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. 2000 Sep;74(17):8065-76.
doi: 10.1128/jvi.74.17.8065-8076.2000.

Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus

M Palmarini  1 , C Hallwirth, D York, C Murgia, T de Oliveira, T Spencer, H Fan
Affiliations

Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus

M Palmarini et al. J Virol. 2000 Sep.

Abstract

Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSVs) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRVs in vivo was studied by in situ hybridization. All of the three enJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV(21). Phylogenetic analysis distinguished five ovine type D retroviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the three enJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.

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Figures

FIG. 1
FIG. 1
Genomic structures of endogenous and exogenous type D retroviruses of sheep. Premature stop codons are indicated by a vertical bar underlined by an asterisk. For convenience, the gag open reading frame has been fixed in the same reading frame of all sequences shown. The numbered bar at the bottom indicates distances in kilobases. The exogenous JSRV and ENTV show the canonical retroviral gag, pro, pol, and env with pro in a different open reading frame from pol, the same for all type D and B retroviruses. An additional open reading frame (orf-x) overlapping pol is present in JSRV but is interrupted by two stop codons in ENTV (8). enJS56A1 is the only one of the three endogenous proviruses cloned in this study to maintain full (or nearly full) open reading frames in all structural genes. enJS59A1 has premature stop codons in gag and pol and a major deletion in env. enJS5F16 has a deletion in pol. Different peptide sequences at the 3′ end of the pol gene in enJS56A1 due to a frameshift are indicated by cross-hatching. The LTRs are indicated by solid boxes.
FIG. 2
FIG. 2
Alignment of deduced gag amino acid sequences of sheep type D retroviruses: exogenous JSRV21 (AF105220), JSRV-SA (M80216), and ENTV (Y16627) and the endogenous enJS5F16 and enJS56A1 that maintain full-length gag open reading frames. Dots refer to identical sequences, while dashes indicate lack of sequence. VR1 and VR2 are underlined. Note that the prolines in VR1 of the exogenous JSRVs and ENTV are absent in the endogenous proviruses. In VR2, ENTV is more similar to enJSRVs than JSRV. The putative CA region and the HpaI site used to generate exogenous-endogenous chimeras (Fig. 4) are indicated.
FIG. 3
FIG. 3
Alignment of deduced env amino acid sequences of sheep type D endogenous retroviruses: exogenous JSRV21, JSRV-SA, and ENTV and the endogenous proviruses (enJS5F16 and enJS56A1) that maintain an open reading frame along the entirety of env. The boundary between the surface (SU) and transmembrane (TM) regions is indicated. VR3 is underlined; note the polymorphism between all sequences in VR3.
FIG. 4
FIG. 4
Virus production by endogenous-exogenous chimeras. (A) Schematic structure of the parental (JSRV21 and enJS56A1) and chimeric plasmids. The restriction enzyme sites used for the cloning are indicated. In pCMV2JS21 and in various chimeric constructs, expression is driven by the CMV immediate-early promoter (indicated by the arrow). (B) Western blot of 300-fold-concentrated supernatant from equal numbers of 293T cells transiently transfected with the constructs shown in panel A. Lung secretions collected from an OPC-affected animal were used as a positive control (LF), while mock-transfected 293T supernatants were used as negative controls. The filters were incubated with a rabbit antiserum against the capsid (CA) protein of JSRV (43). The 26-kDa CA protein is indicated.
FIG. 5
FIG. 5
Nucleotide sequence alignment of the 5′ untranslated regions of sheep type D retroviruses: exogenous JSRV21, JSRV-SA, and ENTV and the endogenous enJS5F16, enJS59A1, and enJS56A1 proviruses. The primer binding site (PBS) is underlined.
FIG. 6
FIG. 6
Phylogenetic analysis of sheep type D retroviruses of sheep. Unrooted phylogenetic trees for U3 (A), env (B), and gag and pol (C) were derived by neighbor joining. To show consistency, all bootstrap values obtained with 1,000 replications of bootstrap sampling are shown. Sequences used for the analysis are termed as in their original references with the exception of loci 1 to 6, which are indicated as L1 to L6 in panel A. GenBank accession numbers: AF105220 (JSRV21); M80216 (JSRV-SA); X95445-X95452 (endogenous loci 1 to 6 and exogenous type I and II LTRs); Y16627 (ENTV); Y18301 to Y18305 (JS7, 809T, 83RS28, and 92K3); Z66531 to Z66533 (enJSRV1 to -3); Z71304 (LTR-UK); (AF136224) enJS5F16; (AF136225) enJS59A1; AF153615 (enJS56A1). In all trees there are five distinct phylogenetic groups: enJSRV-A and -B for the endogenous loci; and the ENTV group and two groups for exogenous JSRV, JSRV-I (African isolates), and JSRV-II (isolates from the United States and United Kingdom).
FIG. 7
FIG. 7
Expression of enJSRVs in vivo. In situ localization of enJSRV env mRNA in selected sheep tissues. Cross-sections of sheep tissues were hybridized with an α-35S-labeled antisense or sense DD54 cRNA probes. Hybridized sections were digested with RNase, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs taken under bright-field or dark-field illumination. (A to C) Day 11 cyclic ovine uterus; (D to F) intestine; (G to I) lung; (J to L) liver. Shown are bright-field exposures (A, D, G, and J); hybridization with antisense probes, dark-field exposures (B, E, H, and K); and hybridizations with sense probes, dark-field exposures (C, F, I, and L). All photomicrographs are shown at a magnification of ×600.
FIG. 8
FIG. 8
enJSRV LTR transcriptional activity. Plasmids penJS56A1-luc, penJS5F16-luc, and penJS59A1-luc were transfected into various cell lines as described in Materials and Methods. Cell lines were derived from mouse differentiated lung epithelial cells (MLE-15 and mtCC1-2) and extrapulmonary tissues such as mouse fibroblasts (NIH 3T3), mouse kidney (TCMK), and sheep endometrium (LE). Luciferase activities of the various endogenous locus LTRs as percentages of the activity of pJS21-luc, a reporter plasmid driven by the JSRV21 LTR, are shown (average of 6 to 12 replicates).
FIG. 9
FIG. 9
Effects of HNF-3α and HNF-3β on enJSRV LTRs. pJS21-luc, penJS56A1-luc, penJS5F16-luc, and penJS59A1-luc were cotransfected into NIH 3T3 cells (that do not efficiently support JSRV enhancer activity) along with an expression plasmid for either HNF-3α or HNF-3β. Different amounts of the transcription factor expression plasmids were cotransfected with a set amount (200 ng) of the reporter plasmid DNA. The amounts of luciferase activity for the different cotransfections are shown as fold activation over the activity of reporter plasmid cotransfected with a plasmid having the CMV promoter but not HNF-3 insert.
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