Hepatitis viruses and signal transduction: true to the core?
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Hepacivirus↗
Biomedical subjects
M M Lai
Publications and source records attributed to M M Lai.
Hepatitis delta virus: the molecular basis of laboratory diagnosis.
Infection with hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is associated with severe and sometimes fulminant hepatitis. The traditional methods for the diagnosis of HDV infection, such as detection of serum anti-HD antibodies, are sufficient for the clinical diagnosis of delta infection. However, such techniques lack the sensitivity and specificity required to more accurately characterize the nature of HDV infection and to assess the efficacy of therapies. Recent improvements in molecular techniques, such as HDV RNA hybridization and RT-PCR, have provided increased diagnostic precision and a more thorough understanding of the natural course of HDV infection. These advances have enhanced the clinician's ability to accurately evaluate the stage of HDV infection, response to therapy, and occurrence of reinfection after orthotopic liver transplant. This review focuses on the recent advances in the understanding of the molecular biology of HDV and in the laboratory diagnosis of HDV infection.
Hepatitis Antibodies↗
The large delta antigen of hepatitis delta virus potently inhibits genomic but not antigenomic RNA synthesis: a mechanism enabling initiation of viral replication.
Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.
Blotting, Northern↗
RNA-Dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases.
Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of alpha-amanitin (<3 microgram/ml) and could be partially restored by an alpha-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by alpha-amanitin to a concentration higher than 25 microgram/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.
Animals↗
Protein 4.1N binding to nuclear mitotic apparatus protein in PC12 cells mediates the antiproliferative actions of nerve growth factor.
Protein 4.1N is a neuronal selective isoform of the erythrocyte membrane cytoskeleton protein 4.1R. In the present study, we demonstrate an interaction between 4.1N and nuclear mitotic apparatus protein (NuMA), a nuclear protein required for mitosis. The binding involves the C-terminal domain of 4.1N. In PC12 cells treatment with nerve growth factor (NGF) elicits translocation of 4. 1N to the nucleus and promotes its association with NuMA. Specific targeting of 4.1N to the nucleus arrests PC12 cells at the G1 phase and produces an aberrant nuclear morphology. Inhibition of 4.1N nuclear translocation prevents the NGF-mediated arrest of cell division, which can be reversed by overexpression of 4.1N. Thus, nuclear 4.1N appears to mediate the antiproliferative actions of NGF by antagonizing the role of NuMA in mitosis.
Animals↗
Mouse hepatitis virus strain JHM infects a human hepatocellular carcinoma cell line.
Mouse hepatitis virus (MHV) strain JHM is a coronavirus that causes encephalitis and demyelination in susceptible rodents. The known receptors for MHV are all members of the carcinoembryonic antigen family. Although human forms of the MHV receptor can function as MHV receptors in some assays, no human cell line has been identified that can support wild-type MHV infection. Here we describe the infection of a human hepatocellular carcinoma cell line, HuH-7, with MHV. HuH-7 cells were susceptible to strains JHM-DL and JHM-DS, yielding virus titers nearly identical to those seen in mouse DBT cells. In contrast, HuH-7 cells were only marginally susceptible or completely resistant to infection by other MHV strains, including A59. JHM produced a strong cytopathic effect in HuH-7 cells with the formation of round plaques. Studies of various recombinant viruses between JHM and A59 strains suggested that the ability of JHM to infect HuH-7 cells was determined by multiple viral genetic elements. Blocking the viral spike (S) protein with a neutralizing antibody or a soluble form of the MHV receptor inhibited infection of HuH-7 cells, suggesting that infection is mediated through the S protein. Transfection with the prototype mouse receptor, biliary glycoprotein, rendered HuH-7 cells susceptible to infection by other MHV strains as well, suggesting that JHM uses a receptor distinct from the classical MHV receptor to infect HuH-7 cells. Possible implications for human disease are discussed.
Animals↗
Formation of a ribonucleoprotein complex of mouse hepatitis virus involving heterogeneous nuclear ribonucleoprotein A1 and transcription-regulatory elements of viral RNA.
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) specifically binds to two transcription-regulatory elements, i.e., the leader and intergenic sequence, of the negative-strand (template-strand) RNA of mouse hepatitis virus (MHV) and may play a role in viral RNA transcription. Previous studies based on the defective-interfering RNAs of MHV suggested that these two RNA elements may interact with each other during transcription, although they do not have complementary sequences. In this study, we showed by an in vitro reconstitution assay that hnRNP A1 could mediate the formation of an RNP complex involving these two RNA elements. Both the RNA-binding domains and protein-interacting domain of hnRNP A1 contributed to the efficient formation of the RNP complex; however, the presence of the two RNA-binding domains alone, without the protein-interacting domain, also resulted in some RNP formation. Omission of hnRNP A1 in the reconstitution reaction abolished the RNP formation, and mutations of the IG sequences significantly inhibited the RNP formation. These findings suggest that the two cis-acting transcription-regulatory sequences of MHV RNA can interact with each other through the formation of an RNP complex involving a cellular protein hnRNP A1. This RNP complex may participate in MHV RNA transcription.
