Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 May;83(9):4591-604.
doi: 10.1128/JVI.01833-08. Epub 2009 Feb 25.

The signal peptide of a simple retrovirus envelope functions as a posttranscriptional regulator of viral gene expression

Marco Caporale  1 , Frederick Arnaud, Manuela Mura, Matthew Golder, Claudio Murgia, Massimo Palmarini
Affiliations

The signal peptide of a simple retrovirus envelope functions as a posttranscriptional regulator of viral gene expression

Marco Caporale et al. J Virol. 2009 May.

Abstract

Retroviruses use different strategies to regulate transcription and translation and exploit the cellular machinery involved in these processes. This study shows that the signal peptide of the envelope glycoprotein (Env) of Jaagsiekte sheep retrovirus (JSRV) plays a major role in posttranscriptional viral gene expression. Expression of the JSRV Env in trans increases viral particle production by mechanisms dependent on (i) its leader sequence, (ii) an intact signal peptide cleavage site, (iii) a cis-acting RNA-responsive element located in the viral genome, (iv) Crm1, and (v) B23. The signal peptide of the JSRV Env (JSE-SP) is 80 amino acid residues in length and contains putative nuclear localization and export signals, in addition to an arginine-rich RNA binding motif. JSE-SP localizes both in the endoplasmic reticulum and in the nucleus, where it colocalizes with nucleolar markers. JSE-SP is a multifunctional protein, as it moderately enhances nuclear export of unspliced viral mRNA and considerably increases viral particle release by favoring a posttranslational step of the replication cycle.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
The N-terminal region of Env facilitates Gag expression and virus particle production in trans. (A) Schematic representation of Env expression plasmids used in this study. Mutants are derived from a JSRV Env (pJSE) expression plasmid. Numbers refer to amino acid residues of the JSRV21 Env (52). Premature termination codons and deletions are indicated with stop signs and dashed lines, respectively. In pJSEHSP-Flag, the signal peptide of the JSRV Env has been replaced by the signal peptide of the human preprotrypsin. The influence of each Env mutant on Gag expression of a JSRV provirus containing two premature termination codons in Env (pJSRVStop1-27) is indicated with a plus or minus sign. (B) Representative Western blots of virus pellets and cell protein extracts of 293T cells cotransfected with pJSRVStop1-27 and various JSE mutants (or the empty plasmid pBS). Cells were analyzed 48 h posttransfection by SDS-PAGE and Western blotting employing an antiserum against the JSRV p23 (matrix). Gag expression of JSRVStop1-27 is increased by all the mutants employed in the experiment. The lack of a suitable antiserum does not allow detection of the truncated JSRV Env proteins, although the ability of each expression plasmid used in this panel to enhance viral particle release of JSRVStop1-27 is indirect evidence of protein expression. Lane JSRV represents virus pellets and protein extracts of cells transfected with the expression plasmid pCMV2JS21 (wild-type JSRV). (C) Representative Western blots, as described for panel B, of virus pellets and protein extracts of cells transfected with pCMV2JS21 (JSRV) or cotransfected with pJSRVStop1-27 and various pJSE truncation mutants (tagged with an HA epitope) or the empty plasmid (pBS). (D) Representative Western blots, as described for panel B, showing that JSESP-Flag (and not JSEHSP-Flag) increases Gag expression of JSRVStop1-27. The Flag epitope in JSEHSP-Flag remains fused to the SU domain of Env.
FIG. 2.
FIG. 2.
The signal peptide cleavage site of the JSRV Env is necessary for optimal Gag expression. (A) Schematic representation of the plasmids used in the experiments shown in this figure. Premature termination codons and a mutated signal peptide cleavage site of the JSRV Env are indicated, as are the epitope tags HA and V5. (B) Representative Western blots of viral pellets and protein extracts of 293T cells transfected with the plasmids indicated in each lane. Samples were analyzed 48 h posttransfection employing an antiserum against the JSRV p23 (matrix) or HA to confirm expression of JSESP-HA. (C) Western blotting of virus pellets and protein extracts of 293T cells transfected with the plasmids indicated above each lane. (D) Immunoprecipitation and Western blotting of protein extracts from 293T cells transfected with the indicated plasmids using V5 and HA antisera. Cell lysates are from the experiments reflected in panel C. In cells transfected with pJSE-34HAV5 or pJSE-V5, both the full-length Env and the TM domain are visible. However, in cells transfected with JSE-34HAV5ΔSPC, only the full-length Env (including the uncleaved signal peptide) is visible.
FIG. 3.
FIG. 3.
