X Region Deletion Mutants Associated With Surface Antigen- Positive Hepatitis B Virus Infections

Transcription

1 GASTROENTEROLOGY 1995;108: X Region Deletion Mutants Associated With Surface Antigen- Positive Hepatitis B Virus Infections MARK A. FEITELSON,* LING-XUN DUAN,* JIANHUI GUO,* and BARUCH S. BLUMBERG *Department of Pathology, Anatomy, and Cell Biology and tdorrance H. Hamilton Laboratories, Section of Molecular Retrovirology, Division of Infectious Diseases, Department of Medicine, Thomas Jefferson University, Philadelphia; and ~Fox Chase Cancer Center, Philadelphia, Pennsylvania Background/Aims: The finding of antibodies against the polymerase of hepatitis B virus in renal dialysis patients before the incubation phase of infection implies underlying virus replication. Hence, the aim of the study was to test for virus during infection. Methods: Viremia was assayed in virus-infected and control patients using the polymerase chain reaction and Southern blotting. Results: Six months before the appearance of surface antigen, most patients had detectable core region, but few patients were X region positive. Three months after surface antigen appeared, most carriers had detectable core and X products. Three years after surface antigen appeared, 5 of 8 carriers with persistent hepatitis B e antigen and I of 8 carriers with corresponding antibody had these products. Cloning and sequencing showed deletions within the X/ precore region of viral DNA. Conclusions: Infection with X region mutants precedes that of wild-type virus, and they reappear after wild-type virus is eliminated in carriers. A hallmark of hepatitis B virus (HBV) infection is the variability in its pathogenesis. This variability ranges from fulminant, acute, or chronic hepatitis to subacute infections and the asymptomatic carrier state. Although many carriers remain asymptomatic, others develop chronic hepatitis, which could resolve or further develop into cirrhosis, dysplasia, and primary hepatocellular carcinoma. *'2 The overlying serological patterns of HBV infection are also complex and are not always reflective of underlying liver disease. Although there is considerable evidence suggesting that the pathogenesis of HBV infection is mediated by immune responses against virus-infected cells, 3'4 virus variants that arise during natural infections may also play important roles in pathogenesis. 5'6 For example, HBV variants with one or more mutations in the precore region of the virus genome seem to be associated with fulminant hepatitis, v'8 Precore mutants are also found in some patients in the course of antiviral chemotherapy. 9'x A point mutation in the surface antigen encoding region is associated with vaccine escape mutants. ~1 Mutations within the pres 12 and core 13 regions have also been described, although the biological significance of such mutants remains to be fully established. In this context, the finding of antibodies against the polymerase of HBV (anti-pol) in patients undergoing renal dialysis prior to the incubation period of infection 14 is consistent with virus replication in the liver and that such an unusual serological presentation may reflect infection with mutant strains of HBV. The fact that anti-pol and/ or hepatitis B x antigen (HBxAg), which are associated with virus replication, ~4-16 may persist after the clearance of wild-type virus and the appearance of antibody to hepatitis B e antigen (anti-hbe) or antibody to hepatitis B surface antigen (anti-hbs) is consistent with continued virus replication, although most of the virus has been cleared from serum The finding of HBxAg-related polypeptides in the replication complex of hepadnaviruses, *9 combined with the apparent inverse relationship between the presence of antibody against the hepatitis B x antigen (anti-hbx) and wild-type virus replication during infection,.7 suggests that immune responses against HBxAg may select for X-negative virus variants. In this study, serial serum samples collected from HBV-infected patients undergoing renal dialysis were tested for the presence of mutants by polymerase chain reaction (PCR) amplification of different regions of the viral DNA. The resuits show the existence of deletion mutants in and around the X open reading frame. Similar mutants have been found recently in multiply transfused children with ~- thalassemia 2 and in some woodchuck hepatitis virusinfected woodchucks. 2~ The relationship of these mutants to the outcome of hepatitis B surface antigen (HBsAg)- positive infections is presented. Abbreviations used in this paper: anti-hbx, antibodies against the hepatitis B x antigen; anti-pol, antibodies against the polymerase product(s) of hepatitis B virus; HBxAg, hepatitis B x antigen; PCR, polymerase chain reaction; prec region, the precore encoding region of hepatitis B virus DNA; pres/s region, region of viral DNA encoding the pres/s envelope polypeptides by the American Gastroenterological Association /95/$3,00

2 W W June 1995 HBV X DELETION MUTANTS 1811 Table 1. Patterns of HBV DNA in Patients Undergoing Renal Dialysis -6 mo +3 mo +3 yr X+ X+ Patient Anti pot Core X core Dot blot Core X core Patient category no. positive a PCR PCR PCR hybridization PCR PCR PCR Dot blot hybridization X Core X core Dot blot PCR PCR PCR hybridization Long-term carriers HBeAg persistent (n = 8) HBeAg to anti- HBe (n = 8) Acute infection Transient HBsAg (n - 7) Dialysis controls No markers and normal ALT (n - 21) 1 After HBsAg w - m - w w w W w - w w + 3 w m - w w w w w w mo w - m w w m + 6 After HBsAg w w w - w w w me w w w - W w w mo w - m - w w w + 9 w - m - w m + 10 w - m - w w w w w w w w w + 13 After HBsAg w - m - w mo w m w w w mo w - w w w + 16 After HBsAg w - m - w w w + 17 After HBsAg w m - w - m mo w - w mo - - w w w w w - - w w w/m mo w w w - w w w mo w m - w w w + w w w/m - w w w + w w w + w w w + w m + w w w + w m - w m - w m w - - w m - w w w w w m - w m - W m w -- m W -- m w w m w w w w w w athe number of months indicates the initial appearance of antbpol relative to HBsAg in each patient. m, faster migrating (mutant) PCR product; w, wild type-sized PCR product. Materials and Methods Patients The patients undergoing renal dialysis used herein have been described previously. 14-1v Briefly, sera from patients undergoing long-term renal dialysis at clinics in the Delaware Valley were collected bimonthly during a 2-10-year duration (spanning ). During the period of collection, a number of patients developed markers of HBV infection with or without elevated transaminases, whereas the remainder seemed uninfected. None of the patients had undergone any form of antiviral treatment. For these studies, a total of 44 patients were studied from the following groups (Table 1). The first group consisted of 8 long-term carriers who were persistently positive for hepatitis B e antigen (HBeAg) (for at least 3 years after the appearance of HBsAg). Five of these patients had one or more episodes of elevated alanine aminotransferase (ALT) levels in blood. The second group consisted of 8 tong-term carriers who seroconverted from HBeAg to anti-hbe during infection. All patients in this group remained HBsAg positive after the appearance of anti-hbe, and all patients had elevated ALT values during the period of observation. The third group consisted of 7 pa- tients with acute HBV infection, indicated by the transient appearance of HBsAg (<6 months). Six of the seven patients had transiently detectable HBeAg, and in 3 cases, the patients seroconverted from HBeAg to anti-hbe. Each of these patients also had an ALT peak during acute infection. The fourth group consisted of 21 patients who had normal transaminases (ALT value of <40) and no detectable HBsAg, anti-hbs, or antibody to hepatitis B core antigen (anti-hbc). Among HBsAg-posirive patients, serum samples selected for PCR analysis were chosen from points in time corresponding to 6 months before the appearance of HBsAg and from 3 months and 3 years after the appearance of HBsAg. Among control patients (group 4), serum samples collected at time 0, 9 months later, and 3.5 years after the start of collection were used for analysis. The serum samples from each of the 44 patients were tested for anti-hbx, anti-pol, and HBV DNA by PCR randomly under code, and the results were then reassembled into the groups described above. HBV Serology All serum samples were analyzed for HBsAg, anti- HBs, HBeAg, anti-hbe, and anti-hbc by using commercially

3 1812 FEITELSON ET AL. GASTROENTEROLOGY Vol. 108, No. 6 available kits and by following the directions of the manufacturer (Abbott Laboratories, Chicago, IL). Determination of Anti-HBx and Anti-pol The enzyme-linked immunosorbent assays for anti- HBx and anti-pol were performed exactly as described previously.14,15,17, is PCR Amplification Primers and conditions for PCR amplification of the different HBV DNA regions have been published recently. 2 Primers were used to amplify the core, X, X plus core, X/ precore, S, or pres/s regions. Amplification was performed for 40 cycles, and the products were analyzed by agarose gel electrophoresis and ethidium bromide staining. Other standard practices, including the use of uracil-n-glycosylase, were used to prevent carryover contamination during sample preparation and amplification. 2 Dot Blot Hybridization Dot blot hybridization was performed with t-btl serum samples.16'~a Southern Blot Hybridization Probes corresponding to the PCR amplified regions were used in Southern blotting to detect the core region (probe spanning residues ), the X region (probe spanning residues ), and the pres/s region (probe spanning residues ). These probes were made by PCR amplification of the cloned HBV DNA with appropriate flanking primers. The fragments were identified by ethidium bromide staining after agarose gel electrophoresis, isolated, and labeled with thymidine [0~-32p]triphosphate by random priming. 22 Cloning and DNA Sequencing Serum samples that were PCR positive for the core gene of HBV DNA were chosen for the cloning and sequencing of X mutants. For these experiments, the X/precore region of HBV DNA from selected samples was PCR amplified 2 and detected by agarose gel electrophoresis and ethidium bromide staining. For cloning, the PCR products were isolated using the PCR Preps DNA Purification System (Promega, Madison, WI). The resulting material was cloned into pt7blue following the directions of the manufacturer (Novagen, Madison, WI). The recombinants were transfected into competent bacteria, and white colonies were selected from 5-bromo-4-chloro- 3 indolyl-~-d-galactoside and isopropylthio-[~-d-galactoside indicator plates according to the recommendations of the manufacturer. Double stranded dideoxy sequencing of supercoiled plasmid DNA obtained using standard minipreps were used to characterize the mutations in the X region on the molecular level. Statistical Analysis Comparison between two characteristics were performed using Fisher's Exact Test. Relationships were consid- ered statistically significant when P < 0.05 in a two-tailed analysis. The relationship of X deletion mutants to HBxAg, to anti-hbx, and to hepatitis, measured by ALT values, were determined by calculating the significance level (P value) between the mean of paired data sets. Results Detection of Anti-pol and HBV DNA Before and During the Incubation Phase of Infection Previous studies from this laboratory have shown that anti-pol often preceded the appearance of HBsAg in serum by weeks or months. 