CONSERVATIVE MUTATIONS IN THE IMMUNOSUPPRESSIVE REGION OF THE BOVINE LEUKEMIA-VIRUS TRANSMEMBRANE PROTEIN AFFECT FUSION BUT NOT INFECTIVITYIN-VIVO

Many retroviruses, including bovine leukemia virus (BLV), contain a hi ghly conserved region located about 40 amino acids downstream from the fusion peptide within the sequence of the external domain of the tran smembrane (TM) protein. This region is notably thought to be involved in the presentation of the NH2-terminal peptide to allow cell fusion, By using hydrophobic cluster analysis and by analogy with the influenz a A hemagglutinin structures, the core of the TM structure including t his particular region was predicted to consist, in the BLV and other r etroviral envelope proteins, of an alpha-helix followed by a loop regi on, both docked against a subsequent alpha-helix that forms a triple-s tranded coiled coil. The loop region could undergo, as in hemagglutini n, a major refolding into an alpha-helix integrating the coiled coil s tructure and putting the fusion peptide to one tip of the molecule. Ba sed on this model, we have identified amino acids that may be essentia l to the BLV TRI structure, and a series of mutations were introduced in the BLV env gene of an infectious molecular clone. A first series o f mutations was designed to disturb the coiled coil structure (substit utions with proline residues), whereas others would maintain the gener al TM structure. When expressed by Semliki Forest virus recombinants, all the mutated envelope proteins were stable and efficiently synthesi zed in baby hamster kidney cells. Both proline-substituted and conserv ative mutants were strongly affected in their capacity to fuse to CC81 indicator cells. In addition, it appeared that the integrity of the T RI coiled coil structure is essential for envelope protein multimeriza tion, as analyzed by metrizamide gradient centrifugation, Finally, to gain insight into the role of this coiled coil in the infectious poten tial of BLV in vivo, the mutated TM genes were introduced in an infect ious and pathogenic molecular clone and injected into sheep. It appear ed that only the conservative mutations (A60V and A64S) allowed mainte nance of viral infectivity in rico, Since these mutations destroyed th e ability to induce syncytia, we conclude that efficient fusion capaci ty of the recombinant envelopes is not a prerequisite for the infectio us potential of BLV in vivo. Viral propagation of these mutants was st rongly affected in some of the infected sheep. However, the proviral l oads within half of the infected animals (2 out of 2 for A60V and 1 ou t of 4 for A64S) were close to the wild-type levels, In these sheep, i t thus appears that the A60V and A64S mutants propagate efficiently de spite being unable to induce syncytia in cell culture.