Translation mapping with the flavivirus Kunjin: gene order and anomalies in translation of Ns5.

Kunjin (KUN) virus-infected cells were synchronized in translation by reversal of hypertonic inhibition; cells were then pulse-labelled with [35S]methionine. Electrophoretic analyses defined the sequence of incorporation of label into all the known KUN gene products shown previously to be unique and unrelated. GP44 or NS1 was inadequately labelled for analysis and was assumed to be translated sequentially after E in the polyprotein sequence. The relationship of the KUN gene order obtained by translation mapping to that proposed by Rice et al. Science (1985) 229, 726-733 based on the nucleotide sequence of yellow fever virus is as follows: KUN: 5'-C.GP20.E.GP44.P19.P10.P71. (?).P21.P98-3' YF: 5'-C.prM.E.NS1.ns2a.ns2b.NS3.ns4a.ns4b. NS5-3'. These results eliminate the ambiguities in identities of the previously hypothetical ns2a, ns2b and ns4b. Although ns4a was not positively identified, a labelled protein of Mr 12,000 to 14,000 was observed in one experiment and it mapped in the appropriate position for ns4a. Variation occurred in translation of NS5 when the hypertonic treatment of 40 min at 37 degrees C was reduced in time or in temperature.