Structure-function analysis of the Sendai virus F and HN cytoplasmic domain: Different role for the two proteins in the production of virus particle

The role of the cytoplasmic domain (cytd) of the Sendai virus HN and F glyc oproteins in the process of virus assembly and budding are evaluated. Recom binant Sendai virus (rSeV) mutants are generated carrying modifications In the cytd of each of the glycoprotein separately. The modifications include increasing truncations and/or amino acid sequence substitutions. Following steady-state (35)[S]methionine/cysteine labeling of the infected cells, the virus particle production is estimated. The radioactive virions in the cel l supernatants are measured relative to the extent of the infection, assess ed by the Intracellular N protein signal. For both the F and HN cytd trunca tion mutants, the largest cytd deletions lead to a 20- to 50-fold reduction in virion production. This reduction cannot be explained by a reduction of the cell surface expression of the glycoproteins. For the F protein mutant s, the virions produced in reduced amount always exhibit a normal F protein composition. It is then concluded that a threshold level of F is required for SeV assembly and budding. The rate or the efficiency with which this th reshold is reached up appears to depend on the nature of the F cytd. A mini mal cytd length is required as well as a specific sequence. The analysis of HN protein mutants brings to light an apparent paradox. The larger cytd tr uncations result In significant reduction of virion production. On the othe r hand, a normal virion production can take place with an underrepresentati on of or, even, an undetectable HN in the particles. The HN uptake in virio n is confirmed to depend on the previously proposed cytd SYWST signal (T. T akimoto, T Bousse, E. C. Coronel, R. A Scroggs, and A. Portner. 1998. J. Vi rol. 72, 9747-9754). (C) 2000 Academic Press.