NUCLEOCAPSID-GLYCOPROTEIN INTERACTIONS REQUIRED FOR ASSEMBLY OF ALPHAVIRUSES

We have studied interactions between nucleocapsids and glycoproteins r equired for budding of alphaviruses, using Ross River virus-Sindbis vi rus chimeras in which the nucleocapsid protein is derived from one vir us and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid prot ein had been replaced with that from Sindbis virus was almost nonviabl e. Nucleocapsids formed in normal numbers in the infected cell, but ve ry little virus was released from the cell. There are 11 amino acid di fferences between Ross River virus and Sindbis virus in their 33-resid ue E2 cytoplasmic domains. Site-specific mutagenesis was used to chang e 9 of these 11 amino acids in the chimera from the Ross River virus t o the Sindbis virus sequence in an attempt to adapt the E2 of the chim era to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provi des direct evidence for a sequence-specific interaction between the nu cleocapsid and the E2 cytoplasmic domain during virus budding. The mut ated chimeric virus readily gave rise to large-plaque variants that gr ew almost as well as Ross River virus, suggesting that additional sing le amino acid substitutions in the structural proteins can further enh ance the interactions between the disparate capsid and the glycoprotei ns. Unexpectedly, change of E2 residue 394 from lysine (Ross River vir us) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interaction s, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glyco protein spikes. The reciprocal chimera, Sindbis virus containing the R oss River virus capsid, also grew poorly. Suppressor mutations arose r eadily in this chimera, producing a virus that grew moderately well an d that formed larger plaques.