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...

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). ...

We examined the full-length hepatitis B virus (HBV) envelope (surface antigen or HBV small surface antigen [HBsAg]) sequences of 12 different liver samples from 10 different hepatoma-containing chronic carriers. Surprisingly, novel and frequent mutations occurred predominantly at amino acids 40 and 47 of HBsAg, in addition to within a known protective B-cell epitope (so-called group a determinant of HBsAg 124-148). Approximately 58% of chronic carriers contain mutations at the group a determinant. The mutation frequency at the hotspot codons 40 and 47 is approximately 83%, 1 order of magnitude higher than at the known polymorphic sites of subtype-specific determinants at codons 122 and 160, which is approximately 4%. This new mutational domain is found to coincide with a major histocompatibility complex class I-restricted T-cell epitope. The potential biological significance of this novel mutation in the immunopathogenesis of HBV chronic carriers is discussed.

Naturally occurring variants of human hepatitis B virus (HBV) containing the core internal deletion (CID) mutation have been found frequently in HBV carriers worldwide. Despite numerous sequence analysis reports of CID variants in patients, in the past decade, CID variants have not been characterized functionally, and thus their biological significance to HBV infection remains unclear. We report here two different CID variants identified from two patients that are replication defective, most likely due to the absence of detectable core protein. In addition, we were unable to detect the presence of the precore protein and e antigen from CID variants. However, the production of polymerase appeared to be normal. The replication defect of the CID variants can be rescued in trans by complementation with wild-type core protein. The rescued CID variant particles, which utilize the wild-type core protein, presumably are enveloped properly since they can be secreted into the medium and band at a position similar to that of mature wild-type Dane particles, as determined by gradient centrifugation analysis. Our results also provide an explanation for the association of CID variants with helper or wild-type HBV in nature. The significance of CID variants in HBV infection and pathogenesis is discussed.

IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad- IrrE in the D. radiodurans irrE-deletion mutant (ΔirrE), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the ΔirrE mutant complemented strain with Drad-irrE, designated ΔirrE-E. The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA, and ddrB, were remarkably induced after UV radiation and MMC shock in the ΔirrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.