JSE-SP function depends on a cis-acting region located in the JSRV genome. (A) Schematic representation of the deletion mutants of pCMV2JS21 used in the experiments reflected in panel B. Premature termination codons and deletions are indicated with stop signs and dashed lines. (B) Representative Western blots of virus pellets and protein extracts of 293T cells transfected with the plasmids indicated in each lane in the absence or presence of JSESP-HA. Samples were analyzed 48 h posttransfection by SDS-PAGE and Western blotting employing an antiserum against the JSRV p23 (matrix) or HA.
FIG. 4.
FIG. 4.
Identification of the SPRE. (A) Schematic representation of the HIV Gag-Pol expression plasmid (pNLgagSty330) and derived constructs. In these plasmids the HIV RRE was replaced by various portions of the JSRV env 3′ UTR or by the MPMV CTE. Dashed lines represent deletions, while numbers refer to the nucleotide sequence of the infectious molecular clone JSRV21 (52). (B) Results of the HIV Gag ELISA performed as described in Materials and Methods. Each plasmid (with the exception of pNLgagSty330) was transfected with pJSESP-HA or with an empty plasmid. pNLgagSty330 was cotransfected with an expression plasmid for the HIV Rev or with an empty plasmid, as described above. Results are averages and standard deviations from at least three independent experiments and were normalized to the values obtained with pNLgagSty330 in the presence of the HIV Rev (414 ± 42.9 pg/ml), which were arbitrarily set at 100%. The minimal region responding to the JSESP-HA is 146 nt in length and is located in the JSRV env 3′ UTR. Plasmid pH-JSSPRE contains the minimal SPRE. (C) The RNA secondary structure of the JSRV and enJS56A1 SPREs as predicted by the Mfold program (version 3.3). Predicated ΔG values are indicated (80). (D) Genomic organization of JSRV and enJS56A1. The relative positions of their respective SPREs are indicated. Numbers refer to nucleotide residues of the JSRV21 and enJS56A1 molecular clones (49, 52).
FIG. 5.
FIG. 5.
The signal peptide of the JSRV envelope colocalizes with nucleolar markers. (A) Schematic representation of JSE-SP. Kyte-Doolittle hydrophobicity plot of JSE-SP. JSE-SP also shows a predicted NLS, an NES, and an ARM. (B) Confocal microscopy of COS cells transfected with pJSESP-HA or with the deletion mutants pJSESPΔNLS-HA and pJSESPΔNES-HA. At 24 h posttransfection, cells were fixed and analyzed by confocal microscopy using antibodies against HA and the nucleolar markers fibrillarin and B23 with the appropriate conjugates, as described in Materials and Methods. Note the nucleolar staining pattern of JSESP-HA. Cells transfected with pJSESPΔNLS-HA still display nuclear staining although this is diffused and does not localize in the nucleoli. Panels in black and white show colocalization using the plug-in of the Image J software (http://rsbweb.nih.gov/ij/). Bars, 10 μm. (C) Representative Western blots of virus pellets and protein extracts of 293T cells transfected with the plasmids indicated in each lane. Samples were analyzed 48 h posttransfection by SDS-PAGE and Western blotting employing an antiserum against the JSRV p23 (matrix) or HA. None of the JSESP-HA mutants substantially increase virus particle release of enJS-2Stop1-27.
FIG. 6.
FIG. 6.
Localization kinetics of JSE-SP. (A) Schematic representation of the JSRV Env expression plasmid pJSE-34HAV5, which bears an HA tag within the signal peptide and a V5 tag at the carboxy-terminal end of the TM domain. (B) COS cells were transfected with pJSE-34HAV5 and fixed at various times posttransfection as indicated on the left. Cells were analyzed by confocal microscopy using antibodies toward HA (green), V5 (red), PDI (an ER marker, shown in red), and fibrillarin (a nucleolar marker, shown in red), and the appropriate conjugates. HA nuclear staining starts to appear as early as 6 h posttransfection. As expected, HA colocalizes with both nucleolar and ER markers, while V5 colocalizes only with ER markers. Panels in black and white show colocalization using the plug-in of the Image J program as in Fig. 5. Bars, 10 μm. (C) Quantification of JSE-SP staining patterns in confocal microscopy of COS cells expressing JSE-34HAV5 at various times posttransfection. Cells were scored as having either exclusively cytoplasmic staining or both cytoplasmic and nuclear staining. Approximately 100 cells were evaluated at each time point. Values are averages from two independent experiments.
FIG. 7.
FIG. 7.
