Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex.
The replicase polyproteins of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are processed by three viral proteases to yield 12 non-structural proteins (nsps). The nsp2 and nsp3 cleavage products have previously been found to interact, a property that allows nsp2 to act as a co-factor in the processing of the downstream part of the polyprotein by the nsp4 protease. Remarkably, upon infection of Vero cells, but not of BHK-21 or RK-13 cells, EAV nsp2 is now shown to be subject to an additional, internal, cleavage. In Vero cells, approximately 50% of nsp2 (61 kDa) was cleaved into an 18 kDa N-terminal part and a 44 kDa C-terminal part, most likely by a host cell protease that is absent in BHK-21 and RK-13 cells. Although the functional consequences of this additional processing step are unknown, the experiments in Vero cells revealed that the C-terminal part of nsp2 interacts with nsp3. Most EAV nsps localize to virus-induced double-membrane structures in the perinuclear region of the infected cell, where virus RNA synthesis takes place. It is now shown that, in an expression system, the co-expression of nsp2 and nsp3 is both necessary and sufficient to induce the formation of double-membrane structures that strikingly resemble those found in infected cells. Thus, the nsp2 and nsp3 cleavage products play a crucial role in two processes that are common to positive-strand RNA viruses that replicate in mammalian cells: controlled proteolysis of replicase precursors and membrane association of the virus replication complex.
Snijder
Eric J
EJ
Department of Virology, Center of Infectious Diseases, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands1.
van Tol
Hans
H
Department of Virology, Center of Infectious Diseases, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands1.
Roos
Norbert
N
Department of Biology, Division of Electron Microscopy, University of Oslo, Norway2.
Pedersen
Ketil W
KW
Department of Biology, Division of Electron Microscopy, University of Oslo, Norway2.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D007240:Equartevirus / Q000201:enzymology* / Q000502:physiology; D015870:Gene Expression; D005822:Genetic Vectors; D006736:Horses; D016366:Open Reading Frames; D020815:Polyproteins / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D011817:Rabbits; D012845:Sindbis Virus; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D020815:Polyproteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase