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. 2004 Jul 27;101(30):11117-22.
doi: 10.1073/pnas.0402877101. Epub 2004 Jul 19.

Late viral interference induced by transdominant Gag of an endogenous retrovirus

Manuela Mura  1 , Pablo Murcia, Marco Caporale, Thomas E Spencer, Kunio Nagashima, Alan Rein, Massimo Palmarini
Affiliations

Late viral interference induced by transdominant Gag of an endogenous retrovirus

Manuela Mura et al. Proc Natl Acad Sci U S A. .

Abstract

The sheep genome harbors approximately 20 copies of endogenous retroviruses (enJSRVs) closely related to the exogenous and oncogenic Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, has a defect for viral exit. We report a previously uncharacterized mechanism of retroviral interference. The defect possessed by enJS56A1 is determined by its Gag protein and is transdominant over the exogenous JSRV. By electron microscopy, cells transfected by enJS56A1, with or without JSRV, show agglomerates of tightly packed intracellular particles most abundant in the perinuclear area. The defect in exit and ability to interfere with JSRV exit could be largely attributed to the presence of tryptophan, rather than arginine, at position 21 of enJS56A1 Gag; C98 and V102 also contribute to these properties. We found that enJS56A1 or similar loci containing W21, C98, and V102 are expressed in sheep endometrium. enJS56A1 is a previously unrecognized example of a naturally occurring endogenous retrovirus expressing a dominant negative Gag acting at a late step of the viral replication cycle. Understanding the late blockade exerted by enJS56A1 could unravel fundamental aspects of retroviral biology and help to devise new antiretroviral strategies.

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Figures

Fig. 1.
Fig. 1.
enJS56A1 specifically inhibits JSRV viral particle release. (A) Western blot analysis of cell lysates (Left) and supernatants (Right) of 293T transfected or cotransfected with enJS56A1 and JSRV expression plasmids. Gag was detected with a JSRV p23 antiserum. Lanes: 1, JSRV; 2, enJS56A1; 3, enJS56A1 + JSRV; M, mock. (B) Western blot analysis of cell lysates and supernatants of 293T cells cotransfected with fixed amounts (14 μg) of JSRV expression plasmid and decreasing amounts of enJS56A1 expression plasmid. (C) JS21Δpro is able to release viral particles in the supernatant of transfected cells. Lanes: M, mock; 1, JS21Δpro; 2, JSRV. (D) enJS56A1 does not inhibit MPMV exit in cotransfection assays. Lanes: 1, MPMV; 2, MPMV+enJS56A1; M, mock. Gag was detected with anti-MPMV CA serum. (E) enJS56A1 does not inhibit M-MLV viral exit. Lanes: 1, M-MLV; 2, M-MLV+enJS56A1; M, mock. Gag was detected with anti-MLV CA serum.
Fig. 2.
Fig. 2.
enJS56A1 assembles intracytoplasmic viral particles. 293T cells were transfected with JSRV (A–D) or enJS56A1 (E and F) or cotransfected (G and H) with both expression plasmids and analyzed by electron microscopy. (A and B) Extracellular complete JSRV particles and particles approaching and budding from the membrane. (C and D) Intracytoplasmic JSRV. (E and F) enJS56A1 particles. (C and F) Arrows, incomplete viral particles. In E, arrows point to apparent projections from the enJS56A1 particles. (G and H) Large clusters of perinuclear viral particles in enJS56A1+JSRV cotransfected cells. N, nucleus.
Fig. 3.
Fig. 3.
Determinants of enJS56A1 defect in viral exit and interference. (A) Schematization of the first 344 Gag amino acid residues. The restriction sites used for the construction of the chimeras, the VR1–VR2 regions, and the amino acid residues found to be critical for the enJS56A1 defect are indicated. enJS59A1 has a premature stop codon in gag upstream of position 88; amino acid residue at position 21 is indicated by *. (B) Schematic representation of the chimeras constructed in this study and results obtained in transfection and cotransfection assays.
Fig. 4.
Fig. 4.
Western blotting analysis of JSRV and enJS56A1 mutants. Supernatants (Upper) and cell lysates (Lower) of 293T cells cotransfected with JSRV and critical mutant expression plasmids were analyzed by Western blotting for the presence of Gag proteins. (A) Lanes: 1, JSRV; 2, JSRV + JSG2A; 3, JSRV+JSR21W; 4, JSRV + JSR98C-L102V; 5, JSRV + JSG2A-R98C-L102V; 6, JSRV + JSR21W-R98C-L102V; 7, JSRV + JSG2V-R21W-R98C-L102V; 8, JSRV + JSR98C. (B) Restoration of enJS56A1 viral exit. Lanes: M, mock; 1, en56W21R-V102L; 2, en56W21R-C98R; 3, en56W21R. Note that the p23 of the endogenous Gag has a slightly lower molecular weight with respect to the homologous JSRV protein, probably because of the lack of proline residues in the VR1 and VR2 regions of the endogenous locus.
Fig. 5.
Fig. 5.
Confocal microscopy of HeLa cells expressing or coexpressing enJS56A1, JSRV, and critical mutants. Positive cells have been quantified according to three phenotypes: diffuse (Diff.), dispersed (Disp.), and concentrated (Conc.). At the top is the quantification of a representative experiment. Values indicate relative percentage of each phenotype, and the last column indicates the total number of cells counted for each transfection. Photomicrographs are representative examples of cells expressing the indicated viruses and mutants. Note that the “dispersed” phenotype for enJS56A1-transfected cells consists of larger foci with a more intense fluorescence than in other samples. Gag staining is in green, and nuclei are in red or with the letter N.
Fig. 6.
Fig. 6.
Modeling of the putative JSRV and enJS56A1 MA proteins. The tertiary structures of the matrix proteins of enJS56A1 and JSRV were modeled by using the Swiss Model Server (http://swissmodel.expasy.org). The matrix protein of MPMV (Protein Data Bank ID code 1BAX) was used as a template. As the available MPMV matrix protein structure at the Protein Data Bank is a carbon trace structure, a tertiary structure including side chains was generated by using the MaxSprout database algorithm (www.ebi.ac.uk/maxsprout). Models are displayed in ribbons, and the W21 and R21 are displayed in green in a ball-and-stick format. The N and C terminals are indicated. The first α-helix is shown in blue. Both models were derived from the amino-terminal 92-aa residues of the JSRV/enJS56A1 Gag.
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