Interfacial domains in Sindbis virus 6K protein. Detection and functional characterization.
Alphavirus 6K is a short, constitutive membrane protein involved in virus glycoprotein processing, membrane permeabilization, and the budding of virus particles. The amino-terminal region that immediately precedes the transmembrane anchor contains a conserved sequence motif consisting of two interfacial domains separated by Asn and Gln residues. The presence of this motif confers on the 6K pretransmembrane region the tendency to partition into the membrane interface. To study the functional importance of the interfacial sequences, three different Sindbis virus 6K variants were obtained with the following modifications: 9YLW11xAAA, 18FWV20xAAA, and 9YLW11xAAA/18FWV20xAAA. Reconstituted mutant viruses were infectious and showed no defects in glycoprotein processing, although virus budding was hampered. Single 6K expression in Escherichia coli cells showed interfacial mutants to have a diminished capacity to modify membrane permeability and to have lower toxicity. In particular, the 9YLW11xAAA/18FWV20xAAA variant was expressed at high levels and did not enhance membrane permeability significantly, although it retained its integral membrane protein condition. Parallel analyses of membrane permeabilization in baby hamster kidney cells were carried out using a Sindbis virus replicon that synthesized both capsid protein and 6K. Transfection of the construct with wild-type 6K strongly increased permeability to the antibiotic hygromycin B. Replicons encoding 6K interfacial mutants induced lower membrane permeabilization. Again, the greatest impairment was observed for the 9YLW11xAAA/18FWV20xAAA variant, permeabilization activity of which was approximately 10% that of wild-type 6K. These findings show the importance of the interfacial 6K sequence for virus budding and modification of membrane permeability.
Sanz
Miguel Angel
MA
Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain. masanz@cbm.uam.es
Madan
Vanessa
V
Carrasco
Luis
L
Nieva
José Luis
JL
D000595:Amino Acid Sequence; D000818:Animals; D002463:Cell Membrane Permeability; D006224:Cricetinae; D018274:Electroporation; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
C092026:6K protein, Sindbis virus; D014759:Viral Envelope Proteins