Animals↗
Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein.
Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.
Animals↗
The calcineurin-dynamin 1 complex as a calcium sensor for synaptic vesicle endocytosis.
Exocytosis of synaptic vesicles is calcium-dependent, with synaptotagmin serving as the calcium sensor. Endocytosis of synaptic vesicles has also been postulated as a calcium-dependent process; however, an endocytic calcium sensor has not been found. We now report a physical association between the calcium-dependent phosphatase calcineurin and dynamin 1, a component of the synaptic endocytic machinery. The calcineurin-dynamin 1 interaction is calcium-dependent, with an EC(50) for calcium in the range of 0.1-0. 4 microM. Disruption of the calcineurin-dynamin 1 interaction inhibits clathrin-mediated endocytosis. Thus, the calcium-dependent formation of the calcineurin-dynamin 1 complex, delivered to the other endocytic coat proteins, provides a calcium-sensing mechanism that facilitates endocytosis.
Animals↗
Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.
Most isolates of hepatitis C virus (HCV) infections are resistant to interferon, the only available therapy, but the mechanism underlying this resistance has not been defined. Here it is shown that the HCV envelope protein E2 contains a sequence identical with phosphorylation sites of the interferon-inducible protein kinase PKR and the translation initiation factor eIF2alpha, a target of PKR. E2 inhibited the kinase activity of PKR and blocked its inhibitory effect on protein synthesis and cell growth. This interaction of E2 and PKR may be one mechanism by which HCV circumvents the antiviral effect of interferon.
Cell Line↗
Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling.
RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)-related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.
Adaptor Proteins, Signal Transducing↗
The nucleocapsid protein of murine hepatitis virus type 3 induces transcription of the novel fgl2 prothrombinase gene.
Using a set of parental and recombinant murine hepatitis virus strains, we demonstrate that the nucleocapsid protein induces transcription of the novel fgl2 prothrombinase gene and elevated procoagulant activity in those strains that produce fulminant hepatitis. Chinese hamster ovary cells cotransfected with a construct expressing nucleocapsid protein from susceptible strains and with a luciferase reporter construct containing the fgl2 promoter showed a 6-fold increase in luciferase activity compared with nontransfected cells or cells cotransfected with a construct expressing nucleocapsid protein from resistant strains. Two deletions found at coding sites 111-123 and 1143-1145 of structural domains I and III, respectively, of the nucleocapsid gene may account for the differences between pathogenic and nonpathogenic strains. Preliminary mapping of the fgl2 promoter has defined a region from -372 to -306 upstream from the ATG translation initiation site to be responsive to nucleocapsid protein. Hence, mapping of genetic determinants in parental and recombinant strains demonstrates that the nucleocapsid protein of strains that induce fulminant hepatitis is responsible for transcription of the fgl2 prothrombinase gene. These studies provide new insights into the role of the nucleocapsid gene in the pathogenesis of viral hepatitis.
Animals↗
An internal polypyrimidine-tract-binding protein-binding site in the hepatitis C virus RNA attenuates translation, which is relieved by the 3'-untranslated sequence.
Hepatitis C virus (HCV) RNA binds to several cellular proteins, which may regulate translation or replication of viral RNA. One of these is polypyrimidine tract-binding protein (PTB), which binds to the 5'-untranslated region (UTR) and the 3'-end 98 nucleotides (nt) (X region) of HCV RNA. Both of these PTB-binding sites regulate HCV translation. In this study, we further investigated the nature of PTB binding on HCV RNA. UV cross-linking studies using HeLa cell extracts and a recombinant PTB showed that the PTB-5'-UTR binding was much weaker than the PTB-3'-UTR binding. Unexpectedly, we found an even stronger PTB-binding site in the core-protein-coding region of HCV RNA. The binding domain was mapped to the 3'-end of this region, which contains a pyrimidine-rich sequence highly conserved among HCV isolates. Using a set of synthetic HCV RNAs with or without this sequence in in vitro translation studies, we showed that the PTB-binding sequence in the core-coding region strongly inhibited translation of HCV RNA. This inhibition was relieved by the presence of the X region at the 3'-end. Furthermore, the previously reported translational enhancement by the HCV 3'-UTR was more pronounced when this PTB-binding site was present in the RNA. These results suggest that PTB binding to an internal site of HCV RNA provides another mechanism for regulation of HCV translation.
Base Sequence↗
Polypyrimidine tract-binding protein binds to the leader RNA of mouse hepatitis virus and serves as a regulator of viral transcription.
