Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus.

The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.