Restoration of Replication Phenotype of Lamivudine-Resistant Hepatitis B Virus Mutants by Compensatory Changes in the “Fingers” Subdomain of the Viral Polymerase Selected as a Consequence of Mutations in the Overlapping S Gene

Abstract

The introduction of lamivudine (LMV) for the treatment of chronic hepatitis B infection has been an important advance in the management of this disease. However, the long-term efficacy of LMV may become limited by the emergence of antiviral-resistant hepatitis B virus (HBV) mutants. The two most common LMV-resistant mutants produce changes in the viral polymerase protein (rt) of rtM204I and rtL180M/M204V (previously rtM550I and rtL526M/M550V). A number of studies have demonstrated that these HBV mutants appear to be replication impaired, both in vitro and in vivo. The detection and selection of compensatory mutations in the polymerase protein that restore the replication phenotype of these HBV mutants have been poorly described to date. The effects of mutations in the fingers subdomain of the viral polymerase protein arising as a consequence of vaccine and hepatitis B immune globulin (HBIg) selected changes in the overlapping envelope gene (S), and a determinant of the hepatitis Bs antigen (HBsAg) were analyzed in vitro. The LMV-resistant HBV mutants rtM204I and rtL180M/M204V produced substantially weaker HBV DNA replicative intermediate signals by Southern blot analysis and less total intracellular HBV DNA by real-time PCR compared to wild-type virus. The viral polymerase protein of these mutants produced little detectable radiolabeled HBV DNA in an endogenous polymerase assay. In contrast, the HBV a determinant HBIg/vaccine escape mutants sP120T, sT123N, sG145R, and sD144E/G145R (that produce rtT128N, Q130P, rtW153Q, and rtG153E respectively) yielded as much virus as wild-type HBV while the sM133L (rtY141S) mutant was replication impaired. Two of these mutants, rtT128N and rtW153Q, when introduced into a replication-competent HBV vector containing the rtL180M/M204V polymerase mutation restored the replication phenotype of this LMV-resistant mutant. These viruses produced levels of intracellular HBV DNA as determined by Southern blot and real-time PCR that were comparable to those of wild-type HBV, indicating that the changes in the fingers subdomain were able to compensate for the reduced replication of the LMV-resistant mutations. Since these viruses carry mutations in the a determinant of HBsAg that may potentially decrease the ability of anti-HBs antibody to neutralize these viruses, these HBV mutants also have the potential to behave as vaccine escape mutants.

Occult hepatitis B in blood donors in Indonesia: altered antigenicity of the hepatitis B virus surface protein

“…These mutations lay between domain A and B of the reversetranscriptase region of polymerase protein, which is crucial for its function in the HBV replication process [35,36]. One or several of the identified mutations could be responsible for diminished rate of replication causing the detection failure of HBsAg.…”

Profile of Mutations in the Reverse Transcriptase and Overlapping Surface Genes of Hepatitis B Virus (HBV) in Treatment-Naïve Indonesian HBV Carriers

“…Mutations at positions rt80, rt173, and rt180 belong to this category (7). Some putative NAr mutations have been reported (8)(9)(10)(11). Unfortunately, not all the effects of these mutations have been verified experimentally in vitro.…”

HBsAg Variants: Common Escape Issues

“…Earlier studies clearly indicated that the transfusion of blood containing HBsAg has been associated with the development of post transfusion hepatitis B (72)(73)(74), and that cases of HBV infection have been identified after transfusion of a HBsAg negative blood supply (75)(76)(77). Moreover, it is well known that mutants of HBsAg are able to cause infection and horizontal transmission despite the presence of anti-HBs (78)(79)(80)(81)(82).…”

“…This suggests that the native conformation of the second ''a'' loop is not essential, at least for the entrance of the virus into the cell. As a consequence it may be argued that the second ''a''-loop related antibodies may not be protective on their own, although they may lower the viral load in the circulation by the formation of aggregates that are easily opsonized (81). Escape mutants may breakthrough vaccine programs and spread locally (85).…”