[Establishment and behavioral evaluation of a mouse model of long-term free-choice alcohol drinking].

OBJECTIVE: To establish a model of long-term free drinking mouse by feeding mice with alcohol to simulate the state of human voluntary long-term drinking, and on this basis, to further discuss the evaluation criteria of long-term free drinking mice model in sports, anxiety and cognitive behavior. METHODS: Forty six-week-old SPF C57BL/6 male mouse were randomly divided into two groups: Long-term free drinking group (n=20) and normal control group (n=20). The two groups were given solid feed normally. The long-term free drinking group was free to take 10% alcohol and water every day, while the normal drinking group only took water every day. The mice were fed for 7 months, and were evaluated by a series of behavioral methods, including Rota-rod test, balance beam test, open filed test, the elevated plus maze, two-box social behavior, new object recognition, Y maze and water maze. RESULTS: With the increase of drinking days, the mice showed significant alcohol addiction in the alcohol preference test. With the increase of alcohol intake, the mice in the long-term free choice drinking group had slightly shiny fur and reduced diet. Compared with the control group, the weight gain began to slow down from the third month, and the weight decreased significantly by the sixth and seventh months (P=0.006, P < 0.001). The mice showed reduced balance locomotion ability (P=0.003, P=0.001) in the rotary bar and balance beam test. In the open field and elevated cross test, the mice had obvious anxiety-like behavior (P < 0.001). The mice showed decreased social ability in the two boxes of social behavior (P < 0.016). In the experiment of new object recognition and Y maze, the exploration of new object decreased (P=0.018, P=0.040). In the water maze, cognitive functions, such as learning and spatial memory were reduced (P < 0.001). CONCLUSION: The successful establishment of the long-term free drinking mouse model is more convenient for us to carry out further research on the neural mechanism of alcohol addiction, and lays an experimental foundation for exploring the neural mechanism of alcohol addiction and related new targets.

[Association between osteoporosis and severe periodontal attachment loss in postmenopausal women].

Objective: To investigate the association between osteoporosis and severe periodontal attachment loss in postmenopausal women. Methods: One hundred and ninety-five postmenopausal women aged 50-65 years old were included in the present study. The participant were recruited from patients in the Peking University International Hospital and through website from March, 2017 to August, 2018. Periodontal examination, filling out a structured questionnaire and bone mass density (BMD) examination were completed to each of the participants. The clinical attachment loss (CAL), oral hygiene index simplified (OHI-S), and bleeding on probing (BOP) were examined and recorded as periodontal parameters. The structured questionnaire was used to collect information of the physical activity, socioeconomic status, marital status, oral health habits, and so on. Bone mass density of the lumbar spine and left hip were scanned by using standardized dual energy X-ray absorptiometry. Based on the T-score of BMD (difference between the measured BMD and the mean value of young white women in terms of standard deviations), osteoporosis was defined as T-score≤-2.5 (according to the World Health Organization criteria). Fourteen risk factors were preliminarily screened by chi-square test, including age, duration of menopause, body mass index (BMI), exercise habit, economic status, marital status, level of education, habit of dental visit, tooth brushing habit, usage of interdental tools, OHI-S, BOP(+)%, osteoporosis in hip and osteoporosis in lumbar. Factors with P<0.05 were selected for multivariate Logistic regression analysis. Logistic regression was used to analysis the risk factors of severe periodontal attachment loss. Results: The mean age of 195 postmenopausal women was (57.8±4.3) years. After adjusting for ages, the economic status, habit of dental visit, OHI-S and BOP(+)%, there was an association of osteoporosis in hip and severe periodontal attachment loss in postmenopausal women [odds ratios (OR)=2.466, 95% confidence intervals (CI) was 1.173-5.185, P=0.017]. Conclusions: Postmenopausal women with osteoporosis in hip has a greater chance of presenting severe periodontal attachment loss.

[Relationship between periodontitis and osteoporosis in postmenopausal women].

