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. 2011 Jul;85(14):7118-28.
doi: 10.1128/JVI.00407-11. Epub 2011 May 18.

The signal peptide of a recently integrated endogenous sheep betaretrovirus envelope plays a major role in eluding gag-mediated late restriction

Alessia Armezzani  1 , Frédérick Arnaud, Marco Caporale, Giuliapia di Meo, Leopoldo Iannuzzi, Claudio Murgia, Massimo Palmarini
Affiliations

The signal peptide of a recently integrated endogenous sheep betaretrovirus envelope plays a major role in eluding gag-mediated late restriction

Alessia Armezzani et al. J Virol. 2011 Jul.

Abstract

The exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with highly related and biologically active endogenous retroviruses (enJSRVs). The endogenous enJS56A1 locus possesses a defective Gag polyprotein which blocks the late replication steps of related exogenous and endogenous retroviruses by a mechanism known as JSRV late restriction (JLR). Conversely, enJSRV-26, which most likely integrated into the sheep genome less than 200 years ago, is able to escape JLR. In this study, we demonstrate that the ability of enJSRV-26 to escape JLR is due to a single-amino-acid substitution in the signal peptide (SP) of its envelope glycoprotein. We show that enJSRV-26 SP does not localize to the nucleolus, unlike the functional SPs of related exogenous and endogenous sheep betaretroviruses. In addition, enJSRV-26 SP function as a posttranscriptional regulator of viral gene expression is impaired. enJSRV-26 JLR escape relies on the presence of the functional enJS56A1 SP. Moreover, we show that the ratio between enJSRV-26 and enJS56A1 Gag is critical to elude JLR. Interestingly, we found that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus. These data further reinforce the notion that transdominant enJSRV proviruses have been positively selected in domestic sheep, and that the coevolution between endogenous and exogenous sheep betaretroviruses and their host is still occurring.

