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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.

No MeSH data available.


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.
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fig1: 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.

Mentions: In order to obtain clear information on the fate and function of glycogag despite its close relationship to Gag, it was essential to control the expression of both proteins and to be able to specifically detect each of them. In the natural setting, glycogag is expressed from a CUG codon in the viral RNA, 88 codons upstream from the normal Gag AUG initiator (Fig. 1A) (1). To generate a glycogag expression plasmid, we began with a codon-optimized expression plasmid for Gag and first inserted the 88 codons on the 5′ side of the AUG (the sequence of the Gag protein here was that of xenotropic murine leukemia virus-related virus [XMRV], a gammaretrovirus very similar to Moloney MLV). We then replaced the CUG glycogag initiator with an AUG. To prevent leaky scanning, leading to Gag synthesis from the plasmid, we also replaced the Gag AUG with the alanine codon GCC (Fig. 1B). Finally, we inserted sequences for a myc epitope tag into the p12 region of the plasmid, at a site previously shown to be tolerant of insertions (12–14). As shown by the results in Fig. 1C, this plasmid, designated pCMV(glycogag), directs the synthesis of glycogag but not that of Gag; the glycogag can be detected with antiserum against either p30CA or Myc.


Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5120145&req=5

fig1: 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.
Mentions: In order to obtain clear information on the fate and function of glycogag despite its close relationship to Gag, it was essential to control the expression of both proteins and to be able to specifically detect each of them. In the natural setting, glycogag is expressed from a CUG codon in the viral RNA, 88 codons upstream from the normal Gag AUG initiator (Fig. 1A) (1). To generate a glycogag expression plasmid, we began with a codon-optimized expression plasmid for Gag and first inserted the 88 codons on the 5′ side of the AUG (the sequence of the Gag protein here was that of xenotropic murine leukemia virus-related virus [XMRV], a gammaretrovirus very similar to Moloney MLV). We then replaced the CUG glycogag initiator with an AUG. To prevent leaky scanning, leading to Gag synthesis from the plasmid, we also replaced the Gag AUG with the alanine codon GCC (Fig. 1B). Finally, we inserted sequences for a myc epitope tag into the p12 region of the plasmid, at a site previously shown to be tolerant of insertions (12–14). As shown by the results in Fig. 1C, this plasmid, designated pCMV(glycogag), directs the synthesis of glycogag but not that of Gag; the glycogag can be detected with antiserum against either p30CA or Myc.

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 “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.

No MeSH data available.