Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

Abstract

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has
pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that
the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and
distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by
unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4
in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length
NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a
HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic
reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins.
Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC)
marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity
and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not
inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular
structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal
markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was
dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal
marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with
viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement
of NSP4 in virus replication.

 


read-online

Post Comment