Naturally occurring deletions within the human hepatitis B virus (HBV) preS2 region have frequently been identified in patients with hepatocellular carcinoma (HCC), while chronic carriers without cirrhosis and HCC contain no detectable preS2 deletion variants. We have characterized two different preS2 internal deletion variants from two patients. In addition to several weak phenotypes, our study revealed three unexpected strong phenotypes: (1) a paradoxical "hypermodification" phenomenon was observed with significantly increased size heterogeneity and molecular weights of the secreted middle (M) envelope proteins containing a preS2 internal deletion. This phenomenon was observed in transient transfection with a human hepatoma Huh7 cell line as well as in stable transfection with a rodent hepatoma cell line 7777. (2) A significantly increased intracellular accumulation of all three envelope proteins (large, middle, and small) was detected by both Western blot analysis and immunofluorescence microscopy. (3) The middle envelope proteins with a preS2 internal deletion were not recognized in vitro by a putative neutralizing antiserum, suggesting that these variants can evade immune recognition in vivo. To our knowledge, this is the first identification and characterization of the M deletion variant protein in HBV natural infection.

Instead of displaying the wild-type selective export of virions containing mature genomes, human hepatitis B virus (HBV) mutant I97L, changing from an isoleucine to a leucine at amino acid 97 of HBV core antigen (HBcAg), lost the high stringency of selectivity in genome maturity during virion export. To understand the structural basis of this so-called "immature secretion" phenomenon, we compared the stability and morphology of self-assembled capsid particles from the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183) or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with little interior space, whereas truncated particles appear as "thin-walled" spherical particles with a much larger inner space. We found no significant differences in capsid stability between wild-type and mutant I97L particles under denaturing pH and temperature in either full-length or truncated core protein contexts. In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at temperatures higher than 75°C, or in 0.1% sodium dodecyl sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated full-length particles during agarose gel electrophoresis is most likely caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids are known to exist in natural infection as dimorphic T‫3؍‬ or T‫4؍‬ icosahedral particles. No difference in the ratio between T‫3؍‬ (78%) and T‫4؍‬ particles (20.3%) are found between wild-type HBV and mutant I97L in the context of HBcAg1-140. In addition, we found no difference in capsid stability between T‫3؍‬ and T‫4؍‬ particles successfully separated by using a novel agarose gel electrophoresis procedure.

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Premedication with pronase or NAC at 20 min before UGI endoscopy improves the mucosal visibility of the stomach. Neither pronase nor NAC produces any obvious interference with the CLO test for the identification of H pylori infection.

We identified two novel naturally occurring mutations (W74L and L77R) in the small S envelope protein of hepatitis B virus (HBV). Mutation L77R alone resulted in >10-fold-reduced secretion of virions. In addition, the 2.8-fold reduction of the extracellular HBV surface antigen (HBsAg) of mutant L77R from transfected Huh7 cells appeared to be correlated with a 1.7-fold reduction of intracellular HBsAg, as measured by enzyme-linked immunosorbent assay (ELISA). Surprisingly, opposite to the ELISA results, Western blot analysis revealed a near-10-fold-increased level of the intracellular mutant small S envelope protein. The discrepancy between ELISA and Western blot data was due to significant accumulation of the mutant L77R HBsAg in the intracellular pellet fraction. In contrast to HBsAg, the secretion of HBeAg was normal in L77R-transfected cells. The wild-type HBsAg was usually more diffuse and evenly distributed in the cytoplasm, often outside the perinuclear endoplasmic reticulum (ER) and Golgi apparatus, as observed by immunofluorescence assay. In contrast, the L77R mutant HBsAg tends to be highly restricted within the ER and Golgi, often accumulated in the Golgi compartments distal from the nucleus. The almost exclusive retention in the ER-Golgi of L77R HBsAg was similar to what was observed when the large envelope protein was overexpressed. These multiple aberrant phenotypes of mutant L77R can be corrected by a second naturally occurring S envelope mutation, W74L. Despite the accumulation of L77R HBsAg in ER-Golgi of transfected Huh7 cells, we detected no increase in Grp78 mRNA and proteins, which are common markers for ER stress response. Hepatitis B virus (HBV) is a major human pathogen.Chronic infection with HBV leads to the development of cirrhosis and hepatocellular carcinoma (2,16,36). HBV variants are often found in chronically infected patients (19,37). The most common naturally occurring mutation in human HBV core protein is at amino acid (aa) 97, changing a highly conserved isoleucine (HBsAg subtype adr) or phenylalanine (HBsAg subtype ayw) to a leucine (L) (3,(12)(13)(14)(15)20). In contrast to the established dogma of preferential virion secretion of mature genome for wild-type (WT) hepadnaviruses (17,33,40,44,47,48), the 97L mutation results in secretion of virions containing an immature genome into the medium and is characterized by excessive amounts of minus-strand DNA (47, 48). Even though the immature secretion phenotype has been observed with woodchuck and snowgoose hepadnaviruses (7, 42), it has not been reported with human patients. This may be due to the presence of naturally occurring compensatory mutations for 97L in the core protein at positions 5 (11) or 130 (49), both changing a highly conserved proline to threonine.HBV surface antigens (HBsAg) consist of three structurally related large (L), middle (M), and small (S) envelope proteins. These proteins share a common carboxyl terminus, with the L protein containing pre-S1, pre-S2, and small S domains, and the M envelope protein conta...