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Figures

Fig. 1.
Fig. 1.
Schematic representation of the plasmids employed in this study. All of the mutants/chimeras used in this study were derived from expression plasmids encoding the full-length enJSRV-26 and enJSRV-18 proviruses (top). Numbers indicate amino acid residues of Env. Premature termination codons are indicated with vertical lines and asterisks. LTR, long terminal repeat; CMV, cytomegalovirus.
Fig. 2.
Fig. 2.
enJSRV-26 env 5′ end contains the determinants necessary to escape JLR. Shown are Western blots of concentrated supernatants (virus) and cell extracts (cells) of 293T cells transfected with the plasmids indicated in each panel. Membranes were incubated with antibodies against the major capsid protein of JSRV (CA) or γ-tubulin as a loading control. Levels of CA associated with viral particles released in the supernatants were quantified by chemifluorescence using ImageQuant TL software (Molecular Dynamics). Graphs represent data obtained from three independent experiments. The values obtained by each chimera expressed in the absence of enJS56A1 (black bars) were arbitrarily set as 100%. Error bars indicate standard errors; statistically significant differences are indicated with one (P < 0.05) or two (P < 0.01) asterisks. (A) Interference assays with wild-type enJSRV-26 and enJSRV-18. (B, C, and D) Interference assays using various enJSRV-26/enJSRV-18 chimeras as indicated in each panel. Only those chimeras containing the 5′ end of the env gene of enJSRV-26 are able to elude enJS56A1 restriction.
Fig. 3.
Fig. 3.
Amino acid residue D6 in the enJSRV-26 Env plays a major role in JLR escape. (A, B, D, and E) Western blots of concentrated supernatants (virus) and cell extracts (cells) of 293T cells transfected with the plasmids indicated in each panel. Membranes were incubated with antibodies against the major capsid protein (CA) of JSRV or γ-tubulin as a loading control. Levels of CA associated with viral particles released in the supernatants were quantified by chemifluorescence using ImageQuant TL software (Molecular Dynamics). Graphs represent data obtained from three independent experiments. The values obtained by each mutant expressed in the absence of enJS56A1 (black bars) were arbitrarily set as 100%. Error bars indicate standard errors; statistically significant differences are indicated with one (P < 0.05) or two (P < 0.01) asterisks. Env expression was controlled by incubating membranes with antibodies against the transmembrane (TM) domain of JSRV. (C) Graphic representation of the alignment of the amino acid sequences of the signal peptides of four insertionally polymorphic enJSRV proviruses and the exogenous JSRV. Lines represent identical residues in the sequences, while letters indicate differences in the amino acid residues. Numbering corresponds to amino acid residues in Env. ARM, arginine-rich motif; NLS, nuclear localization signal; NES, nuclear export signal.
Fig. 4.
Fig. 4.
Signal peptide (SP) of enJSRV-26 does not localize in the nucleoli. The intracellular localization of the signal peptide of enJSRV-18 and enJSRV-26 is shown. COS cells were transfected with expression plasmids for the SPs of enJSRV-18 or enJSRV-26 (tagged with the HA epitope), fixed 24 h posttransfection, and incubated with anti-HA (top) and fibrillarin (middle) antibodies. Nuclei are shown in blue. Both SPs display nuclear localization, but only the enJSRV-18 SP shows a strong colocalization with nucleolar markers such as fibrillarin. Bars correspond to 10 μm.
Fig. 5.
Fig. 5.
Signal peptide of enJSRV-26 does not function as a posttranscriptional regulator of viral gene expression and is essential to escape JLR. (A and C) Western blots of concentrated supernatants (virus) and cell extracts (cells) of 293T cells transfected with the plasmids indicated in each panel. Membranes were incubated with antibodies against the JSRV major capsid protein (CA), the HA or FLAG epitopes (to detect SPs or Env, respectively), or γ-tubulin as a loading control. Levels of CA associated with viral particles released in the supernatants were quantified by chemifluorescence using ImageQuant TL software (Molecular Dynamics). Graphs represent data obtained from three independent experiments. The values obtained by enJSRV-26 or related mutants in the absence of enJS56A1 (black bars) were arbitrarily set as 100%. Error bars indicate standard errors; statistically significant differences are indicated with one (P < 0.05) or two (P < 0.01) asterisks. (B) HIV Gag ELISAs were performed on supernatants of 293T cells transfected with HIV-1 Gag-Pol expression plasmids (pHIV-SPRE26, pHIV-SPRE18, and pHIV-SPRE56) in the presence or absence of expression plasmids for the enJSRV-26, enJSRV-18, and enJS56A1 SPs (SP26-HA, SP18-HA, and SP56-HA). Controls included supernatants of cells transfected with the HIV-1 Gag-Pol expression plasmid containing HIV-1 RRE (pHIV1-RRE) in the presence or absence of the HIV-1 Rev (pRev).
Fig. 6.
Fig. 6.
enJS56A1 Env expression is critical to escape JLR. (A and B) Western blots of concentrated supernatants (virus) and cell extracts (cells) of 293T cells transfected with the plasmids indicated in each panel. Membranes were incubated with antibodies against the JSRV major capsid protein (CA) and γ-tubulin as a loading control. Note that enJS56A1-4CTE lacks the viral Env and is able to block enJSRV-26. Levels of CA associated with viral particles released in the supernatants were quantified by chemifluorescence using ImageQuant TL software (Molecular Dynamics). Graphs represent data obtained from three independent experiments. The values obtained by enJSRV-26 were arbitrarily set as 100%. Error bars indicate standard errors; statistically significant differences are indicated with one (P < 0.05) or two (P < 0.01) asterisks. (C) Western blot analysis of concentrated supernatants of 293T cells transfected with the indicated plasmids at 3, 6, 12, and 24 h posttransfection. The bottom panel represents the quantification of blots by chemifluorescence as described for panels A and B. Values are expressed as arbitrary pixel units derived from three independent experiments.
Fig. 7.
Fig. 7.
enJS56A1 is amplified in the domestic sheep genome. (A) Graph representing qPCR used to estimate the gene dosage, in wild and domestic sheep, of the transdominant proviruses enJSRV-6, enJSRV-18, and SOX9. Each bar represents an individual animal, and each letter represents a different species/breed. Gray boxes indicate assays that could not be performed due to the exhaustion of sample DNA. The absence of a bar indicates no amplification of the corresponding enJSRV locus. Samples tested included genomic DNA collected from B. taxicolor (a), P. nayaur (b), O. dalli (c), O. canadensis (d), O. ammon (e), O. vignei (f), O. orientalis (g), and various breeds of domestic sheep (O. aries), such as Soay (h), Norway (i), Dorset (j), Suffolk (k), Texel (l), Jacob (m), Red Maasai (n), Finsheep (o), Merino (p), Xalda (q), and Rambouillet (r). (B) Fluorescent in situ hybridization of metaphase R-banded chromosomes derived from a Merino sheep (mixed breed). Fluorescent probes were derived from BAC clones containing the enJSRV-20, enJS56A1, or enJSRV-6 proviruses as described in Materials and Methods. The green fluorescent signals, indicated by arrows, are specific for the two transdominant proviruses (both located on chromosome 6 at band 6q13) and the enJSRV-6 locus (situated on chromosome 1 at band 1q41). Ideograms of Ovis aries chromosomes with R-banding patterns also are shown. (C) Relative frequency of the wild-type arginine (R) or the transdominant tryptophan (W) Gag residue at position 21 of the enJS56A1-like proviruses. The 5′ region of gag of enJS56A1 and enJSRV-20 was amplified by PCR from genomic DNA collected from the species indicated in the panel. Note that letters in parentheses (c, d, e, etc.) refer to the code used for panel A. PCR products were cloned into the pCR4-TOPO vector (Invitrogen), and 40 individual clones for each PCR product were sequenced to determine the relative presence of an arginine or trypthophan residue at position 21 in Gag.
Fig. 8.
Fig. 8.
Model of enJSRV-26 JLR escape. The ability of enJSRV-26 to elude JLR restriction is dependent on the impaired function of its SP. Consequently, the signal peptide responsive element (SPRE) of enJSRV-26 competes with the SPRE of enJS56A1 for the functional SP of the latter, resulting in the reduced expression of the transdominant Gag.
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