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Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

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ABSTRACT

Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.

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Glycogag and EIAV S2 modulate MLV entry. Specific infectivity (left) and entry (right) of MLV with wild-type Gag-Pol (blue bars), mutant Gag-Pol lacking gGag (red bars), mutant Gag-Pol with Y36A mutation in gGag (green bars), and mutant Gag-Pol lacking gGag produced in the presence of S2 expression plasmid (grey bars). The viruses were produced with Xeno (xenotropic) envelope (A, B, E, and F) or EbΔMuc (C, D, G, and H) and assayed on the Cre reporter cell line. The S2/Gag-Pol plasmid ratios used in the experiments whose results are shown in panels E to H were 1:27 and 1:9. *, P < 0.0001.
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fig7: Glycogag and EIAV S2 modulate MLV entry. Specific infectivity (left) and entry (right) of MLV with wild-type Gag-Pol (blue bars), mutant Gag-Pol lacking gGag (red bars), mutant Gag-Pol with Y36A mutation in gGag (green bars), and mutant Gag-Pol lacking gGag produced in the presence of S2 expression plasmid (grey bars). The viruses were produced with Xeno (xenotropic) envelope (A, B, E, and F) or EbΔMuc (C, D, G, and H) and assayed on the Cre reporter cell line. The S2/Gag-Pol plasmid ratios used in the experiments whose results are shown in panels E to H were 1:27 and 1:9. *, P < 0.0001.

Mentions: We then used this assay to determine whether virions that were deficient in infectivity because they lacked glycogag were capable of entering new host cells. Viruses carrying a xenotropic Env either with or without glycogag were produced in the presence of the Gag-Cre fusion protein. As the entry assay uses firefly luciferase, infectivities in this experiment were measured using a NanoLuc luciferase reporter vector, rather than the firefly luciferase vector used in the majority of our experiments. As shown by the results in Fig. 7A, the glycogag-containing virus had a specific infectivity (as measured on HT1080 cells) that was significantly higher than that of the virus lacking glycogag, as expected. Figure 7B shows the results of the entry assay with these two virus preparations, as assayed on HT1080 cells containing the Cre-dependent luciferase reporter. It can be seen that the entry capability of the glycogag-containing virus was correspondingly higher than that of the virus lacking glycogag. Therefore, virions which cannot infect cells because they lack glycogag and carry a xenotropic Env are blocked at entry into new host cells.


Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins
Glycogag and EIAV S2 modulate MLV entry. Specific infectivity (left) and entry (right) of MLV with wild-type Gag-Pol (blue bars), mutant Gag-Pol lacking gGag (red bars), mutant Gag-Pol with Y36A mutation in gGag (green bars), and mutant Gag-Pol lacking gGag produced in the presence of S2 expression plasmid (grey bars). The viruses were produced with Xeno (xenotropic) envelope (A, B, E, and F) or EbΔMuc (C, D, G, and H) and assayed on the Cre reporter cell line. The S2/Gag-Pol plasmid ratios used in the experiments whose results are shown in panels E to H were 1:27 and 1:9. *, P < 0.0001.
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fig7: Glycogag and EIAV S2 modulate MLV entry. Specific infectivity (left) and entry (right) of MLV with wild-type Gag-Pol (blue bars), mutant Gag-Pol lacking gGag (red bars), mutant Gag-Pol with Y36A mutation in gGag (green bars), and mutant Gag-Pol lacking gGag produced in the presence of S2 expression plasmid (grey bars). The viruses were produced with Xeno (xenotropic) envelope (A, B, E, and F) or EbΔMuc (C, D, G, and H) and assayed on the Cre reporter cell line. The S2/Gag-Pol plasmid ratios used in the experiments whose results are shown in panels E to H were 1:27 and 1:9. *, P < 0.0001.
Mentions: We then used this assay to determine whether virions that were deficient in infectivity because they lacked glycogag were capable of entering new host cells. Viruses carrying a xenotropic Env either with or without glycogag were produced in the presence of the Gag-Cre fusion protein. As the entry assay uses firefly luciferase, infectivities in this experiment were measured using a NanoLuc luciferase reporter vector, rather than the firefly luciferase vector used in the majority of our experiments. As shown by the results in Fig. 7A, the glycogag-containing virus had a specific infectivity (as measured on HT1080 cells) that was significantly higher than that of the virus lacking glycogag, as expected. Figure 7B shows the results of the entry assay with these two virus preparations, as assayed on HT1080 cells containing the Cre-dependent luciferase reporter. It can be seen that the entry capability of the glycogag-containing virus was correspondingly higher than that of the virus lacking glycogag. Therefore, virions which cannot infect cells because they lack glycogag and carry a xenotropic Env are blocked at entry into new host cells.

View Article: PubMed Central - PubMed

ABSTRACT

Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called &ldquo;glycogag&rdquo; (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.

No MeSH data available.


Related in: MedlinePlus