Structure and expression of the 27.7 KDA protein of the transmissible gastroenteritis coronavirus

Coronaviruses are important pathogens of wild and domestic mammals and birds, and humans, causing primarily acute infections of the respiratory and gastroenteric systems. To understand its role in the replication of porcine transmissible gastroenteritis virus, a detailed study was made of gene 3b, and its product, a protein of 27.7 kilodaltons. In vitro, the 27.7 kilodalton protein expressed from the cloned gene becomes partially translocated into microsomes apparently through the use of a noncleaved hydrophobic N-terminal signal sequence, becomes glycosylated on two of three potential asparagine linkage sites, and remains anchored by a centrally located hydrophobic domain. Immunofluorescence studies show the protein is made in infected cells and localizes primarily at concentrated sites in the perinuclear region where virus is known to assemble. It therefore must function as an integral membrane protein at some stage in the process of virus replication. An unusual feature of gene 3b is that it is translated as the 3' gene from the unique region of bicistronic mRNA3. To investigate how ribosomes approach the downstream open reading frame, s)mthetic transcripts of the wild-type mRNA 3 and mRNA 3 modified to carry the chloramphenicol acetyl transferase gene upstream of gene 3a were studied. Deletion analyses indicated that an internal ribosomal entry region exists very near the translation initiation site for 3b and ruled out the likelihood that ribosomes scan through gene 3a to reach gene 3b. These experiments indicate that ribosomes approach gene 3b by downstream entry, possibly by the newly described mechanism of ribosomal shunting.