A cellular protein, previously described as p55, binds specifically to the plus strand of the mouse hepatitis virus (MHV) leader RNA. We have purified this protein and determined by partial peptide sequencing that it is polypyrimidine tract-binding protein (PTB) (also known as heterogeneous nuclear ribonucleoprotein [hnRNP] I), a nuclear protein which shuttles between the nucleus and cytoplasm. PTB plays a role in the regulation of alternative splicing of pre-mRNAs in normal cells and translation of several viruses. By UV cross-linking and immunoprecipitation studies using cellular extracts and a recombinant PTB, we have established that PTB binds to the MHV plus-strand leader RNA specifically. Deletion analyses of the leader RNA mapped the PTB-binding site to the UCUAA pentanucleotide repeats. Using a defective-interfering RNA reporter system, we have further shown that the PTB-binding site in the leader RNA is critical for MHV RNA synthesis. This and our previous study (H.-P. Li, X. Zhang, R. Duncan, L. Comai, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 94:9544-9549, 1997) combined thus show that two cellular hnRNPs, PTB and hnRNP A1, bind to the transcription-regulatory sequences of MHV RNA and may participate in its transcription.
5' Untranslated Regions↗
Polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3' untranslated region, thereby altering RNA conformation.
Mouse hepatitis virus (MHV) RNA transcription is regulated mainly by the leader and intergenic (IG) sequences. However, a previous study has shown that the 3' untranslated region (3'-UTR) of the viral genome is also required for subgenomic mRNA transcription; deletion of nucleotides (nt) 270 to 305 from the 3'-UTR completely abolished subgenomic mRNA transcription without affecting minus-strand RNA synthesis (Y.-J. Lin, X. Zhang, R.-C. Wu, and M. M. C. Lai, J. Virol. 70:7236-7240, 1996), suggesting that the 3'-UTR affects positive-strand RNA synthesis. In this study, by UV-cross-linking experiments, we found that several cellular proteins bind specifically to the minus-strand 350 nucleotides complementary to the 3'-UTR of the viral genome. The major protein species, p55, was identified as the polypyrimidine tract-binding protein (PTB, also known as heterogeneous nuclear RNP I) by immunoprecipitation of the UV-cross-linked protein and binding of the recombinant PTB. A strong PTB-binding site was mapped to nt 53 to 149, and another weak binding site was mapped to nt 270 to 307 on the complementary strand of the 3'-UTR (c3'-UTR). Partial substitutions of the PTB-binding nucleotides reduced PTB binding in vitro. Furthermore, defective interfering (DI) RNAs harboring these mutations showed a substantially reduced ability to synthesize subgenomic mRNA. By enzymatic and chemical probing, we found that PTB binding to nt 53 to 149 caused a conformational change in the neighboring RNA region. Partial deletions within the PTB-binding sequence completely abolished the PTB-induced conformational change in the mutant RNA even when the RNA retained partial PTB-binding activity. Correspondingly, the MHV DI RNAs containing these deletions completely lost their ability to transcribe mRNAs. Thus, the conformational change in the c3'-UTR caused by PTB binding may play a role in mRNA transcription.
3' Untranslated Regions↗
Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells.
Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.
Animals↗
A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA.
All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3'-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5' end or 45 nt from the 3' end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3'-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3' end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3' end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3' end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.
3' Untranslated Regions↗
Acinetobacter baumannii bloodstream infection: clinical features and antimicrobial susceptibilities of isolates.
The number of nosocomial infections caused by Acinetobacter baumannii has increased in recent years. The purposes of this study are to discover the risk factors of transmission to prevent the nosocomial infection of A. baumannii. We retrospectively studied 36 patients with A. baumannii bacteremia at China Medical College Hospital from January 1996 to December 1997. There were 23 males and 13 females. All bacteremia were acquired nosocomially. Malignancy (n = 8) and intracranial hemorrhage (n = 6) were the most common underlying diseases. Only one patient on arterial line disclosed intraarterial catheter-related A. baumannii bacteremia and 3 patients had evidence of A. baumannii pneumonia. Twenty-one patients (58%) had central venous catheters in place at the onset of bacteremia, but none was proven to be catheter-related infection. There were 32 patients (89%) with unknown portal of entry. Multivariate logistic regression analysis revealed that potential risk factors related to A. baumannii bacteremia were prior antimicrobial therapy (P < 0.05). The most common clinical features of A. baumannii bacteremia were, in descending order, fever, leukocytosis, thrombocytopenia and hypotension. Eleven patients (30.6%) died directly from A. baumannii bacteremia. All isolates were resistant to ampicillin, cephalothin, cefonicid and moxalactam. The most alarming evidence was that 19% of isolates showed resistance to imipenem. Our findings emphasized that A. baumannii bacteremia had the following characteristics: usually acquired nosocomially, unknown portal of entry, and high multiresistance, especially the increasing resistance rate to imipenem. Imipenem must be reserved as a last-line agent to treat A. baumannii infections, so we want to suggest that the treatment of choice for A. baumannii is gentamicin, amikacin or ceftazidime.
Acinetobacter Infections↗