Extragenic and intragenic suppression of a transport mutation in the hemagglutinin gene of an influenza A virus as revealed by backcross and sequence determination.

Cooperation of viral proteins, or functional domains within a protein, can be studied by analyzing temperature-sensitive (ts) mutants and revertants carrying suppressor mutations. Accordingly, we have sequenced the hemagglutinin (HA) genes of a ts mutant of fowl plague virus (FPV), with a transport defect in the HA, and of five independent ts+ revertants (R1, R3, R4, R5, and R9). The amino acid replacement in position 480 from Thr to Ile, leading to the loss of a complex carbohydrate side chain, is responsible for the ts phenotype. R3, R4, and R5 are true revertants in that they have Thr in position 480, while R1 and R9 have kept Ile. The sequence of the HA of R1 is exactly the same as that of the ts mutant, while the R9 HA has two additional amino acid replacements in positions 91 (Lys-Thr) and 104 (Gly-Val). By doing a backcross with wild-type virus, it was shown that R1 carries an extragenic suppressor mutation, while R9 is intragenically suppressed. We conclude that the HA is transported from the site of its synthesis in the rough endoplasmic reticulum (RER) to the plasma membrane along with another viral gene product, which by mutation can complement the ts defect. An alternative interpretation is that the ts mutation results from a change in HA which allows an interacting protein to bind HA too soon, holding it back in the RER. The suppressor mutation may remove this premature interaction.