14 The finding of anti-pol before the incubation phase of infection (usually 6-26 weeks) (Table 1) in 4 of the 16 patients (25%) who developed into long-term carriers and in 3 of 7 transiently infected individuals (43%) implies HBV infection 1 year or more before the appearance of HBsAg. This possibility was tested by dot blot hybridization, but the results were uniformly negativ e (Table 1). Hence, antipol may be an early marker of HBV infection, but it is not accompanied by high levels of viremia 6 or more months before the appearance of HBsAg. If anti-pol reflects amplification of virus replicative complexes in the liver, as suggested earlier, then very low amounts of HBV may be present in serum during or before the incubation phase of infection. When serum samples collected approximately 6 months before the appearance of HBsAg were tested for HBV DNA by PCR amplification using primers from different regions of the HBV genome, 2 18 of 23 samples (78%) were positive (Table 1). The origin of the ethidium bromidestained band at 0.53 kilobase was confirmed by Southern blot hybridization using a core region probe (see below). Similar analysis of the S and pres/s regions yielded only the expected sized bands in patients who were core region PCR positive (data not shown). Hence, PCR amplification showed the presence of HBV in serum collected at least 6 months before the appearance of HBsAg. In contrast to these results, amplification of the same samples with the X primers, followed by Southern blot hybridization using an X region probe, resulted in the expected band in only 4 of 23 patients (17%) (Table 1). Three of these cases were among patients who became carriers, an example of which is presented in Figure I and the other from a patient who developed transient infection. These results were not caused by differences in PCR amplification of core compared with X sequences because control experiments with known amounts of template showed no difference in the efficiency of PCR amplification (data not shown). Attempts to use other

4 - June 1995 HBV X DELETION MUTANTS 1813 primer pairs within the X region for PCR failed to yield any products (data not shown). The core positive and X negative PCR finding suggests either that there were point mutations within one or both of the primers used for X region amplification or that one or both primer regions were rearranged or deleted. To test this possibility, amplification of the X plus core region was performed. Among the 14 serum samples that were core positive but X negative by PCR amplification (Table 1), 11 samples (79%) showed a smear of products (e.g., Figure 1). These products were mostly --<1.2 kilobases, the size expected for the X plus core amplification product, and strongly hybridized to the core region probe. However, the X region probe failed to hybridize (data not A HBsAg - ~ HBV DNA (dot) - - ~/ anti-hbc HBeAg anti-hbe HBxAg anti-hbx anti-pc[ HBV DNA: (core PCR) (X PCR) - - ~ (X + core PCR) - m m w/m w/rn w/re w/m w/m months B X + core PCR shown). In Figure l, a fast migrating smear, Jacking the expected product of 1.2 kilobases, was found 18 months before HBsAg. The 1.2-kilobase band occurred only when the serum became dot blot positive. Control experiments showed that these results were not caused by different concentrations of virus particles from serum or from serial dilutions of the supernatant from the virus producing Hep G cell line (Figure 2) and were not caused by the efficiency of X plus core region amplification compared with that of the core or X alone (data not shown). These results suggest the presence of HBV variants before and during the incubation phase of infection whose X region seems to be heterogeneous in size. Appearance of X Region Mutants in Chronic HBV Infection Serum samples collected 3 months after the appearance of HBsAg in carriers were tested for HBV DNA by dot blot hybridization and then by PCR amplification. At this time, 15 of the 16 carriers (94%) were dot hybridization positive (Table 1). All carriers with persistent A B C kb ~i!!}~ i ii!i "~ -~ ~ii!i!~i IIIQ ~>~ C X PCR O!! J ~ O kb D CorePCR kb Figure 1. (A) Summary of serological markers and PCR results from a dialysis patient who developed the chronic carrier state (patient 5; Table 1). The PCR products from the X core, X, and core regions are presented in B, C, and D, respectively. Note the appearance of anti-pol and HBV DNA by core region PCR 12 and 18 months, respectively, before the appearance of HBsAg. The X region PCR signal appeared approximately 4 months before the initial detection of HBsAg. A PCR signal for the X plus core region was observed 18 months before the appearance of HBsAg. Although the initial serum samples from this patient were PCR negative (i.e., within the first 4 months of collection), the PCR products observed at 4 and 10 months after the start of collection consisted of fast migrating smears and little or no signal at the expected size of 1.2 kilobases (kb), suggesting early infection with X deletion mutants of HBV. Figure 2. Effect of HBV DNA template concentration on the X plus core region products resulting from PCR amplification. Products were analyzed by agarose gel electrophoresis and Southern blotting using the core region sequences as probe. (A) The X plus core PCR products from increasing lo-fold dilutions (lanes 4-1, respectively) of HBV partially purified from the supernatant of the virus producing Hep G cell line. (B) Increasing lo-fold dilutions (lanes 4-1, respectively) of HBV from an HBeAg-positive serum sample. C is the X plus core PCR product from 20 copies of the recombinant plasmid ptkhh2, which lacks the nick plus gap structure of virion DNA. Note that the expected band of 1.2 kilobases does not cause faster migrating material at increasingly dilute template concentrations, despite the nickgap structure of v#ion DNA.