OBJECTIVE: To explore the relationships of periodontal parameters, cortical width on mental foramen and osteoporotic condition in postmenopausal women. METHODS: Ninetyeight postmenopausal women between 50 to 65 years old were recruited. General conditions, such as age, menopausal age, duration of menopause, and body mass index (BMI) were recorded. Periodontal parameters were examined, including oral hygiene index simplified (OHI-S), probing depth (PD), clinical attachment loss (CAL), gingival recession (GR) and bleeding on probing (BOP). Panoramic radiograph was taken and the cortical width (CW) of mental foramen was measured on images. The examiner was celebrated. Bone mass density (BMD) of left hip and lumbar spine was assessed using standardized dual energy X-ray absorptiometry. According to World Health Organization, based on the T-score of BMD (difference of the measured BMD and the mean value of young white women in terms of standard deviations), the subjects were divided into osteoporotic group (T-score<-2.5) and non-osteoporotic group (T-score≥-2.5). These parameters were compared between the groups. RESULTS: The number of osteoporotic group was 47 (47.96%). Ages and duration of menopause were significantly different between the groups. Osteoporotic group presented older ages [(59.64±4.58) years vs. (56.94 ± 4.26) years, P<0.05], and longer duration of menopause [(10.17± 5.37) years vs. (6.02 ±4.48) years, P<0.05]. There was no significant difference in menopausal age and BMI between the groups. BOP% was statistically significantly higher in osteoporotic group (29.43±21.12) than in non-osteoporotic group (21.43±17.09), with a P-value of 0.046. The other periodontal parameters, including OHI-S, PD, CAL, and GR were not statistically significantly different in the groups. The CWs were statistically significantly lower in osteoporotic group compared with non-osteoporotic group, with a P-value of 0.001. The mean values of CWs were (3.61±1.04) mm (osteoporotic group) and (4.25±0.77) mm (non-osteoporotic group), respectively. CONCLUSION: The study demonstrated absence of a significant association between periodontal parameters and BMD. However, the CWs were found to be related with the BMD, which may be used to detect BMD abnormal in maxillofacial imaging. The dentists should pay attention not only to the oral health, but also to the general bone mass density, which may be detected on panoramic images.

[Serum status and their changes of some vitamin substances and homocysteine in healthy subjects in Beijing].

Objective: To investigate serum status of folate, vitamin B(12), homocysteine (Hcy) and hydroxyvitamin D (25OHD) and their trends in different gender and age groups. Methods: This was a cross-sectional study. The enrolled subjects were those received medical examination in Beijing Hospital from September to November 2018 and were identified as appeared healthy persons. 1220 subjects were recruited and were divided into groups of young and middle age group (30-49 years, 50-59 years) and the elderly group (60-69 years, 70-79 years and ≥80 years). We measured folate, vitamin B(12), and 25OHD using electrochemiluminescence by chemiluminescence immunoassay. Hcy was measured by autobiochemical analyzer. Results: Total folate levels in male and female subjects were 7.16 (4.74-10.75) and 9.17 (6.49-13.55) µg/L respectively. Total vitamin B(12) levels in the male and female were 505.60 (386.80-700.90) and 582.60 (430.70-846.98) ng/L respectively. Hcy levels were 14.68 (12.25-18.58) and 11.29 (9.65-13.58) µmol/L. 25OHD levels were 21.60 (16.40-28.70) and 16.80 (12.30-24.15) µg/L respectively. Total folate and vitamin B(12) levels in female were higher than that in male subjects (Z=-7.796, -4.772, P<0.001). However, total Hcy and 25OHD levels in male were higher than that in female subjects (Z=-15.230, -8.447, P<0.001). Comparing with the substances in the above age groups, folate level in the elderly was lower than that in the younger age and middle age groups.However, vitamin B(12), 25OHD and Hcy levels were higher in the elderly groups. Furthermore, the levels of folate, vitamin B(12) and 25OHD were getting higher in the group of ≥80 years female compared with the rest of the age groups, but it turned lower in the male group of ≥80 years. Conclusions: There are some differences in the serum values of folate, vitamin B(12), Hcy and 25OHD among various age groups as well as between males and females. These should be considered in the development of national reference ranges.

The downregulation of onzin expression by PKCepsilon-ERK2 signaling and its potential role in AML cell differentiation.

