Venezuelan equine encephalitis virus non-structural protein 3 dictates superinfection exclusion in mammalian cells

Superinfection exclusion (SIE) prevents secondary infections of already infected cells. Arthritogenic alphaviruses induce SIE via early proteolytical cleavage of replicase precursor by non-structural protein 2 (nsP2). Here, we explore the SIE mechanism of the encephalitic Venezuelan equine encephalitis virus (VEEV). Using single-cell imaging techniques and VEEV replicons encoding green or red fluorescent proteins, we observed full SIE capacity in three hours. Transient expression of VEEV nsP3, but not nsP2, reduced alphavirus replication, suggesting a key role for VEEV nsP3 in the SIE mechanism. In particular, the VEEV nsP3 C-terminal hypervariable domain (HVD) was found to be required and sufficient for the SIE of VEEV and the more distantly related Sindbis virus. As the nsP3 HVD is known to bind multiple host proteins to form RNA replication complexes and modulate the cellular stress response, we propose that sequestering essential host protein(s) by VEEV nsP3 interferes with RNA replication of the superinfecting alphavirus.