5 1814 FEITELSON ET AL. GASTROENTEROLOGY Vol. 108, No. 6 HBeAg had the expected sized product resulting from PCR amplification of the core, X, and the X plus core regions. Among patients who later seroconverted from HBeAg to anti-hbe, 6 patients had the expected PCR products from these regions, whereas the other 2 patients (no. 9 and 13) were X PCR negative. In patient 9, X plus core amplification yielded a smear of products --< 1.2 kilobases, whereas in patient 13, no products were observed (Table 1). Hence, 14 of the 16 carriers (88%) with HBV DNA detectable by dot blot hybridization also had PCR products compatible with wild-type HBV infection, in contrast to the -6-month time point analyzed above. Changes were observed in the dot blot hybridization and PCR results from sera collected approximately 3 years, compared with 3 months, after the appearance of HBsAg. Among the carriers with persistent HBeAg, 3 patients (38%) lost HBV DNA by dot blot hybridization (Table 1). Two of the latter 3 patients became X region PCR negative and developed a smear of hybridizable material of < 1.2 kilobases after X plus core region amplification. The PCR profiles from the other 5 carriers who remained dot blot positive did not change, suggesting that most carriers with persistent HBeAg also circulate the virus with a wild-type-sized X gene. Among the carriers who seroconverted from HBeAg to anti-hbe, only 1 patient (13%) was dot blot negative at 3 months, whereas 7 of 8 patients (88%) were dot blot negative by 3 years (Table 1). Six of the carriers in this group (75%) had the expected X and X plus core region PCR products at 3 months; only 1 of these carriers had the same products at 3 years (Table 1). The others had either no X plus core PCR product or a smear of < 1.2 kilobases. Hence, the clearance of HBV DNA, as measured by dot blot hybridization, seems to correlate with the loss of X and X plus core region PCR signals. An example of this pattern is presented in Figure 3. Similar PCR patterns were observed in 4 additional carriers (patients 10, 12, 15, and 16) (data not shown). Hence, putative X region mutants seem to accumulate during chronic infection in patients in which wild-type HBV DNA is cleared. This event is often, but not always, associated with seroconversion from HBeAg to anti-hbe. Detection of X Region Mutants in Acute Infections Among 7 acutely infected patients, all were HBsAg positive at 3 months, and 6 of the 7 patients (86%) seroconverted to anti-hbs by 3 years. Five of the seven patients (71%) were PCR positive for the core region 6 months before the appearance of HBsAg; only kb HBsAg HBV DNA a anti-hbc HBeAg anti-hbe HBxAg antfhbx anti-pol ALT HBV DNA ~ (core PCR) (X PCR) (X+ O PCR) wt a b c d e f g h i ///// \ \'>'> , w w W W W rrt gel Jane: a b c d e f g h i months Figure 3. X plus core (c) PCR amplification products from an HBV carrier (patient 1; Table 1) analyzed by agarose gel electrophoresis and ethidium bromide staining. This patient was persistently HBeAg positive but lost HBV DNA by dot blot hybridization approximately 2 years after the appearance of HBsAg. In parallel, the PCR signal for the wild-type (wt) X region became undetectable. X plus core region amplification showed the disappearance of the expected wild-typesized band and the appearance (or intensification) of several faster migrating bands. The results suggest the clearance of wild-type HBV DNA from serum and the appearance of X region mutants in its place. kb, kilobase. 1 of these 5 patients had the expected products following the X and X plus core PCR reaction (Table 1). Three months after the appearance of HBsAg, all 7 patients were core region positive by PCR, 3 of the 7 patients (43%) lacked the X region product by PCR, and 1 of the 7 had a mixed infection. At 3 years after the appearance of HBsAg, low levels of HBV DNA persisted in 6 of the 7 patients (86%) by PCR. Seroconversion from HBsAg to anti-hbs resulted in the clearance of wild-type HBV in 1 patient, but either wild-type or apparently mutant HBV persisted among the other patients (e.g., Figure 4). Unlike HBV carriers in which wild-type HBV is often eliminated and is later replaced by X region mutants (Figure 3 and Table 1), seroconversion from HBsAg to anti-hbs in acute infections did not result in the appearance of X region mutants (Figure 4 and Table 1). Apparently, X mutants are either not generated or selected for in acute HBV infections.

6 June 1995 HI3V X DELETION MUTANTS 1815 HBsAg HBV DNA (dot) + - anti-hbs anti-hbc HBeAg anti-hbe HBxAg anti-hbx anti-pol ALT HBV DNA (core PCR) (X PCR) - (X+C PCR) m m m m // months '0' Figure 4. Serological and PCR profile from one acutely infected patient. Anti-pol became detectable approximately 3 months before the appearance of HBsAg, viral DNA, and HBeAg. PCR analysis showed the presence of the core region (c) of HBV DNA in all but the first serum sample. However, the X region was not detected in any of the serum samples tested. Amplification of the X plus core region of HBV DNA yielded no signal from the first serum collected but resulted in a smear of hybridizable material in serum samples collected several months after the appearance of HBsAg. These results are consistent with heterogeneity within the X region and little or no wild-type HBV in this infection. Cloning and Sequencing of X Region Mutants Further characterization of the X/precore PCR products by cloning and sequencing showed two distinct types of mutations. One type consisted of in-frame missense mutations, whereas the other type was characterized by deletions (Figure 5). Among clones with missense mutations, one had a G to C transition at base 1608 that changed the arginine to a proline at that position. The same clone had an A to G mutation at position 1610 that converted a highly conserved methionine to a valine residue. The possibility that this methionine may be used for translation of functional HBxAg polypeptides 23 and that the proline substitution could alter the conformation of HBxAg