PATTERNS OF INTEGRATION AND EXPRESSION OF RETROVIRAL, NONREPLICATIVE VECTORS IN AVIAN EMBRYOS - EMBRYO DEVELOPMENTAL STAGE AND VIRUS SUBGROUP ENVELOPE MODULATE TISSUE-TROPISM

We previously demonstrated that Avian Leukemia Viruses (ALV) carrying the v-myc gene specifically induce two types of tumors, cardiomyocytic tumors when the virus is injected before embryonic day 3 (E3), skin t umors when the virus is injected at E3 or E5. Aiming to elucidate the mechanisms which determine this time-dependent change in target, we in fected chick and quail embryos at E3 and E5 with replication-deficient , lacZ gene-carrying, ALV-based viruses produced by a packaging cell l ine. Three constructs driven by 3 different Long Terminal Repeats (LTR s) were tested and yielded similar results. When the constructs were i noculated at E3 and the lacZ gene product revealed 5 days later, aroun d 70% of the embryos carried lacZ+ clones in the heart, around 50% had positive clones in the skin anywhere on the body, while a few embryos displayed clones in internal organs (liver, stomach, lungs). Immunocy tological identification of the heart cell type(s) expressing the viru s revealed that the only cells infected were cardiomyocytes. When the constructs were inoculated at E5, no lacZ+ clones appeared in the hear t but all were located in the cephalic skin. In order to examine the r elationship between viral integration and expression, DNA of different organs or tissues from lacZ stained embryos was analyzed by PCR. A ti ght correlation between integration and expression in the heart and in the skin was revealed in most cases. In contrast, a significant PCR s ignal was often detected in the liver or the stomach despite weak or a bsent expression as revealed by lacZ+ clones. We then investigated the influence of envelope glycoprotein subgroups on the tropism of these constructs. The lacZ vector driven by RAV-2 LTRs was packaged as subgr oups A, B or E viral particles. The A subgroup, used in the part of th e study described above, infects both chick and quail while the B and E subgroups are specific for chick or quail respectively. These B and E subgroups induced lacZ+ clones in the heart (after E3 injection) whi le no clones or only a few were detected in the skin either after E3 o r E5 injection. The following conclusions can be drawn: 1) cardiomyocy tes are at E3 the major target for integration and expression of ALV-d erived viruses in vivo; 2) targets change rapidly with embryonic age; and 3) tissue-specific infections depend on the envelope subgroup, thu s presumably on the presence of the cognate receptor. This study clear ly indicates that E3 inoculation of ALV-based retroviral vectors is a simple and powerful method to transfer gene sequences into cardiomyocy tes and epidermal cells.