Onzin is a small, novel, and highly conserved protein with unique structure and tissue-restricted expression. The regulation of its expression and biological roles remain greatly elusive. Here, we provide the first demonstration that onzin expression is significantly downregulated during differentiation induction of acute myeloid leukemic (AML) cell lines and primary cells by all-trans retinoic acid (ATRA) and especially by phorbol 12-myristate 13-acetate (PMA). Applying chemical inhibitions, RNA interferences, and transfected expressions of dominant negative mutants or constitutive catalytic forms of the related kinases, we show that protein kinase C-epsilon (PKCepsilon)-extracellular signal-regulated protein kinase 2 (ERK2) signaling axis is required for PMA-induced downregulation of onzin expression. The ectopic expression of onzin partially inhibits PMA-induced monocytic differentiation of AML cells, whereas suppression of onzin by specific short hairpin RNAs enhances PMA-induced differentiation to a degree. Furthermore, onzin partially inhibits the transcriptional activity of hematopoiesis-related important transcription factor PU.1 via their interaction. Taken together, our results show that PMA downregulates onzin expression through PKCepsilon-ERK2 signaling pathway, which favors monocytic differentiation of leukemic cells.

Multi-sites cleavage of leukemogenic AML1-ETO fusion protein by caspase-3 and its contribution to increased apoptotic sensitivity.

Leukemia-associated fusion protein AML1-ETO is a product of the chromosome translocation (8;21) frequently occurred in acute myeloid leukemia (AML). The fusion oncoprotein blocks leukemic cell differentiation, and it also induces growth arrest with increased sensitivity to apoptosis induction. Such dichotomous functions make it difficult to clarify the role of AML1-ETO in leukemogenesis. Here, we systematically showed that constitutively and overexpressed AML1-ETO protein was cleaved to four fragments of 70, 49, 40 and 25 kDa by activated caspase-3 during apoptosis induction by extrinsic mitochondrial and death receptor signaling pathways. The in vitro proteolytic system combined with MALDI-TOF/TOF mass spectrometer confirmed that AML1-ETO and wild-type ETO but not RUNX1 (AML1) proteins were direct substrates of apoptosis executioner caspase-3. Site-directed mutagenesis analyses identified two nonclassical aspartates (TMPD188 and LLLD368) as caspase-3-targeted sites in the AML1-ETO sequence. When these two aspartates were mutated into alanines, more intriguingly, the apoptosis-amplified action of AML1-ETO induction completely disappeared, while inducible expression of the caspase-3-cleaved 70 kDa fragment of AML1-ETO after tetracycline removal is sufficient to enhance apoptotic sensitivity. Further investigations on the potential in vivo effects of such a cleavage and its possible role in leukemogenesis would provide new insights for understanding the biology and treatment of AML1-ETO-associated leukemia.

Inducible expression of AML1-ETO fusion protein endows leukemic cells with susceptibility to extrinsic and intrinsic apoptosis.

AML1-ETO, a leukemia-associated fusion protein generated by the frequently occurred chromosome translocation t(8;21) in acute myeloid leukemia, was shown to exert dichotomous functions in leukemic cells, that is, growth arrest versus differentiation block. By the analysis of oligonucleotide microarray, AML1-ETO was shown to modulate the expressions of an impressive array of pro- and anti-apoptotic genes. Here, we investigate potential effects of the ecdysone inducible AML1-ETO expression on apoptosis of leukemic U937 cell line. We show that AML1-ETO significantly stabilizes death receptor Fas protein and increases proapoptotic Bak in addition to reducing Bcl-2 expression. Accordingly, inducible AML1-ETO expression is followed by apoptosis to a lower degree. Especially, AML1-ETO endows leukemic cells with the susceptibility to anti-Fas agonist antibody, ultraviolet light and camptothecin analog NSC606985-induced apoptosis with increased activation of caspase-3/8. Considering that apoptosis-enhancing effect of AML1-ETO would not be favorable to the leukemogenesis harboring the t(8;21) translocation, it must be overcome to fulfill their leukemogenic potential. Complementary to this prediction is that two AML1-ETO-carrying leukemic cells, Kasumi-1 and SKNO-1, present similar sensitivity to apoptosis induction with AML1-ETO-negative leukemic cells. Therefore, genetic and/or epigenetic screenings of apoptosis-related genes modulated by AML1-ETO deserve to be explored for understanding the mechanisms of AML1-ETO-induced leukemogenesis.

Cloning and genetic characterization of an evolutionarily conserved human olfactory receptor that is differentially expressed across species.