JSE-SP facilitates cytoplasmic accumulation of unspliced viral RNA. (A) Schematic representation of the plasmids used in these experiments. pJSRVSPRE contains a large deletion within env but maintains the minimal SPRE identified in Fig. 4. The dashed line in pJSRVSPRE indicates a large deletion in env. (B) qRT-PCR of nuclear and cytoplasmic RNA fractions of cells transfected with the plasmids shown in panel A. Samples were analyzed as described in Materials and Methods. The number of samples analyzed is indicated within each bar, and results are expressed as the relative expression ratio of Gag RNA in the presence or absence of JSESP-HA. Each sample was analyzed in triplicate, and values were normalized to pre-GAPDH (nuclear fraction) or GAPDH (cytoplasmic fraction) values, taking into consideration the efficiency of each PCR using the formula indicated in Materials and Methods. Statistical analysis of the data was performed using the software REST (56). P values indicating a statistically significant difference between samples in the presence or absence of JSE-SP are indicated above each bar. (C) Northern blotting (top) using a JSRV env U3 probe showing the unspliced full-length RNAs for JSRVSPRE, JSRVΔSPC, and JSRV. The expected length of full-length unspliced RNA for JSRV and JSRVΔSPC is 7.5 kb, while that for JSRVSPRE is 5.8 kb. Positive signals were quantified by phosphorimaging (bottom) and normalized with signals obtained with a probe for GAPDH after subtracting the background of each individual lane. Values are expressed as percentages of the value obtained by the cytoplasmic fraction of the full-length JSRV RNA, which was arbitrarily assigned a value of 100%. Molecular sizes of the JSRV RNAs are given. (D) Protein extracts and virus pellets from culture supernatants from the same cells were analyzed by Western blotting employing an antiserum against JSRV p23 (matrix). Positive signals were quantified by phosphorimaging and reported as percentages of the arbitrary chemifluorescence units of JSRV Gag, which was arbitrarily assigned a value of 100%.
FIG. 8.
FIG. 8.
JSE-SP function is dependent on Crm1 and B23. (A) COS cells were cotransfected with 1 μg of the expression plasmid for JSRV, MPMV, or HIV (the latter including a Rev expression plasmid) and increasing amounts of dominant negative forms of Crm1 (0.5 to 5 μg). Virus pellets from supernatants of transfected cells were analyzed 48 h posttransfection by Western blotting employing an antiserum against the JSRV CA or HIV p55/p24. Results were quantified by phosphorimaging and are shown as arbitrary chemifluorescence units relative to those for JSRV, MPMV, or HIV (which were arbitrarily assigned a value of 100%). Values are averages and standard deviations from three independent experiments. (B) 293T cells were transfected with pH-JSSPRE plus pJSESP-HA, with pH-MPMV-CTE, or with pNLgagSty330 plus pCMVsRev. In each transfection set, cells were cotransfected with an expression plasmid of the dominant negative Tap/NXF1 (0.5 to 5 μg) or with the empty expression plasmid as a control. At 48 h posttransfection, Gag was measured in the supernatant of transfected cells by ELISA, as described in Materials and Methods. Results are expressed relative to the values obtained with pH-JSSPRE, pH-MPMV-CTE, or pNLgagSty330 plus pCMVsRev cotransfected with the empty expression plasmid in three independent experiments. (C) COS cells were cotransfected with 1 μg of the expression plasmids for JSRV (pCMV2JS21) or MPMV (pSARM4) and increasing amounts of dominant negative forms of B23 (0.5 to 5 μg). Virus pellets from supernatants of transfected cells were analyzed as described for panel A. (D) 293T cells were transfected with JSESP-Flag or an empty plasmid (Mock), and at 48 h posttransfection, proteins were extracted from different subcellular compartments (cytosol, nucleus, and membranes and organelles). Protein extracts were coimmunoprecipitated with a Flag antibody and analyzed by Western blotting using a B23 antibody. Controls included Western blots of the same protein lysates using antibodies against Flag, B23, calnexin, GAPDH, and histone H3. B23 coimmunoprecipitates with JSESP-Flag, especially in the membrane/organelle fraction, although it is clearly less abundant in this fraction than in the other two fractions.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adachi, Y., T. D. Copeland, M. Hatanaka, and S. Oroszlan. 1993. Nucleolar targeting signal of Rex protein of human T-cell leukemia virus type I specifically binds to nucleolar shuttle protein B-23. J. Biol. Chem. 26813930-13934. - PubMed
    1. Alberti, A., C. Murgia, S. L. Liu, M. Mura, C. Cousens, M. Sharp, A. D. Miller, and M. Palmarini. 2002. Envelope-induced cell transformation by ovine betaretroviruses. J. Virol. 765387-5394. - PMC - PubMed
    1. Arnaud, F., M. Caporale, M. Varela, R. Biek, B. Chessa, A. Alberti, M. Golder, M. Mura, Y.-P. Zhang, L. Yu, F. Pereira, J. C. DeMartini, K. Leymaster, T. E. Spencer, and M. Palmarini. 2007. A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses. PLoS Pathog. 3e170. - PMC - PubMed
    1. Arnaud, F., P. R. Murcia, and M. Palmarini. 2007. Mechanisms of late restriction induced by an endogenous retrovirus. J. Virol. 8111441-11451. - PMC - PubMed
    1. Arrigo, S. J., and I. S. Chen. 1991. Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 5808-819. - PubMed

Publication types

MeSH terms

LinkOut - more resources