Env protein containing R peptide (R(+) Env), which is known to suppress the fusogenic potential of the Env protein in other susceptible cells. To analyze the ecotropic receptor, CAT1, in XC cells, a mouse CAT1 tagged with the influenza virus hemagglutinin epitope (mCAT1-HA)-expressing retroviral vector was inoculated into XC and NIH 3T3 cells. The molecular size of the mCAT1-HA protein expressed in XC cells was smaller than that in NIH 3T3 cells due to altered N glycosylation in XC cells. Treatment of XC cells with tunicamycin significantly suppressed the formation of XC cell syncytia induced by the R(+) Env protein but not that induced by the R(-) Env protein. This result indicates that N glycosylation is required for XC cell-specific syncytium formation by the R(+) Env protein. The R(+) Env protein induced syncytia in XC cells expressing a mutant mCAT1 lacking both of two N glycosylation sites, and tunicamycin treatment suppressed syncytium formation by R(+) Env in those cells. This suggests that N glycosylation of a molecule(s) other than the receptor is required for the induction of XC cell syncytia by the R(+) Env protein.     

Cells transformed by certain strains of Moloney sarcoma virus contain murine p60.

It was previously demonstrated that the 60,000 dalton (p60) precursor-like polyprotein containing murine p30 was a constituent of the feline leukemia virus pseudotype of Moloney sarcoma virus [m1MSV(FeLV)]. It is now shown that p60 is detected in cells of five mammalian species transformed by m1MSV, indicating that p60 is specified by this genome. Moreover, little or no murine p30 is detected in the m1MSV-transformed cells, suggesting that the murine group p30 antigenic reactivity of S + L- cells is ude to p60. Pulse-chase studies in cells producing m1MSV(FeLV) show that p60 is the largest polypeptide detectable during the pulse, and that intracellular p60 is not cleaved into smaller (for example, p30) polypeptides during chase periods of up to 10 hr. The lack of cleavage of p60 is in contrast to the properties of p30 precursors detected in cells containing replicating avian or mammalian RNA tumor viruses. The inefficient cleavage of intracellular p60 and the kinetics of appearance of murine p30 in extracellular m1MSV(FeLV) suggest that p60 cleavage to p30 occurs in cells shortly before virus release. While only p60 was detected in the m1MSV-transformed cells, p60 and p70 were detected in m3MSV-transformed cells, and no immunoprecipitable polypeptides were detected in HT-1 MSV-transformed cells. The observed differences in the intracellular polypeptide expression by each of the strains of MSV suggests differences in genetic content.     

Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5

Human SERINC5 (SER5) protein is a recently described restriction factor against human immunodeficiency virus-1 (HIV-1), which is antagonized by HIV-1 Nef protein. Other retroviral accessory proteins such as the glycosylated Gag (glycoGag) from the murine leukemia virus (MLV) can also antagonize SER5. In addition, some viruses escape SER5 restriction by expressing a SER5-insensitive envelope (Env) glycoprotein. Here, we studied the activity of human and feline SER5 on HIV-1 and on the two pathogenic retroviruses in cats, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). HIV-1 in absence of Nef is restricted by SER5 from domestic cats and protected by its Nef protein. The sensitivity of feline retroviruses FIV and FeLV to human and feline SER5 is considerably different: FIV is sensitive to feline and human SER5 and lacks an obvious mechanism to counteract SER5 activity, while FeLV is relatively resistant to SER5 inhibition. We speculated that similar to MLV, FeLV-A or FeLV-B express glycoGag proteins and investigated their function against human and feline SER5 in wild type and envelope deficient virus variants. We found that the endogenous FeLV recombinant virus, FeLV-B but not wild type exogenous FeLV-A envelope mediates a strong resistance against human and feline SER5. GlycoGag has an additional but moderate role to enhance viral infectivity in the presence of SER5 that seems to be dependent on the FeLV envelope. These findings may explain, why in vivo FeLV-B has a selective advantage and causes higher FeLV levels in infected cats compared to infections of FeLV-A only.     

Properties of a murine retroviral recombinant of avian acute leukemia virus E26: a murine fibroblast assay for v-ets function.

A replication-defective murine retroviral construct, termed pME26, was generated by inserting avian gag-myb-ets sequences derived from the cloned avian acute leukemia virus E26 into an Abelson murine leukemia virus-derived retroviral vector. ME26 virus can be rescued efficiently from transfected NIH 3T3 cells by replicating murine leukemia viruses. Either pME26-transfected nonproducers or ME26 virus-infected NIH 3T3 cells expressed a 135-kilodalton fusion protein (p135) which was detectable by immunoprecipitation with antiserum directed against avian leukemia virus p27gag, myb or ets oncogene protein, or murine leukemia virus p15gag and was principally localized in the nucleus. NIH 3T3 cells infected with ME26 exhibited morphological alterations and increased proliferation in reduced serum and formed small colonies in agar suspension. Discrete foci could be readily recognized in cells maintained in a defined medium containing 0.03 to 0.1% calf serum. In newborn NFS/N mice, ME26 induced a significantly higher mortality and incidence of erythroid and myeloid leukemias. Analysis of a series of mutants affecting the expression of various portions of p135 indicated that the v-ets gene acts to mitogenically stimulate the proliferation of NIH 3T3 fibroblasts and reduces or abolishes their serum dependence. These properties provide an assay system to study functions of the ets gene family.