ZFP36 family members include ZFP36, ZFP36L1, and ZFP36L2, which belong to CCCH-type zinc finger proteins with two tandem zinc finger (TZF) regions. Whether ZFP36L1 and ZFP36L2 have antiproliferative activities similar to that of ZFP36 is unclear. In this study, when ZFP36L1 or ZFP36L2 was overexpressed in T-REx-293 cells, cell proliferation was dramatically inhibited and the cell cycle was arrested at the G1 phase. The levels of cell-cycle-related proteins, including cyclin B, cyclin D, cyclin A, and p21, decreased; however, p53 increased in ZFP36L1-or ZFP36L2-overexpressing T-REx-293 cells. Forced expression of ZFP36L1 or ZFP36L2 also inhibited cell proliferation and cyclin D gene expression in three human colorectal cancer cell lines: HCT116 p53+/+, HCT116 p53−/−, and SW620 (mutated p53) cells. However, it increased p53 and p21 expression only in HCT116 p53+/+ cells. Knockdown of ZFP36L1 or ZFP36L2 increased cell proliferation and cyclin D expression; furthermore, the mutation of the TZF of ZFP36L1 or ZFP36L2 caused them to lose their antiproliferative ability, to the extent that they could not inhibit cyclin D expression in these three cell lines. The results indicated that ZFP36L1 and ZFP36L2 play a negative role in cell proliferation; the underlying mechanisms might be mediated through a cyclin D-dependent and p53-independent pathway.

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Mutations of human hepatitis B virus (HBV) occur frequently within the capsid (core) protein in natural infections. The most frequent mutation of the core protein in HBV from Southeast Asia occurs at amino acid 97, changing an isoleucine (I) to a leucine (L). In our systematic study of virus-host interactions, we have examined the replication efficiency of a site-directed mutant, I97L, and its parental wild-type HBV in several different hepatoma cell lines. Interestingly, we found that this capsid variant replicated in human Huh7 hepatoma cells approximately 4.8-fold better than its parental wild-type HBV. A similar phenomenon was observed in another hepatoma cell line, J3. In addition, the level of encapsidated RNA pregenome in mutant I97L was about 5.7-fold higher than that of the wild-type HBV in Huh7 cells. Unlike Huh7 cells, no significant difference in viral DNA replication between the same I97L mutant and its parental wild-type HBV was observed in HepG2, a human hepatoblastoma cell line. This finding of a profound replication advantage for mutant I97L in Huh7 and J3 cells but not in HepG2 cells may have important implications for the emergence of this mutant in chronic HBV carriers. We speculate here that the mutation confers a host factor-independent growth advantage for the survival of HBV variants in gradually dedifferentiating hepatocytes and thus helps prolong viral persistence.Human hepatitis B virus (HBV) can cause acute and chronic hepatitis in humans, with the latter often resulting in cirrhosis and hepatocellular carcinoma (6,11,55,56). HBV is an enveloped virus, which consists of an outer lipoprotein envelope and an inner nucleocapsid containing a 3.2-kb partially doublestranded DNA genome. The nucleocapsid, formed by a 183-amino-acid core antigen, assembles in the cytoplasm with a 3.2-kb pregenomic RNA and the viral polymerase. The encapsidated pregenomic RNA is then retrotranscribed into viral DNA (20,53).Because the polymerase of HBV lacks a proofreading function, HBV has a low fidelity in replication and thus tends to produce sequence variants at a high frequency. Naturally occurring mutations in HBV have been hypothesized to play a role in the pathology of HBV-related diseases and in the persistence of HBV infection (23, 27, 59, 60, 68, 69). As a major T-cell target (13, 42), the core protein accumulates frequent mutations in chronic carrier patients with active liver disease (1,5,8,14,15,16,27).Within the core protein, the most frequent mutation occurs at amino acid 97 (1,5,14,15,16,17,21,22,27,29,33,40,41,43,48,50,61,64,65,70). The codon 97 mutation changes the wild-type amino acid from a phenylalanine to a leucine in the ayw subtype (F97L) or from an isoleucine to a leucine in the adr subtype (I97L). Previously, we identified an "immature secretion" phenotype for capsid variant 97L (referring to both I97L and F97L), which is characterized by secretion of an excessive amount of Dane particles containing immature singlestranded DNA intermediates in both the ayw and adr subtypes (70, 71). ...