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

Integrated into the sheep genome are 15 to 20 copies of type D endogenous l oci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenou s viruses cause infectious neoplasms of the respiratory tract in small rumi nants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively calle d enJRSVs) and analyzed their genomic structures, their phylogenies with re spect to their exogenous counterparts, their capacity to form viral particl es, and the expression specificities of their long terminal repeats (LTRs), In addition, the pattern of expression of enJSRVs in vivo was studied by i n situ hybridization, All of the three enJSRV proviruses had open reading f rames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble vi ral particles when highly expressed in human 293T cells. We localized the d efect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecula r clone JSRV(21). Phylogenetic analysis distinguished five ovine type D ret roviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (Afri can versus United Kingdom/North America isolates). Transient transfection a ssays indicated that the LTRs of the three enJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulm onary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of nor mal ovine tissues revealed high expression of enJSRV mRNA in the luminal ep ithelium and glandular epithelium of the uterus; lower expression was local ized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.