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. 2016 Nov 22;7(6):e01985-16.
doi: 10.1128/mBio.01985-16.

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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Free PMC article

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

Yadvinder S Ahi et al. mBio. .
Free PMC article

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.

Importance: Many murine leukemia viruses (MLVs) encode a protein called "glycogag." The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5.

Figures

FIG 1
FIG 1
Glycogag expression plasmid and Glycogag-negative MLV clone. (A) Schematic of translation of glycogag and Gag from the viral genome. *, cleavage site in glycogag. Precise location of cleavage site is not known. (B) Schematic of pCMV(glycogag), the glycogag expression plasmid. (C) Lysates of cells transfected with pCMV(glycogag) or Gag expression plasmids were probed with anti-p30CA or anti-Myc antibodies. gGag and Gag bands are indicated. *, cleavage product of gGag. (D) Schematic of glycogag-negative Moloney MLV clone. (E) Lysates of cells transfected with wild-type (+gGag) or glycogag-negative (−gGag) Moloney MLV clones or of mock-transfected cells were probed with anti-p30CA antibody at 48 h posttransfection. gGag, Gag, and capsid (CA) bands are indicated.
FIG 2
FIG 2
Glycogag traffics through endoplasmic reticulum and Golgi apparatus. (A) Confocal microscopy of HeLa/gGag cells and control HeLa/vector cells cultured in the presence of 10-ng/ml doxycycline for 24 h and stained with anti-Myc antibody for gGag and anti-GM130 for Golgi apparatus. (B) Confocal microscopy of HeLa/gGag cells transiently expressing Sec61-mCherry fusion protein cultured in the presence of 10-ng/ml doxycycline for 24 h. The cells were treated with 200-ng/ml brefeldin A for 3 h, followed by fixation and immunostaining with anti-Myc antibody (for detection of gGag). DAPI was used for staining nuclei. The arrow in panel A indicates localization of gGag in Golgi apparatus.
FIG 3
FIG 3
Effect of gGag on infectivity of MLV(Xeno). Specific infectivities (luciferase activity units divided by relative amounts of p30CA) of MLV(Xeno) with wild-type Gag-Pol (blue bars) or mutant Gag-Pol lacking gGag (red bars) produced in 293T cells and assayed on the indicated cell lines. NIH, NIH/3T3 mouse cells; hXPR1, human XPR1; *, P < 0.0001.
FIG 4
FIG 4
Effect of gGag on MLV infectivity is determined by Env protein. Specific infectivities of MLV with indicated Env glycoproteins produced in the presence or absence of gGag. Viruses were produced with wild-type or gGag-deficient Gag-Pol together with the indicated Env expression clones. The x axis shows the type of Env glycoprotein on the virus. For each Env, the red dots represent the ratios of specific infectivities of virus with gGag to virus without gGag in individual experiments. The plus signs show the geometric means of these values, and the bars at the top and bottom of each vertical line show associated 95% confidence intervals for each Env. Eb-FL, full-length Ebola glycoprotein; EbΔMuc, Ebola glycoprotein with deletion of mucinlike domain. The target cell line used for viruses with VSV(g), Ampho (amphotropic), 10A1, GALV, RD114, Xeno (xenotropic), Eb-FL, and EbΔMuc glycoproteins was HT1080, the target cell line used for viruses with Eco (ecotropic) and BLV glycoprotein was HT1080/mCAT1, and the target cell line used for viruses with RSV Env A was D17 cells expressing subgroup A receptor.
FIG 5
FIG 5
Effect of MLV gGag and EIAV S2 expressed in trans on infectivity of MLV(Xeno) and MLV(Ebola). (A) Specific infectivities of MLV(Xeno) with wild-type Gag-Pol (blue bar) or mutant Gag-Pol lacking gGag (red bar), and MLV(Xeno) with mutant Gag-Pol cotransfected with increasing amounts of pCMV(glycogag) (green bars). (B and C) Specific infectivity of MLV(Eb-FL) (B) and MLV(Xeno) (C) with wild-type Gag-Pol (blue bar) or with mutant Gag-Pol lacking gGag (red bar), together with pCMV(glycogag) or S2 expression plasmid. The pCMV(glycogag)/Gag-Pol plasmid ratios used were increased by threefold increments from 1:6,561 to 1:3 in the experiment whose results are shown in panel A and from 1:243 to 1:3 in the experiment whose results are shown in panel B; in the experiment whose results are shown in panel C, the ratio used was 1:27. The S2/gag-Pol plasmid ratios used were increased by threefold increments from 1:243 to 1:3 in the experiment whose results are shown in panel B and from 1:81 to 1:3 in the experiment whose results are shown in panel C. The target cell line used in these experiments was HT1080/mCAT1. *, P < 0.0001.
FIG 6
FIG 6
Y36A mutant of gGag is partially active in enhancing MLV(Xeno) infectivity. Specific infectivity of MLV(Xeno) with wild-type Gag-Pol (blue bar) or with mutant Gag-Pol lacking gGag (red bar), and MLV(Xeno) with mutant Gag-Pol cotransfected with increasing amounts of wild-type (green bars) or Y36A mutant gGag (purple bars) pCMV(glycogag). The wild-type or Y36A mutant gGag/Gag-Pol plasmid ratios used were increased by threefold increments from 1:243 to 1:3. The target cell line used in these experiments was HT1080/mCAT1. *, P < 0.0001.
FIG 7
FIG 7
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.
FIG 8
FIG 8
Antagonism between Serinc5 and glycogag with respect to MLV(Xeno) and MLV(Ebola) infectivity. Specific infectivities of MLV with wild-type Gag-Pol (blue bars) or mutant Gag-Pol lacking gGag (red bars) produced in the absence or presence of increasing amounts of Serinc5 expression plasmid and with Xeno (A) or Eb-FL (B) glycoprotein are shown. The Serinc5/Gag-Pol plasmid ratios used were increased by threefold increments from 1:81 to 1:3. The infectivity measurements were performed on the HT1080/mCAT1 cell line.
FIG 9
FIG 9
Serinc5 modulates the entry of MLV(Xeno) and MLV(Ebola). Specific infectivities (left) and entry (right) of MLV with wild-type Gag-Pol (blue bars) or mutant Gag-Pol lacking gGag (red bars) produced with Xeno (A and B) or EbΔmuc (C and D) glycoprotein together with control plasmid (none) or Serinc5 expression plasmid (Serinc5), with or without S2 expression plasmid used at a 1:27 or 1:9 ratio to Gag-Pol plasmid. The viruses for specific infectivity and entry measurements were produced in parallel transfections. Specific infectivity and entry measurements were performed on HT1080/mCAT1 cells and Cre reporter cells, respectively.

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