THE 6-KILODALTON MEMBRANE-PROTEIN OF SEMLIKI FOREST VIRUS IS INVOLVEDIN THE BUDDING PROCESS

Alphavirus genomes encode a small hydrophobic protein of 6 kDa (the 6K protein) that is expressed as part of a large polyprotein containing the sequences of the two virus transmembranal glycoproteins which form the spikes of the infectious particle. Although made in amounts equiv alent to those of the glycoproteins, very little of the 6K protein is found in secreted infectious virions. The role of this protein in viru s replication and structure has been studied by use of-a variety of mu tationally altered forms of 6K, which yield phenotypically distinct vi ruses. A complete deletion of the gene encoding the 6K protein (Delta 6K) of Semliki Forest Virus (SFV) has been constructed from an SFV inf ectious cDNA and the transcribed RNA-produced progeny virus that close ly resembled the normal virus (P. Liljestrom, S. Lusa, D. Huylebroeck, and H. Garoff, J. Virol. 65:4107-4113, 1991). Further studies of this mutant have now been performed, and they show that growth of Delta 6K has a strong dependency on its host cell, varying from 2 to 50% of th e rate of formation of the wild-type SFV. Mammalian cells are much mor e defective than insect and avian cells in replication of the Delta 6K mutant. This mutant is not defective in formation and transport of th e glycoproteins or in production of nucleocapsids, which accumulate at the plasma cell membrane in infected BHK cells. The major defect, thu s, is in the final assembly and budding of new virus. In BHK cells inf ected with the Delta 6K strain, a relatively large fraction of the tot al infectious virus formed can be recovered by osmotic lysis of exhaus tively washed cells. Infectious SFV totally lacking 6K is identical to wild-type SFV in the early stages of virus replication, i.e., binding and uptake. The particles themselves are more thermolabile than those of wild-type SFV, suggesting that the 6K protein may be a part of the structure of mild-type virus or that the slower budding leads to an a ltered configuration of the trimeric spikes. These data support other studies that implicate the 6K protein as an important but nonessential component in the assembly and budding of the alphavirus particle, per haps by affecting the packing of the glycoproteins and their interacti ons with membrane lipid.