We have cloned the full-length cDNA and genomic region of a human prostate specific G-protein coupled receptor with properties characteristic of an olfactory receptor. A partial cDNA sequence of this gene, called PSGR, was recently cloned. The gene contains two exons and one intron of 14.9 kb in its 5'untranslated region, and was mapped to human chromosome 11p15.2. A cluster of transcription initiation sites for the 2.8 kb PSGR mRNA was identified. Cloning of the homologous gene from the mouse revealed 93% amino acid homology between the human and mouse or rat (previously cloned as RA1c) proteins, and 99% identity between the rat and mouse homologs. Although northern analysis indicated expression of the human PSGR homolog was prostate specific, its mRNA could also be detected in the olfactory zone and the medulla oblongata of the human brain. In the mouse, the PSGR gene is predominantly expressed in the brain and colon. In the rat, the PSGR homolog is expressed in the liver in addition to the brain. These data add to the growing body of evidence suggesting that olfactory receptors may have functional roles in tissues other than the olfactory organ, and further, suggest that these functions may vary across species.

Low-level secretion of human hepatitis B virus virions caused by two independent, naturally occurring mutations (P5T and L60V) in the capsid protein.

The functional significance of naturally occurring variants of human hepatitis B virus (HBV) remains largely unknown. Previously, we reported an immature secretion phenotype caused by a highly frequent mutation at amino acid 97 of the HBV core (capsid) protein (HBcAg). This phenotype is characterized by a nonselective and excessive secretion of virions containing an immature genome of single-stranded viral DNA. To extend our study of virion secretion to other naturally occurring variants, we have characterized mutations at HBcAg codons 5, 38, and 60 via site-directed mutagenesis. Although the phenotype of the mutation at codon 38 is nearly identical to that for the wild-type virus, our study reveals that a single mutation at codon 5 or 60 exhibits a new extracellular phenotype with significantly reduced virion secretion yet maintains normal intracellular viral DNA replication. A complementation study indicates that the mutant core protein alone is sufficient for the "low-secretion" phenotype. Furthermore, the low-secretion phenotype of the codon 5 mutant appears to be induced by the loss of a parental proline residue, rather than by the gain of a new amino acid. Our study underscores the core protein as another crucial determinant in virion secretion, in addition to the known envelope proteins. Our present results suggest that a very precise structure of both alpha-helical and nonhelical loop regions of the entire HBcAg molecule is important for virion secretion. The low-secretion variants may contribute to the phenomenon of gradually decreasing viremia in chronic carriers during the late phase of persistent infection.

Vibration arthrometry in patients with knee joint disorders.

Physiological patellofemoral crepitus (PPC) is the vibration signal produced by the knee joint during slow motion (less than 5 degrees per second), which can be measured by vibration arthrometry (VAM). By using the autoregressive (AR) model for the PPC signals of patients with knee osteoarthritis, the study analyzes the PPC signals to evaluate the condition of patellar-femoral joint cartilage. Accordingly, we can divide osteoarthritis into three types, type 1: the cartilage of patellar-femoral joint is intact, the osteoarthritis found in the femoral-tibial joint surface; type 2: degeneration occurs in the surface cartilage of both the femoral-tibial joint and the femoral trochlea, but not on the patellar surface; type 3: both patellar-femoral and femoral-tibial joints have osteoarthritis. For the analysis, the intraclass distance of AR coefficients and spectral power ratio of dominant poles are adopted. Based on the proposed method, two cases of type 1, six of type 2, and 28 of type 3 were found in 36 cases of knee osteoarthritis. This is in agreement with the operative findings. For comparison, the PPC signals of 10 subjects with normal knees (without pain or wound history) were also measured. The results of analysis of the 10 normal subjects were consistent and clearly differentiable from those of the osteoarthritis patients. Therefore, the proposed method is efficient for the analysis of the condition of patellar-femoral joint cartilage and VAM may become an alternative way of noninvasive diagnosis of knee osteoarthritis.

A frequent, naturally occurring mutation (P130T) of human hepatitis B virus core antigen is compensatory for immature secretion phenotype of another frequent variant (I97L).

A frequent mutation at codon 97 of human hepatitis B virus core antigen has been shown to cause an "immature secretion" phenotype, featuring nonselective and excessive secretions of virions containing immature viral genome. Our current study demonstrates that this abnormality can be efficiently offset by another frequent core mutation, P130T.

Vibration arthrometry in the patients with failed total knee replacement.

