Testing the balanced electrostatic interaction hypothesis of hepatitis B virus DNA synthesis by using an in vivo charge rebalance approach.

Previously, a charge balance hypothesis was proposed to explain hepatitis B virus (HBV) capsid stability, assembly, RNA encapsidation, and DNA replication. This hypothesis emphasized the importance of a balanced electrostatic interaction between the positive charge from the arginine-rich domain (ARD) of the core protein (HBc) and the negative charge from the encapsidated nucleic acid. It remains unclear if any of the negative charge involved in this electrostatic interaction could come from the HBc protein per se, in addition to the encapsidated nucleic acid. HBc ARD IV mutant 173GG and ARD II mutant 173RR/R157A/R158A are arginine deficient and replication defective. Not surprisingly, the replication defect of ARD IV mutant 173GG can be rescued by restoring positively charged amino acids at the adjacent positions 174 and 175. However, most interestingly, it can be at least partially rescued by reducing negatively charged residues in the assembly domain, such as by glutamic acid-to-alanine (E-to-A) substitutions at position 46 or 117 and to a much lesser extent at position 113. Similar results were obtained for ARD II mutant 173RR/R157A/R158A. These amino acids are located on the inner surfaces of HBc icosahedral particles, and their acidic side chains point toward the capsid interior. For HBV DNA synthesis, the relative amount of positive versus negative charge in the electrostatic interactions is more important than the absolute amount of positive or negative charge. These results support the concept that balanced electrostatic interaction is important during the viral life cycle.

Testing an electrostatic interaction hypothesis of hepatitis B virus capsid stability by using an in vitro capsid disassembly/reassembly system.

To test a previously coined "charge balance hypothesis" of human hepatitis B virus (HBV) capsid stability, we established an in vitro disassembly and reassembly system using bacterially expressed HBV capsids. Capsid disassembly can be induced by micrococcal nuclease digestion of encapsidated RNA. HBV core protein (HBc) mutants containing various amounts of arginine were constructed by serial truncations at the C terminus. Capsids containing smaller amounts of arginine (HBc 149, 154, and 157) remained intact after micrococcal nuclease digestion by native gel electrophoresis. Capsids containing larger amounts of arginine (HBc 159, 164, 169, and 171) exhibited reduced and more diffuse banding intensity and slightly upshifted mobility (HBc 159 and 164). Capsids containing the largest amounts of arginine (HBc 173, 175, and 183), as well as HBc 167, exhibited no detectable banding signal, indicating loss of capsid integrity or stability. Interestingly, capsid reassembly can be induced by polyanions, including oligonucleotides, poly-glutamic acid, and nonbiological polymer (polyacrylic acid). In contrast, polycations (polylysine and polyethylenimine) and low-molecular-weight anions (inositol triphosphate) induced no capsid reassembly. Results obtained by gel assay were confirmed by electron microscopy. Reassembled capsids comigrated with undigested parental capsids on agarose gels and cosedimented with undigested capsids by sucrose gradient ultracentrifugation. Taken together, the results indicate that HBV capsid assembly and integrity depend on polyanions, which probably can help minimize intersubunit charge repulsion caused mainly by arginine-rich domain III or IV in close contact. The exact structure of polyanions is not important for in vitro capsid reassembly. A large amount of independent experimental evidence for this newly coined "electrostatic interaction hypothesis" is discussed.

Modulation of alpha interferon anti-hepatitis C virus activity by ISG15.

ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-alpha) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-alpha, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-alpha-specific effect, which was not observed with IFN-gamma, correlated with an increase in expression of the IFN-alpha-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-alpha-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-alpha after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-alpha-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-alpha treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-alpha pathway and its antiviral effect.

Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5'-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-beta expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.

Exposure of RNA templates and encapsidation of spliced viral RNA are influenced by the arginine-rich domain of human hepatitis B virus core antigen (HBcAg 165-173).

Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nuclease-resistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.

Reduced secretion of virions and hepatitis B virus (HBV) surface antigen of a naturally occurring HBV variant correlates with the accumulation of the small S envelope protein in the endoplasmic reticulum and Golgi apparatus.

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.

Coexistence of two distinct secretion mutations (P5T and I97L) in hepatitis B virus core produces a wild-type pattern of secretion.

Unlike a Tokyo isolate of hepatitis B virus variants, we found a Shanghai isolate that secretes few virions with an immature genome despite its core I97L mutation. Core mutations P5T and I97L were found to be mutually compensatory in offsetting their respective distinct effects on virion secretion.

Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins.

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 degrees 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 T=3 or T=4 icosahedral particles. No difference in the ratio between T=3 (78%) and T=4 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 T=3 and T=4 particles successfully separated by using a novel agarose gel electrophoresis procedure.

Elevated levels of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 during the acute phase of Kawasaki disease.

Kawasaki disease (KD) is an acute, self-limiting, multisystem vasculitis of unknown etiology affecting infants and young children. Unless treated promptly with high-dose intravenous gamma globulin and aspirin, patients frequently develop coronary aneurysms. Previously, matrix metalloproteinase 9 (MMP-9), which is secreted complexed to tissue inhibitor of metalloproteinase 1 (TIMP-1), has been implicated in abdominal aortic aneurysm formation. Since the clinical and pathological features of KD include inflammation and weakening of blood vessels, we analyzed acute- and convalescent-phase paired plasma or serum samples from 31 KD patients, 7 patients who did not completely meet the criteria for KD, and 26 non-KD controls (9 febrile and 17 afebrile patients) for pro-MMP-9 (92 kDa) enzyme activity by gelatin zymography and for active MMP-9 (83 kDa), pro-MMP-9, and TIMP-1 protein levels by enzyme-linked immunosorbent assay. Statistical analysis was performed by using Student t tests, linear regression, and the Wilcoxon rank-sum test. Markedly elevated pro-MMP-9 enzymatic activity, pro-MMP-9 protein levels, and TIMP-1 protein levels were found during the acute phase of illness in patients with clinically established KD and in patients who were suspected of having KD but did not meet all of the criteria. There was no significant difference in active MMP-9 levels. Furthermore, pro-MMP-9 and TIMP-1 protein levels were significantly elevated among KD patients, compared to those of febrile and afebrile non-KD controls. The significantly elevated pro-MMP-9 enzyme and protein levels during the acute phase of KD may reflect vascular remodeling or an inflammatory response to a microbial agent, suggesting a pathophysiological role for MMP-9 in coronary aneurysm formation.