The alphavirus TF protein plays a critical role in promoting viral propagation by altering cell-cell boundaries

Abstract

During cellular infections, alphaviruses produce a 6 kDa membrane protein (6K) and a transframe variant (TF) of 6K through ribosomal frameshifting. The role of these proteins in alphavirus biology is largely unknown. Here, we show that TF of Chikungunya virus plays a unique and critical role in promoting virus propagation by manipulating host cell boundaries. TF achieves this by targeting and downregulating human Scribble, a component of the polarity complex that controls cellular morphology and apoptosis, leading to its punctate localization, ubiquitination, and subsequent degradation via the proteasomal pathway. The TF-Scribble interaction is mediated by a newly discovered PDZ-domain binding motif (PBM) in TF, with the PDZ domains of Scribble, as demonstrated by co-localization during viral infection and further by co-immunoprecipitation and proteomics analysis. This PBM is unique to TF and is absent in 6K; thus, 6K cannot modulate Scribble localization in infected cells. shRNA-mediated knockdown of Scribble potentiates CHIKV propagation highlighting the requirement of TF for the downregulation of Scribble. While wildtype CHIKV drastically alters cell-cell boundaries, a mutated version of CHIKV generated through reverse genetics ({Delta}TF CHIKV), which produces 6K but not TF, is unable to trigger similar morphological alterations due to its inability to engage Scribble, ultimately resulting in suboptimal virus propagation. A cryoEM reconstruction of {Delta}TF CHIKV at a global resolution of 3.6 [A], one of the highest reported among all alphavirus structures, indicates that the assembly of glycoprotein spikes and nucleocapsid is unaffected. Thus, the loss of TF affects the ultimate stage of cellular egress in the virus life cycle, and not earlier stages such as entry, replication or assembly. Our work thus establishes a novel function for the TF component of alphaviruses in modulating host morphology, potentiating virus transmission, and also distinguishes the functionality of TF from that of 6K in alphavirus biology. TeaserA novel functionality of Chikungunya Virus in modulating cell boundaries to allow efficient virus propagation