suggests that these mutants may compromise HBxAg function. Other point mutations within the middle of the X gene were either silent or resulted in amino acid changes that were conservative replacements. The other type of mutation involved deletions mostly within the X open reading frame. One clone (Figure 5) had a deletion that extended from base 1303 through base This mutation not only resulted in the deletion of the entire X region but also in the loss of the prec translation start codon, the core promoter and enhancer, 24 the origin of plus strand DNA synthesis (DR2), and more than 100 carboxy terminal amino acid residues of the viral polymerase. The fact that these mutation types were not caused by an artifact of PCR amplification and cloning was controlled for by PCR amplifying, cloning, and sequencing the X/preC region from an HBV DNA plasmid of known sequence, 25 and by showing that the sequences obtained were identical to those of the input clone (data not shown). Possible PCR artifacts associated with amplification from alternate priming sites on viral DNA, the use of multiple primer pairs in and around the X region for amplification, and the possibility that the secondary structure or the nick-gap structure of HBV DNA yields artifically truncated amplification products have been excluded by the controls in this and an earlier report. 2 Relationship of X Region Mutants to Other Markers of Infection The frequent detection of X region mutants in HBsAg-negative serum samples (Table l) suggest that the mutants predominate during the HBsAg-negative phases of infection. To examine this more closely, PCR analysis of the core region showed that it was present in 38 of 39 patients' HBsAg-positive serum samples (97%) and in 24 of 30 HBsAg-negative serum samples (80%), showing that viremia was common in both phases of these infections. The finding of core PCR signals in most HBsAg-negative serum samples was not caused by contamination during sample collection or handling because none of the 21 apparently uninfected patients undergoing renal dialysis from the same units (with normal ALT values and no markers of HBV infection) were PCR positive (Table 1). Despite the presence of HBV DNA in most HBsAg-positive and -negative serum samples (by core PCR), the majority of HBsAg-positive serum samples (62%) had wild-type X region by PCR, whereas a majority of HBsAg-negative serum samples (77%) were X region PCR negative (P = ). These results suggest an inverse relationship between wild-type markers of infection and X deletion mutants. The relationship between the integrity of the X region and an elevated ALT value was examined next. Given that the wild-type X region predominated in HBsAg-

7 1816 FEITELSON ET AL. GASTROENTEROLOGY Vol. 108, No. 6 MF24 AGCGACTGCG TGGAACCTI~ TCGGCTCCTC TGCCOATCCA TACTGCGGAA AGCGACTGCG TGGAACCTIT TCGGCTCCTC TGCCGATCCA TACTGCGGAA AGCGACTGCG TGGAACCTTT TCGGCTCCTC TGCCGATCCA TACTGCGGAA 12~1 CTCCTAGCCO CTrGTTT]'GC TCGCAGCAGG TCTGGAGCAA ACATTATCGG CTCCTAGCCG CTTGTr'I'FGC TCGCAGCAGG TCTGGAGCAA ACATTATCGG CTCCTAGCCG CTTGTTITGC TC START X 1331 GACTGATAAC TCTGTTGTCC TATCCCGCAA ATATACATCG "[TrCCATGGC GACTGATAAC TCTGTTGTCC TATCCCGCAA ATATACATCG TI"rCCATGGC 1381 TGCTAGGCTG TGCTGCCAAC TGGATCCTGC GCGGGACGTC CTI~GT'ITAC TGCTAGGCTG TGCTGCCAAC TGGATCCTGC GCGGGACGTC CTTTGTrTAC 1431 GTCCCGTCGG CGCTGAATCC TGCGGACGAC CCTI'CTCGGG GTCGC~UrGGG GTCCCGTCGG CGCTGAATCC TGCGGACGAC CCT]'CTCGGG GTCGC~I'FGGG 1481 ACTCTCTCGT CCCC'FI'CTCC GTCTGCCGTT CCGACCGACC ACGGGGCGCA ACTCTCTCGT CCCCTrCTCC GTCTGCCGTT CCGACCGACC ACGGGGCGCA 1831 CCTCTCTTTA CGCGGACTCC CCOTCTGTGC ctrctcatct GCCGGACCGT CCTCTCTTTA CGCGGACTCC CCGTCTGTGC CTTCTCATCT GCCGGACCGT DR2 STOP POL 1581 GTOCACTrCG CTTCACCTCT GCACGTCGCA TGGAGACCAC CGTGAACGCC GTGCACTTCG CTTCACCTCT C, CACGACCCG TGGAGACCAC CGTGAACGCC 1631 CACCAAATAT TGCCCAAGGT C'FTACATAAG AGGACTCTI'G GACTCTCAGC CACCAA_TTCT TGCCCAAGGT c~racataag AGGACTCTTG GACTCTC!G~ 1681 AATGTCAACO ACCGACCTTO AGGCATAC~r CAAAGACTGT TTGTTrAAAG AATGTCAACG ACCGACCTTG AGGCATACTr CAAAGACTGT TrGTTTAAAG 1731 ACTGGGAGGA GTTGGGGOAG GAGAI"rAGOT TAAAGOTCTT TGTACTAGOA ACTGGGAGGA GTTGGGGGAG GAGATrAGGT TAAAGGTCTT TOTACTAGGA START PREC DRI 1781 OGCTGTAOGC ATAAATYGGT CTOCGCACCA GCACCATGCA AC~qq~TCAC OGCTGTAGGC ATAAA'i~G OT CTGCflCACCA GCACCATGCA ACT]'FI~CAC A ACTrTTTCAC STOP X 1831 CTCTGCCTAA TCATCTCTrG TTCATGTCCT /~ CTG'ITCAAG CCTCCAAGCT CTCTGCCTAA TCATCTCTI'G TTCATGTCCT ACTGTTCAAG CCTCCAAGCT CTCTGCCTAA TCATCTCTTG TTCATGTCCT ACTG~I'rCAAG CCTCCAAGCT MF GTGCCTT(3GG TGGCTITGGG GCATGGACAT CGACCCT~AT A GTOCCTTt]GG TGGCTTTGGO GCATGGACAT CGACCCTrAT A GTGCCTTGGG TOGCTI~GGG GCATGGACAT CGACCCTTAT A Figure 5. Representative mutation types within the X/preC region of HBV DNA found within HBsAg-positive blood samples. The first sequence in each row is from an infectious clone of wild-type HBV DNA. _,5 The second sequence in each row is from a clone that contains multiple point mutations within the middle region of the X gene. The third sequence in each row is from a clone that contains a deletion that spans the X region and adjacent upstream residues (from nucleotide 1301 to 1819, inclusive). The base designated as position 1 is the start of the unique EcoR1 site within HBV DNA. The primers used for PCR amplification are named, and the sequences are identified by a double underline. The translation start and stop codons for the appropriate genes, as well as the direct repeat I and 2, are identified and indicated in bold. Point mutations within the X gene are indicated by a changed base that is underlined in each case. The X region deletion is indicated by a gap in the sequence of the appropriate clone. The clones shown were from HBsAg-positive serum samples of patient 5 presented in Figure I and are representative of mutations found in other patients in this study and elsewhere. 