X-deficient woodchuck hepatitis virus mutants behave like attenuated viruses and induce protective immunity in vivo
Zhensheng Zhang, … , James Jacob, T. Jake Liang
Zhensheng Zhang, … , James Jacob, T. Jake Liang
Published November 15, 2001
Citation Information: J Clin Invest. 2001;108(10):1523-1531. https://doi.org/10.1172/JCI13787.
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Article

X-deficient woodchuck hepatitis virus mutants behave like attenuated viruses and induce protective immunity in vivo

Abstract

The X protein (HBX) of the hepatitis B virus (HBV) has been shown to be important for the establishment of HBV infection in vivo. Our previous studies suggested that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. In this study, we generated a series of woodchuck hepatitis virus (WHV) X mutants, including mutants of the domain interacting with the proteasome, and studied their infectivity in woodchucks. Many of the mutants were defective in transactivation but none of them were completely replication defective in vitro. In vivo, all the wild-type and some X mutant–transfected animals demonstrated evidence of infection with anti-WHc and/or anti-WHs seroconversion. Most of the wild-type– and X mutant–transfected animals had transient viremia. Some animals were later challenged with infectious WHV. Animals inoculated with X mutants, including those with no serologic evidence of infection, were protected from the challenge, suggesting previous infection with resulting protective immunity. Our study demonstrates that the previously described functional domains of HBX are biologically important and the X-defective mutants, possibly as attenuated viruses, are not completely replication defective in vivo.

Authors

Zhensheng Zhang, Nobuyuki Torii, Zongyi Hu, James Jacob, T. Jake Liang

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Figure 4

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PCR analyses of WHV DNAs. (a) Standard PCRs were performed with three pa...
PCR analyses of WHV DNAs. (a) Standard PCRs were performed with three pairs of primers: Pre-X.F and X.R pair (NX pair) to amplify N terminus of X with expected PCR product of 543 bp’s; X.F and C.R pair (CX pair) to selectively amplify C terminus of X of viral DNA (expected PCR product: 597 bp’s); CMV.F and C.R pair (CMVX pair) to amplify only cloned DNA (expected PCR product: 504 bp’s). The positions of these primers in the viral DNA and cloned DNA are shown. (b) Amplification refractory mutation detection system for wild-type sequence in X mutant inoculum DNA. The position of the wild-type–specific primer Xwt.F is shown in a. Wild-type and Xlg– constructs were mixed at ratios of 1:10 to 1:10,000. One nanogram of each mixture and the mutant inoculum was amplified for 30 cycles as described in Methods. (c) PCR-Southern analyses of viral and cloned WHV DNAs with three pairs of primers: lanes 1 and 4 with NX pair; lanes 2 and 5 with CX pair; lanes 3 and 6 with CMVX pair. The viral DNA was extracted from WHV-containing serum of known titer. About 1 pg of viral or cloned DNA was used for the analyses.