This is a preliminary research on the vibration arthrometry of artificial knee joint in vivo. Analyzing the vibration signals measured from the accelerometer on patella, there are two speed protocols in knee kinematics: 1) 2 degrees/s, the signal is called "physiological patellofemoral crepitus (PPC)", and 2) 67 degrees/s, the signal is called "vibration signal in rapid knee motion". The study has collected 14 patients who had revision total knee arthroplasty due to prosthetic wear or malalignment represent the failed total knee replacement (FTKR), and 12 patients who had just undergone the primary total knee arthroplasty in the past two to six months and have currently no knee pain represent the normal total knee replacement (NTKR). FTKR is clinically divided into three categories: metal wear, polyethylene wear of the patellar component, and no wear but with prosthesis malalignment. In PPC, the value of root mean square (rms) is used as a parameter; in vibration signals in rapid knee motion, autoregressive modeling is used for adaptive segmentation and extracting the dominant pole of each signal segment to calculate the spectral power ratios in f < 100 Hz and f > 500 Hz. It was found that in the case of metal wear, the rms value of PPC signal is far greater than a knee joint with polyethylene wear and without wear, i.e., PPC signal appears only in metal wear. As for vibration signals in rapid knee motion, prominent time-domain vibration signals could be found in the FTKR patients with either polyethylene or metal wear of the patellar component. We also found that for normal knee joint, the spectral power ratio of dominant poles has nearly 80% distribution in f < 100 Hz, is between 50% and 70% for knee with polyethylene wear and below 30% for metal wear, whereas in f > 500 Hz, spectral power ratio of dominant poles has over 30% distribution in metal wear but only nonsignificant distribution in polyethylene wear, no wear, and normal knee. The results show that vibration signals in rapid knee motion can be used for effectively detecting polyethylene wear of the patellar component in the early stage, while PPC signals can only be used to detect prosthetic metal wear in the late stage.

Subtype-independent immature secretion and subtype-dependent replication deficiency of a highly frequent, naturally occurring mutation of human hepatitis B virus core antigen.

The most frequent mutation of the human hepatitis B virus (HBV) core antigen occurs at amino acid 97. Recently, a phenylalanine (F)-to-leucine (L) mutation at this position (mutant F97L) in HBV surface antigen subtype ayw has been shown to result in an immature secretion phenotype, which is characterized by the nonselective export of an excessive amount of virions containing minus-strand, single-stranded HBV DNA. While subtype ayw mutant F97L has been found in Europe, the major reservoir of HBV resides in Asia and Africa. We report here that the immature secretion phenotype indeed can be found in an HBV strain (subtype adr) prevalent in Asia, changing from an isoleucine (I) to a leucine (mutant I97L). Despite its immature secretion phenotype, the adr variant I97L replicates as well as its parental adr wild-type I97I, supporting the conclusion that the extracellular phenotype of immature secretion is not a consequence of the intracellular HBV DNA replication defect. Further studies demonstrated that it is the acquisition of a leucine, rather than the loss of a wild-type amino acid at codon 97, that is important for immature secretion. We conclude that immature secretion is a subtype-independent phenotype and deficiency in intracellular DNA synthesis is a subtype-dependent phenotype. The former is caused by the trans-acting effect of a mutant core protein, while the latter by a cis-acting effect of a mutated nucleotide on the ayw genome. These immature secretion variants provide an important tool for studying the regulation of HBV virion assembly and secretion.

The mechanism of an immature secretion phenotype of a highly frequent naturally occurring missense mutation at codon 97 of human hepatitis B virus core antigen.

A very frequent missense mutation at codon 97 of human hepatitis B virus (HBV) core antigen (HBcAg) has been found in chronic carriers worldwide. Functional characterization of this mutant revealed one intracellular and two extracellular phenotypes in contrast to wild-type HBV: (i) a 6- to 12-fold decrease in the level of the full-length relaxed circular DNA, a 4- to 5-fold decrease in the plus-strand DNA, and an approximately 1.8-fold decrease in the minus-strand and overall DNA levels in the intracellular viral core particles; (ii) a 5.7-fold increase in the immature secretion of Dane particles, containing minus-strand, single-stranded virion DNA; and (iii) a significant reduction of nonenveloped core particles in the medium. The steady-state levels of mutant and wild-type core proteins expressed from the same vector appeared to be similar. Using a complementation assay and gradient centrifugation analysis, we demonstrated that this mutant core protein alone is necessary and sufficient for immature secretion. The decreased level of intracellular HBV DNA is caused by both the cis defect of the mutant genome and the trans defect of the mutant core protein. We have dissected further the relationship between the intracellular and extracellular phenotypes of mutant F97L. The pleiotropic effects of the HBcAg codon 97 mutation were observed consistently in several different experimental settings. The mechanism and biological significance of these findings are discussed.