2 positive serum samples and that episodes of elevated ALT levels occurred during the HBsAg-positive phase of these infections, it was no surprise that a highly significant correlation was observed between wild-type-sized X region and elevated ALT value (P = ). The presence of putative X mutants during the incubation phase of infection did not predict whether the infection was going to be acute or chronic or whether an elevated ALT value was going to develop (Table 1) (P > 0.1). However, seroconversion from HBxAg to anti-hbx after the appearance of HBsAg was associated with the loss of wild- type-sized X region and the appearance of putative X mutants only among carriers (P = ) (Table 2 and Figure 3). This observation is consistent with previous results showing that HBxAg and anti-hbx seroconversion is associated with the loss of HBV DNA by dot blot hybridization and then by seroconversion from HBeAg to anti-hbej 7 Because seroconversion from HBxAg to anti-hbx is also associated with an acute exacerbation of chronic hepatitis,.5 it was important to test whether the clearance of wt HBV during such exacerbations resuited in the appearance of X region mutants. Accordingly, the mean ALT values of chronically infected patients who cleared wild-type HBV DNA and gave rise to mutants was significantly higher than the mean ALT values of patients who did not give rise to mutants (P = 0.013). These results suggest that immune responses that are involved in the seroconversion from HBxAg to anti- HBx, and in the short-term exacerbation of chronic liver disease that accompanies elimination of wild-type HBV, may select for HBV X region variants. Discussion The results from this study indicate the presence of HBV variants in patients undergoing renal dialysis who developed HBsAg-positive infections. Characterization of these variants showed multiple point (missense) mutations and/or deletions within the X region (Figure 5). These findings are similar to those recently reported in children with ~-thalassemia. 2 In both studies, the X deletion mutants were defined as being core positive but X negative by PCR. Using these criteria, X region variants were found in HBsAg-negative serum samples collected from many of the same patients before the incubation period of infection (Table 1 and Figure 1). The previous finding of anti-pol in serum samples collected from these patients more than 1 year before the appearance of HBsAg.4 might now be explained by the presence of X region variants. Infection with these variants before the incubation phase of wild-type HBV does not seem to cause immunity capable of eliminating wild-type HBV because these patients later develop other markers of wild-type infection (e.g., HBsAg, HBeAg, and anti- HBc). This also seems to be the case in transiently infected patients who develop anti-hbs and remain PCR positive (Table 1). The parallel finding of X deletion mutants in multiply transfused children with ~-thalassemia who were successfully vaccinated against HBV also suggests that immunity to wild-type virus is not cross protective. 2 Given these observations, it is possible that the infections described above are characterized by vaccine escape mutants 1~ that also have mutations within

8 June 1995 HBV X DELETION MUTANTS 1817 Table 2. Relationship of X Region Mutants to HBxAg, anti-hbx, and ALT Status +3 me +3 yr Patient category Patient no. HBx anti-hbx ALT HBx anti-hbx ALT Long-term carriers HBeAg persistent (n = 8) HBeAg to anti-hbe (n = 8) Acute fnfection Transient HBsAg (n = 7) the X region. Therefore, a novel aspect of this study is the finding that these infections include the presence of X region variants of HBV that appear and predominate in sera before wild-type HBV infection. The data presented above also suggest that X region mutants predominate in carriers who have cleared the wild-type virus, and in some cases, seroconverted from HBeAg to anti-hbe (Figure 3 and Table 1). This would be selectively advantageous to the virus in that it could continue to replicate and persist for years even under conditions in which wild-type HBV is eliminated. The inverse relationship between anti-hbx in serum and the detection of wild-type HBV DNA 17 may be such a set of conditions in chronic infection because HBxAg is a component associated with the replication complexes of HBV 19 and because anti-hbx and/or immune responses associated with it may target hepatocyres replicating wild-type HBV DNA. 26 The result would be the selection of X region variants under conditions in which the replication complex of wild-type HBV would be targeted by the host. In addition, it is possible that the low levels of such variants may eventually recombine to cause wildtype infection at a later time. Among long-term carriers, this may explain the phenomenon of virus reactivation, 27 outbreaks of "sporadic" hepatitis B, or the appearance of HBsAg and markers of virus replication among patients with anti-hbs. 28'29 It is not clear from this study that the wild-type HBV infections in patients undergoing renal dialysis develop from de novo infection or by recombination of complementary X region variants. The finding of X region variants in HBsAg-positive serum samples collected 3 months after the appearance of HBsAg in 4 of the 7 transiently infected patients (57%) compared with only 3 of 16 carriers (19%) may contribute to the outcome of infection. This finding is consistent with the possibility that these variants may not be capable of establishing the HBsAg-positive long-term carrier state. Because the host-vires relationship that evolves after virus exposure is partially dependent on the nature and kinetics of antiviral immune responses, 3'3 infections consisting mostly of poorly replicating variants may result in acute infections more often than in infections consisting of mostly wild-type HBV. Although not shown here, larger numbers of patients may indicate that the wild type/mutant ratio associated with infection has prognostic value. The molecular basis for HBV attenuation in this region of the virus genome is probably multifold. First, HBxAg is a regulatory protein in that it has well documented transactivating properties. 3. Although it has been reported that HBxAg is capable of trans-activating a variety of cellular

