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

Superinfection exclusion (SIE) is a well-known phenomenon induced by a broad spectrum of viruses to hinder a virus from the same virus family to establish a secondary infection in an already infected cell. Despite many years of study, the molecular mechanism(s) of alphavirus SIE remain enigmatic. Alphaviruses are arthropod-borne viruses that cause arthritogenic or encephalitic diseases in vertebrates, depending on the viral species. Several arthritogenic alphaviruses are known to block RNA replication of a superinfecting alphavirus via early proteolytic cleavage by non-structural protein 2 (nsP2). Here, we explore for the first time the SIE mechanism of an encephalitic alphavirus, Venezuelan equine encephalitis virus (VEEV). Using single-cell imaging techniques and encapsidated VEEV replicons encoding green or red fluorescent proteins, we observed immediate onset of VEEV interference, which increases to nearly full SIE capacity in three hours. In a series of experiments, we observed that transient expression of VEEV nsP3, but not nsP2, reduced alphavirus replication in the same mammalian cell, suggesting a key role for VEEV nsP3 in the mechanism of SIE. In particular, the VEEV nsP3 C-terminal hypervariable domain (HVD) was found to be required and sufficient for 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 of essential host protein(s) by VEEV nsP3 interferes with RNA replication of the superinfecting alphavirus.