Interactions between PE2, E1, and 6K required for assembly of alphaviruses studied with chimeric viruses.
During the assembly of alphaviruses, a preassembled nucleocapsid buds through the cell plasma membrane to acquire an envelope containing two virally encoded glycoproteins, E2 and E1. Using two chimeric viruses, we have studied interactions between E1, E2, and a viral peptide called 6K, which are required for budding. A chimeric Sindbis virus (SIN) in which the 6K gene had been replaced with that from Ross River virus (RR) produced wild-type levels of nucleocapsids and abundant PE2/E1 heterodimers that were processed and transported to the cell surface. However, only about 10% as much chimeric virus as wild-type virus was assembled, demonstrating that there is a sequence-specific interaction between 6K and the glycoproteins required for efficient virus assembly. In addition, the conformation of E1 in the E2/E1 heterodimer on the cell surface was different for the chimeric virus from that for the wild type, suggesting that one function of 6K is to promote proper folding of E1 in the heterodimer. A second chimeric SIN, in which both the 6K and E1 genes, as well as the 3' nontranslated region, were replaced with the corresponding regions of RR also resulted in the production of large numbers of intracellular nucleocapsids and of PE2/E1 heterodimers that were cleaved and transported to the cell surface. Budding of this chimera was severely impaired, however, and the yield of the chimera was only approximately 10(-7) of the SIN yield in a parallel infection. The conformation of the SIN E2/RR E1 heterodimer on the cell surface was different from that of the SIN E2/SIN E1 heterodimer, and no interaction between viral glycoproteins and nucleocapsids at the cell plasma membrane could be detected in the electron microscope. We suggest that proper folding of the E2/E1 heterodimer must occur before the E2 tail is positioned properly in the cytoplasm for budding and before heterodimer trimerization can occur to drive virus budding.
Yao
J S
JS
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Strauss
E G
EG
Strauss
J H
JH
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D016865:Reassortant Viruses / Q000502:physiology*; D012398:Ross River virus / Q000502:physiology*; D012845:Sindbis Virus / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins; D019065:Virus Assembly
C092026:6K protein, Sindbis virus; D008562:Membrane Glycoproteins; D011498:Protein Precursors; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C022012:glycoprotein E1, Sindbis virus; C064950:glycoprotein E2, Sindbis virus; C102373:glycoprotein PE2, Sindbis virus