9 1818 FEITELSON ET AL. GASTROENTEROLOGY Vol. 108, No. 6 gene promoters, 32'33 a major site of HBxAg-mediated regulation during infection may be at the level of virus gene expression and replication. 34 Under these circumstances, point mutations or deletions within the X open reading frame would produce a phenotype in which the levels of virus gene expression and replication is a small fraction of that expected. The result would be detection of such variants in serum samples negative for most or all HBV markers. The finding of most X deletion mutants in HBsAg-negative serum samples in this report (Table 1) and elsewhere 2 is compatible with this idea. Second, mutations within the X region may also result in the deletion of the one or both direct-repeat sequences, which are important for the initiation of minus and plus strand DNA synthesis, respectively, during replication. 35 Recent data have shown that the direct-repeat sequences could be mutated or deleted and that the virus is still capable of replication. 36 Third, 3' deletions in the X open reading frame eliminate the core promoter, 2<37 although the core polypeptide may still be made from the X promoter and upstream virus enhancer. 38'39 Fourth, the fact that deletion of the prec sequences does not affect virus replication 4 suggests that deletion of this region, per se, may not contribute to virus attenuation. However, if the deletion extends through the entire prec region, it may affect the pregenomic binding site for the viral polymerase 4. and the pregenomic packaging signal. 42 Fifth, deletions that extend upstream from the middle of the X open reading frame also include the 3' end of the polymerase open reading frame. Partial deletion of the polymerase may also contribute to the attenuation of these variants and may explain their distribution in nature. There are several considerations that may contribute to the generation and persistence of X region variants during infection. It is possible that the cellular enzyme topoisomerase 1, which has a number of recognition sites within the cohesive overlap region of viral DNA (spanning the directrepeat sequences), cleaves the viral DNA within this region. 43 The supercoiled DNA that appears within the nucleus of infected cells would then transcribe truncated pregenomic RNA molecules. Encapsidation and reverse transcription of these RNAs in the cytoplasm would result in the propagation of virus variants having deletions in and around the X region. The apparently large number of different deletion mutants observed in the sera of single patients (e.g., Figure 1B) may arise from recombination at these preferred cleavage sites. Alternatively or in addition, the characteristics of reverse transcriptases, which HBV uses for replication, may be responsible for the rapid generation of mutants here, as it does in retroviruses. 44'45 The lack of reverse transcriptase proofreading capability results in the accumulation of base pair changes during minus-strand DNA synthesis, resulting in the appearance of point mutations. Misincorporation of bases may promote the appearance of other mutations, including the insertions, deletions, as well as a high rate of homologous recombination among the variants that arise during replication, documented in this and previous reports. 2 In the context of the HBV replication cycle, the sequences in the packaging signal of the pregenomic RNA are important for the appropriate priming of minus strand synthesis and translocation of the polymerase-primer complex to the 3' direct repeat 1 sequences, where minus strand elongation then occurs. 46 Point mutations within the packaging signal may affect the fidelity of transfer to the 3' direct repeat 1, resulting in the appearance of upstream priming sites (within the X region) from which minus-strand synthesis would continue. The result would be the generation of a family of pregenomic RNAs truncated at various positions within their 3' ends, which corresponds to heterogeneous deletions within the X region, as observed in these studies. Although the relative contribution of topoisomerase and reverse transcriptases to the genetic variation within this region of viral DNA remains to be elucidated, their combined action would promote a high rate of mutation within this part of the viral genome at different points of the virus life cycle. References 1. Feitelson MA. Hepatitis B virus infection and primary hepatocellular carcinoma. Clin Microbiol Rev 1992;5: Hoofnagie JH, Shafritz DA, Popper H. Chronic type B hepatitis and the "healthy" HBsAg carrier state. Hepatology 1987; 7: Mondelli M, Eddleston ALWF. Mechanisms of liver cell injury in acute and chronic hepatitis B. Semin Liver Dis 1984;4: Dudley FJ, Fox RA, Sherlock S. Cellular immunity and hepatitisassociated Australia antigen liver diseases. Lancet 1972; 1: Alberti A. Do single nucleotide mutations result in clinically significant changes in hepatitis B virus pathogenicity? J Hepatol 1990; 10: Brown JL, Carman WF, Thomas HC. The clinical significance of molecular variation with the hepatitis B virus genome. Hepatology 1992; 15: Liang T J, Hasegawa K, Rimon N, Wands JR, Ben-Porath E. A hepatitis virus mutant associated with an epidemic of fulminant hepatitis. N Engl J Med 1991;324: Omata M, Ehata T, Yokosuka O, Hosoda K, Ohto M. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med 1991;324: Gunter S, Meisel H, Reip A, Miska S, Kruger DH, Will H. Frequent and rapid emergence of mutated pre-c sequences in HBV from e-antigen positive carriers who seroconvert to anti-hbe during interferon treatment. Virology 1992; 187: Brunetto MR, Giarin M, Saracco G, Oliveri F, Calvo P, Capra G, Randone A, Abate ML, Manzini P, Capalbo M, Piantino P, Verme G, Bonino F. Hepatitis B virus unable to secrete e antigen and response to interferon in chronic hepatitis 8. Gastroenterology 1993; 105:

