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. 2007 Feb;81(4):1762-72.
doi: 10.1128/JVI.01859-06. Epub 2006 Nov 29.

The transdominant endogenous retrovirus enJS56A1 associates with and blocks intracellular trafficking of Jaagsiekte sheep retrovirus Gag

Pablo R Murcia  1 , Frederick Arnaud, Massimo Palmarini
Affiliations

The transdominant endogenous retrovirus enJS56A1 associates with and blocks intracellular trafficking of Jaagsiekte sheep retrovirus Gag

Pablo R Murcia et al. J Virol. 2007 Feb.

Abstract

The sheep genome harbors approximately 20 endogenous retroviruses (enJSRVs) highly related to the exogenous Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, acts as a unique restriction factor by blocking JSRV in a transdominant fashion at a late stage of the retroviral cycle. To better understand the molecular basis of this restriction (termed JLR, for JSRV late restriction), we functionally characterized JSRV and enJS56A1 Gag proteins. We identified the putative JSRV Gag membrane binding and late domains and determined their lack of involvement in JLR. In addition, by using enJS56A1 truncation mutants, we established that the entire Gag protein is necessary to restrict JSRV exit. By using differentially tagged viruses, we observed, by confocal microscopy, colocalization between JSRV and enJS56A1 Gag proteins. By coimmunoprecipitation and molecular complementation analyses, we also revealed intracellular association and likely coassembly between JSRV and enJS56A1 Gag proteins. Interestingly, JSRV and enJS56A1 Gag proteins showed distinct intracellular targeting: JSRV exhibited pericentrosomal accumulation of Gag staining, while enJS56A1 Gag did not accumulate in this region. Furthermore, the number of cells displaying pericentrosomal JSRV Gag was drastically reduced in the presence of enJS56A1. We identified amino acid residue R21 in JSRV Gag as the primary determinant of centrosome targeting. We concluded that JLR is dependent on a Gag-Gag interaction between enJS56A1 and JSRV leading to altered cellular localization of the latter.

