The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without Ha-ras co-transfection

Abstract

Hepatitis C virus (HCV) is an important cause of chronic liver disease and is complicated by hepatocellular carcinoma (HCC). Mechanisms whereby the virus promotes cellular transformation are poorly understood. We hypothesized that the guanosine triphosphatase activity encoded in the HCV NS4B protein's nucleotide binding motif (NBM) might play a role in the transformation process. Here we report that NS4B can transform NIH-3T3 cells, leading to tumor formation in vivo. This transformation was independent of co-transfection with activated Haras. Detailed analyses of NS4B mutants revealed that this transforming activity could be progressively inhibited and completely abrogated by increasing genetic impairment of the NS4B nucleotide binding motif. Conclusion: NS4B has in vitro and in vivo tumorigenic potential, and the NS4B transforming activity is indeed mediated by its NBM. Moreover, our results suggest that pharmacological inhibition of the latter might inhibit not only HCV replication but also the associated HCC. (HEPATOLOGY 2008;47:827-835.) C hronic infection with the hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The incidence of HCC and the mortality rate associated with it are increasing dramatically. 1,2 Although chronic inflammation, fibrosis, and liver cell proliferation are considered the major factors contributing to the development of HCC, accumulating data support direct viral effects as well. 3 HCV is a positive single-stranded RNA virus. Its 9.6-kb genome encodes a single ϳ3000-amino-acid polyprotein, which is proteolytically processed into structural proteins, which are components of the mature virus, and nonstructural proteins, which are involved in replicating the viral genome. 4 Several viral proteins including NS3, NS5A, and core have been implicated in cellular transformation. 5-8 NS4B, a 27-kDa membrane protein, has been similarly shown by Park et al 9 to transform NIH 3T3 cells. 9 Transformation, however, only occurred when NS4B was co-transfected with the activated Ha-ras gene. The mechanism by which NS4B mediates its transformation potential remains unknown.We have recently reported the identification of a nucleotide-binding motif (NBM) within NS4B and shown that this motif mediates both binding and hydrolysis of guanosine triphosphate (GTP) and HCV RNA replication. 10 The NBMs of human oncogenes, such as ras, mediate their malignant transformation. 11,12 We thus hypothesized that the NBM of NS4B may similarly mediate its role in transformation. Here we report that NS4B of the Con 1 isolate transforms NIH3T3 cells independently of exogenous Ha-ras, the transformed cells lead to tumor formation in nude mice, and the NBM of NS4B mediates the latter's role in transformation, with exciting implications for novel therapies. Materials and MethodsPlasmids. Standard recombinant DNA technology was used to construct and purify all plasmids. All regions

Conserved GXXXG- and S/T-Like Motifs in the Transmembrane Domains of NS4B Protein Are Required for Hepatitis C Virus Replication

“…Thus, the findings that the G125L and G129L mutations lead to severe decreases in JFH1 replication efficiency suggest that Gly 125 and Gly 129 may be part of a GXXXG-like motif. However, G 129 is also the first residue of a previously reported Walker A motif (G 129 SIGLGK 135 ) (19). Therefore, the phenotype of G129L mutation may just reflect the loss of NS4B GTPase activity.…”

Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory

“…A recent study using microfluidic affinity analysis described an interaction between HCV RNA and in vitro transcribed and translated NS4B [89]. This interaction appears to be selective for the 3 0 end of the viral negative-strand RNA and involves several Arg residues located predominantly in the C-terminal part of NS4B.…”

Hepatitis c virus and host cell lipids: An intimate connection

“…49 Ectopic expression of NS4B in mammalian cells leads to membrane alterations resembling the so called "membranous web" (MW) that is induced upon HCV infection. 50 It has been reported that NS4B can directly bind to HCV RNA, 51 and possesses ATPase and GTPase activities for which an NTPbinding site, located in a cytosolic loop between TM helices 2 and 3 appears to be required. 52 However, the importance of these properties of NS4B for the viral replication cycle is still not clear.…”