Functional characterization of naturally occurring variants of human hepatitis B virus containing the core internal deletion mutation.

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.

A defective interference-like phenomenon of human hepatitis B virus in chronic carriers.

Defective interfering (DI) particles have been found in many RNA and DNA viruses of bacteria, plants, and animals since their first discovery in influenza virus. However, this fundamental phenomenon has not been demonstrated in human natural infections. Using a new approach, here we provide the first experimental evidence for the existence of DI-like viruses in human chronic carriers of hepatitis B virus (HBV). Functional characterization of naturally occurring core internal deletion (CID) variants of HBV revealed all of the features of DI particles. When equal amounts of wild-type and CID variant DNAs were cotransfected into a human hepatoma cell line, Huh7, a three- to fivefold enrichment of CID variants was most often observed. The fluctuations of the virus populations between CID variants and helper HBV in three chronic carriers are reminiscent of the cycling phenomenon in other DI viral systems. This finding has important implications for chronic viral hepatitis and other chronic progressive viral diseases.

Core antigen mutations of human hepatitis B virus in hepatomas accumulate in MHC class II-restricted T cell epitopes.

Despite the extensive molecular information on serum-derived human hepatitis B viruses (HBV), liver-derived replicative HBV genomes have remained largely uninvestigated. We have examined the sequences of the entire core antigen (nucleocapsid) of liver-derived HBVs in 15 different hepatoma patients. Bona fide mutations, rather than subtype polymorphism, have been identified based on the high-frequency occurrence of structural differences from wild type at the highly evolutionarily conserved positions, instead of at the positions known to contain genetic heterogeneity among different isolates from different geographic locations. The distribution of these naturally occurring mutations of HBV core gene appears to be nonrandom and is found predominantly within three major (I, IV, and V) and four minor domains (II, III, VI, and VII). In general, domain IV mutations correlate with domain V mutations. The replicative HBV DNAs tend to accumulate a higher number of mutated core domains than the integrated HBV DNAs. At the domain level, there is no significant difference in HBV core mutation frequencies between the liver tumors and the adjacent nontumorous livers. Strikingly, domains I, III, and V coincide with three major known T cell epitopes within the core protein in acute and chronic hepatitis B patients. Furthermore, these domains coincide with HLA class II-restricted T cell epitopes, rather than with the conventional HLA class I-restricted epitopes of cytotoxic T lymphocytes. Our results support the hypothesis that HBV core antigen variants can accomplish immunoevasion via accumulated escape mutations. In addition, they also provide a potential molecular explanation for the maintenance of persistent infection of human hepatitis B virus in chronic carriers.

The mechanism of natural occurrence of two closely linked HBV precore predominant mutations.

Two precore predominant mutations of human hepatitis B virus (HBV) at either nucleotide (nt) 1896 or nt 1899 often occur in combination. At nt 1896, a G to A mutation creates a TAG stop codon at codon 28 of precore protein. At nt 1899, a G to A mutation changes glycine at codon 29 to aspartic acid. To assess the effect of each individual mutation as well as any interaction between these two mutations, HBV derivatives bearing one or both precore predominant mutations have been constructed. HBV e-Ag-negative mutants bearing a TAG stop codon mutation at codon 28 uniformly replicate at least 20-fold better than mutants bearing a TGA stop codon at the same amino acid position, irrespective of the sequence context at nt 1899. A single mutation at nt 1899, changing the wild-type G to a pyrimidine (T or C) is deleterious to viral RNA encapsidation and DNA replication. Our results explain in part why only a purine (G or A) at nt 1899, never a pyrimidine, is observed in natural HBV genomes. The effects caused by these two closely linked mutations on viral replication are not independent of each other. The stringent selection for a highly efficient RNA encapsidation element may play a crucial role in the natural occurrence of these two closely linked precore mutations. The putative 27-amino-acid peptide resulting from the truncation of precore by the nt 1896 mutation has no apparent effect on viral replication. The preferential occurrence of the G to A mutation at nt 1896 and 1899, instead of at other nonpredominant positions, is likely to be a combined consequence of both selection and higher intrinsic mutation frequency at these positions.