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Figures

FIG. 1.
FIG. 1.
Mutational analysis of JSRV Gag amino acid residues 19, 20, and 21. (A) Alignment of the N-terminal Gag peptides from the indicated viruses, using ClustalW (39). R21 and G22 are highly conserved among betaretroviruses. Highly conserved residues are highlighted in bold. R21 and G22 are indicated with vertical arrows. K19 and H20 are underlined. Consensus symbols are displayed below the alignment, as follows: “*” indicates identical residues in all sequences in the alignment, “:” indicates conserved substitutions, and “.” indicates semiconserved substitutions. ENTV, enzootic nasal tumor virus; M-PMV, Mason-Pfizer monkey virus; SRV-2, simian retrovirus 2; TV-ervD, brushtail possum type D endogenous retrovirus; MMTV, mouse mammary tumor virus; HERV-K, human endogenous retrovirus K. (B) Analysis of JSRV mutants bearing mutations in the highly basic region of the M domain. Viral pellets (upper panel) and lysates (lower panel) of cells transfected with the indicated plasmids were resolved by SDS-PAGE and immunoblotted with a JSRV CA antiserum. (C) Western blot analysis of viral pellets (upper panel) and lysates (lower panel) of cells cotransfected with expression plasmids for JSRV and mutants, as indicated.
FIG. 2.
FIG. 2.
Patterns of Gag staining by confocal microscopy of cells expressing JSRV, enJS56A1, and M domain mutants. (A) By confocal microscopy, Gag staining in HeLa cells expressing JSRV and enJS56A1 is described as diffuse (panel a), dispersed (panel b), or concentrated (panel c) (19). The figure represents typical examples of enJS56A1-expressing cells; anti-MA staining is displayed in gray, and the letter “N” indicates the location of the nucleus. (B) Gag staining patterns of JSRV and enJS56A1 M domain mutants in a representative experiment. The number on top of each bar indicates the total number of cells counted for each virus/mutant.
FIG. 3.
FIG. 3.
Analysis of JSRV L domains. (A) Viral pellets from cells transfected with increasing amounts of the indicated plasmids were resolved by SDS-PAGE and immunoblotted with an antiserum towards JSRV MA. The names of the transfected constructs are indicated below the panels, while the amounts of plasmid DNA transfected (in 10-cm dishes) are shown above the upper panel. (B) Lysates of cells transfected with the indicated plasmids were resolved by SDS-PAGE and immunoblotted with an antiserum towards JSRV MA. Cells expressing JSRVΔLD201-7 showed more intense bands for both immature and mature Gag, indicative of a defect in viral exit for this virus. (C) Confocal microscopy of HeLa cells expressing JSRV and JSRV L domain mutants. Anti-MA staining is displayed in green, and nuclei are shown in blue. Cells expressing JSRVΔLD201-7 show a characteristic accumulation of Gag staining at the cell membrane. Bar, 5 μm. (D) Viral pellets (upper panel) and lysates (lower panel) of cells transfected with the indicated plasmids were resolved by SDS-PAGE and immunoblotted with an antiserum towards JSRV MA. JSRVΔLD201-7 is defective for viral exit but is not transdominant over wild-type JSRV.
FIG. 4.
FIG. 4.
Truncated enJS56A1 Gag mutants do not block JSRV exit. (A) Organization of JSRV Gag. The names of the Gag cleavage products are displayed inside the boxes (with the exception of p4, which is indicated below). Vertical lines indicate the cleavage sites. The numbers above the bar refer to the positions of the boundaries in mature Gag of the JSRV21 infectious molecular clone (20). Myristate is represented by a gray circle. The relative positions of the L and M domains are also indicated. The apparent molecular weights of MA, CA, and NC are indicated below the bar. (B) Western blot analysis of enJS56A1 deletion mutants. Viral pellets (upper panel) and lysates (lower panel) of cells transfected with the indicated plasmids were resolved by SDS-PAGE and immunoblotted with a JSRV MA antiserum. Truncated constructs did not interfere with JSRV exit.
FIG. 5.
FIG. 5.
JSRV and enJS56A1 Gag proteins colocalize. (A) Schematic representation of JSRV and enJS56A1 tagged constructs. (B) Representative images of confocal microscopy of HeLa cells coexpressing either JSRVFLAG-C and enJS56A1HA-C, JSRVHA-C and enJS56A1FLAG-C, or JSRVHA-MA and enJS56A1FLAG-MA. Strong colocalization of JSRV and enJS56A1 is observed in all cases. Both dispersed and concentrated phenotypes are represented (see Results). FLAG staining is displayed in green, HA staining is shown in red, and nuclei are shown in blue. Bar, 5 μm.
FIG. 6.
FIG. 6.
Association between JSRV and enJS56A1 Gag proteins. (A) 293T cells were cotransfected with enJS56A1HA-MA and JSRVFLAG-MA. At 48 h posttransfection, cell lysates were immunoprecipitated (IP) and analyzed by SDS-PAGE/Western blotting (WB) as indicated beside each panel. Gag expression was assessed by Western blotting using a JSRV MA antiserum. enJS56A1-JSRV Gag association is evident in lysates from cells cotransfected with both viruses (lane 4). Note that the JSRV MA antiserum is a polyclonal serum and is much more sensitive in Western blotting than the anti-HA or anti-FLAG monoclonal antibody. (B) Supernatants of cells transfected with the indicated plasmids were resolved by SDS-PAGE and immunoblotted with an antiserum towards JSRV CA. Note that JSRVΔpro releases virus particles into the supernatant with an immature Gag (lane 5) because of the lack of a functional protease in this virus. Coexpression of JSRVΔpro and enJS56A1ΔNC2 (lane 8) leads to the release of viral particles with both immature and mature Gag. (C) Supernatants and lysates of cells transfected with the indicated plasmids were resolved by SDS-PAGE and immunoblotted with an antiserum towards JSRV MA. Note that enJS56A1ΔMHR is highly expressed (lane 4) but does not interfere with JSRV exit (lane 6).
FIG. 7.
FIG. 7.
Intracellular targeting of JSRV and enJS56A1 Gag proteins. (A) HeLa cells were transfected with JSRV (panels a to c), JSRVHA-MA (panels d to f), or enJS56A1 (panels g to i) or cotransfected with JSRVHA-MA and enJS56A1 (panels j to l) and then stained with anti-γ-tubulin antibodies and antiserum to either JSRV MA (panels a to c and g to i) or the HA tag (panels d to f and j to l). Gag and HA staining is shown in green, while γ-tubulin staining is shown in red. Centrosomes are indicated with white arrows. (B and C) Quantification of centrosomal targeting in cells expressing concentrated Gag staining (see Results). Viruses/mutants are indicated on the right. The number on top of each bar indicates the total number of cells counted per sample.
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