10 lune 1995 HBV X DELETION MUTANTS 1819 I_1. Carman WF, Zanetti AR, Karayiannis P, Waters J, Manzillo G, Tanzi E, Zuckerman A J, Thomas HC. Vaccine-induced escape mutant of hepatitis B virus. Lancet 1990;336: L2. Gerken G, Kremsdorf D, Capel F, Petit MA, Dauguet C, Manns MP, Meyer zum Buschenfelde K-H, Brechot C. Hepatitis B defective virus with rearrangements in the pres gene during chronic HBV infection. Virology 1991; 183: Okamoto H, Tsuda F, Mayumi M. Defective mutants of hepatitis B virus in the circulation of symptom-free carriers. Jpn J Exp Med 1987;57: Feitelson MA, Millman I, Duncan GD, Blumberg BS. Presence of antibodies to the polymerase gene product(s) of hepatitis B and woodchuck hepatitis virus in natural and experimental infections. J Med Virol 1988;24: Horiike N, Blumberg BS, Feitelson MA. Characteristics of hepatitis B x antigen, antibodies to X antigen, and antibodies to the viral polymerase during hepatitis B virus infection. J Infect Dis 1991; 164: Feiteison MA, Clayton MM, Blumberg BS. X antigen/antibody markers in hepadnavirus infections. Presence and significance of hepadnavirus X gene product(s) in serum. Gastroenterology 1990; 98: Feitelson MA, Clayton MM. X antigen/antibody markers in hepadnavirus infections. Antibodies to the X gene product(s). Gastroenterology 1990;99: Lega L, Vierucci A, Blumberg BS, Saracco G, Rizzetto M, Zhu M, Feitelson MA. Hepatitis B x antigen and polymerase antibodies in the serum of hepatitis B carriers with or without hepatitis delta virus infection. Effects of interferon treatment. J Hepatol 1992;14: Feitelson MA. Products of the "X" gene in hepatitis B and related viruses. Hepatology 1986;6: Feitelson MA, Lega L, Guo J, Resti M, Rossi ME, Azzari C, 81umberg BS, Vierucci A. Pathogenesis of posttransfusion viral hepatitis in children with ~-thalassemia. Hepatology 1994;19: Kew MC, Miller RH, Chen H-S, Tennant BC, Purcell RH. Mutant woodchuck hepatitis virus genomes from virions resemble rearranged hepadnaviral integrants in hepatocellular carcinoma. Proc Natl Acad Sci USA 1993;90: Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132: Kwee L, Lucite R, Aufiero B, Schneider RJ. Alternate translation initiation on hepatitis B virus X mrna produces multiple polypeptides that differentially transactivate class II and III promoters. J Virol 1992;66: Yee J-K. A liver-specific enhancer in the core promoter region of human hepatitis B virus. Science 1989;246: Will H, Cattaneo R, Darai G, Deinhardt F, Schellekens H, Schaller H. Infectious hepatitis B virus from cloned DNA of known nucleotide sequence. Proc Natl Acad Sci USA 1985;82: Katayama K, Hayashi N, Sasaki Y, Kasahara A, Ueda K, Fusamote H, Sate N, Chisaka O, Matsubara K, Kamada T. Detection of hepatitis B virus X gene protein and antibody in type B chronic liver disease. Gastroenterology 1989; 97: Davis GL, Hoofnagle JH, Waggoner JG. Spontaneous reactivation of chronic hepatitis B virus infection. Gastroenterology 1984; 86: Koziol DE, Alter H J, Kirchner JP, Holland PV. The development of HBsAg-positive hepatitis despite the previous existence of antibody to HBsAg. J Immunol 1976; 117: Heijtink RA, van Hattum J, Schalm SW, Masurel N. Co-occurrence of HBsAg and anti-hbs: two consecutive infections or a sign of advanced chronic liver disease? J Med Virol 1982; 10: Feitelson MA. Hepatitis B virus gene products as immunological targets in chronic infection. Mol Biol Med 1989;6: Twu J-S, Schioemer RH. Transcriptional trans-activating function of hepatitis B virus. J Virol 1987;61: Aufiero B, Schneider RJ. The hepatitis B X-gene product transactivates both RNA polymerase II and III promoters. EMBO J 1990; 9: Seto E, Mitchell R J, Yen TSB. Trans-activation by the hepatitis B virus X protein depends on AP-2 and other transcription factors. Nature 1990;344: Colgrove R, Simon G, Ganem D. Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication. J Virol 1989; 63: Molnar-Kimber KL, Summers J, Mason WS. Mapping of the cohesive overlap of duck hepatitis B virus DNA and of the site of initiation of reverse-transcription. J Virol 1984;51: Condreay LD, Wu T-T, Aldrich CE, Delaney MA, Summers J, Seeger C, Mason WS. Replication of DHBV genomes with mutations at the sites of initiation of minus- and plus-strand DNA synthesis. Virology 1992; 188: Roossinck MF, Jameef S, Loukin SH, Siddiqui A. Expression of hepatitis B viral core region in mammalian cells, Mol Cell Biol 1986; 6: Shaul Y, Rutter W J, Laub O. A human hepatitis B viral enhancer element. EMBO J 1985;4: Treinin M, Laub O. Identification of a promoter element located upstream from the hepatitis B virus X gene. Mol Cell Biol 1987; 7: Chert H-S, Kew MC, Hornbuckle WE, Tennant BC, Cote P J, Gerin JL, Purcell RH, Miller RH. The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host. J Virol 1992;66: Kochel HG, Kann M, Thomssen R. Identification of a binding site in the hepatitis B virus RNA pregenome for the viral pol gene product. Virology 1991; 182: Junker-Niepmann M, Bartenschlager R, Schaller H. A short cisacting sequence is required for hepatitis B virus pregenome encapsidatien and sufficient for packaging of foreign RNA. EMBO J 1990; 9: Wang H-S, Rogler CE. Topoisomerase 1-mediated integration of hepadnavirus DNA in vitro. J Virol 1991;65: Temin, HM. Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus gene variation. Proc Natl Acad Sci USA 1993;90: Zhang J, Temin HM. Rate and mechanism of nonhomologous recombination during a single cycle of retroviral replication. Science 1993;259: Wang G-H, Seeger C. Novel mechanism for reverse transcription in hepatitis B viruses. J Virol 1993;67: Received July 26, Accepted January 26, Address requests for reprints to: Mark A. Feitelson, Ph.D., Department of Pathology, Anatomy, and Cell Biology, Room 219 Alumni Hall, Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pennsylvania Fax: (215) Supported by United States Public Health Service grants CA , RR-05895, CA-06927, and CA and by a Focused Giving Grant from Johnson and Johnson. The authors thank Marcia M. Clayton for excellent technical assistance and Darin Trelka and Dr. Bill Sun for performing some of the control experiments.