Transmissible gastroenteritis coronavirus genome packaging signal is located at the 5' end of the genome and promotes viral RNA incorporation into virions in a replication independent process

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nt from 5' end of its RNA genome, using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at position 106-136 nt. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of genome 3' end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging, were not sufficient to direct packaging of an heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well defined RNA motifs within TGEV RNA genome that are essential for packaging will be useful in the design of packaging-deficient biosafe coronavirus derived-vectors and to provide new targets for antiviral therapies.This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-167075) and the European Community's Seventh Framework Programme (FP7/2007-2013), under the project PoRRSCon (EC grant agreement 245141). L.M. and P.A.M.-G. received predoctoral fellowships from the Ministry of Science and Innovation of Spain (BES-2011-043489 and BES-2008-001932, respectively). L.D.P. received a predoctoral fellowship from the National Institute of Health (ISCIII) of Spain (FI08/00109)