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M M Lai

Publications and source records attributed to M M Lai.

At least 91 records · Page 5Linked to original sources
PubMed1996

A pseudoknot ribozyme structure is active in vivo and required for hepatitis delta virus RNA replication.

The ribozymes of hepatitis delta virus (HDV) have so far been studied primarily in vitro. Several structural models for HDV ribozymes based on truncated HDV RNA fragments, which are different from the hammerhead or the hairpin/paperclip ribozyme model proposed for plant viroid or virusoid RNAs, have been proposed. Whether these structures actually exist in vivo and whether ribozymes actually function in the HDV replication cycle have not been demonstrated. We have now developed an in vivo ribozyme self-cleavage assay capable of detecting self-cleavage of dimer or trimer HDV RNA in vivo. By site-directed mutagenesis and compensatory mutations to disrupt and restore potential base pairing in the ribozyme domain of the full-length HDV RNA according to the various structural models, a close correlation between the detected in vivo and the predicted in vitro ribozyme activities of various mutant RNAs was demonstrated. These results suggest that the proposed in vitro ribozyme structure likely exists and functions during the HDV replication cycle in vivo. Furthermore, the pseudoknot model most likely represents the structure responsible for the ribozyme activity in vivo. All of the mutants that had lost the ribozyme activity could not replicate, indicating that the ribozyme activities are indeed required for HDV RNA replication. However, some of the compensatory mutants which have restored both the cleavage and ligation activities could not replicate, suggesting that the ribozyme domains are also involved in other unidentified functions or in the formation of an alternative structure that is required for HDV RNA replication. This study thus established that the ribozyme has important biological functions in the HDV life cycle.

Base Sequence↗
PubMed1996

Hepatitis delta antigens enhance the ribozyme activities of hepatitis delta virus RNA in vivo.

The mechanism of regulation for the ribozyme activity of hepatitis delta virus (HDV) RNA in infected cells is unknown. Previously, we developed a direct assay capable of detecting the ribozyme activity of HDV dimer or trimer RNAs in vivo (K.-S. Jeng, A. Daniel, and M. M. C. Lai, J. Virol, 70:2403-2410, 1996). In this study, we used this method to examine the effects of hepatitis delta antigen (HDAg) on the ribozyme activities of HDV RNA in vivo. The HDV multimer cDNAs were cotransfected with plasmids encoding either HDV small delta antigen (SHDAg) or large delta antigen (LHDAg), and the self-cleavage of the primary transcripts from the HDV cDNA was analyzed at day 2 postransfection. The results were as follows. (i) Both HDAgs, particularly LHDAg, enhanced the self-cleavage activity of HDV RNA; however, HDAgs are not required for HDV RNA cleavage. (ii) HDAg could not restore the ribozyme activity of mutant HDV RNAs which have lost the ribozyme function. (iii) The enhancement of ribozyme activity by HDAg does not require HDV RNA replication. (iv) RNA-binding activity of HDAg is required for the enhancement of RNA cleavage. (v) The self-ligation activities of HDV ribozyme also were enhanced by HDAg. These results suggest that HDAg can regulate the cleavage and ligation of HDV RNA during the HDV life cycle.

Cell Line↗
PubMed1996

Epitopes exposed on hepatitis delta virus ribonucleoproteins.

A total of 17 antibodies, raised in several nonhuman species and specific for different regions on the delta antigen (delta Ag), were used to map, via immunoprecipitation, those domains exposed on the surface of the viral ribonucleoprotein (RNP). These studies showed that the domains for the nuclear localization signal and the C-terminal extension, unique to the large form of delta Ag, are exposed. Also exposed is the C-terminal region of the small form of delta Ag. In contrast, reactivity was not found with the coiled-coil domain needed for protein dimerization. When the hepatitis delta virus (HDV) RNA was released by treatment of viral RNP with vanadyl ribonucleoside complexes, no change in the pattern of delta Ag epitope presentation was detected, consistent with the interpretation that a multimeric protein structure persists in the absence of RNA. These RNP studies have implications not only for understanding of the process of HDV assembly but also for evaluation of the immune responses of an infected host to HDV replication.

Animals↗
PubMed1995

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus.

Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low-stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules.

Amino Acid Sequence↗
PubMed1995

Differential subcellular localization of hepatitis C virus core gene products.

The expression of the core gene of two different hepatitis C virus (HCV) isolates was analyzed. In the presence of its downstream E1 envelope protein sequence, two major core protein products with molecular masses of 21 kDa (P21) and 19 kDa (P19) and a minor protein product with molecular mass of 16 kDa (P16) were detected. In the absence of its downstream E1 envelope protein sequence, P21 and P19 remained the major protein products expressed from the core gene of the HCV-RH isolate, whereas P16 became the major protein product of the core gene of the HCV-1 isolate. Analysis of the amino-terminal sequences of P21 and P16 expressed in Escherichia coli revealed that P21 and P16 were co-amino terminal. Deletion-mapping analysis indicated that P16 lacked the carboxy-terminal sequence of P21. Immunofluorescence analysis of the subcellular localization of different HCV core proteins indicated that P21 and P19 displayed a reticular and punctate staining pattern typical of endoplasmic reticulum-associated proteins, while P16 was localized to the nucleus. The distinct subcellular localization of P16 raises the possibility that P16 may have a biological function very different from those of P21 and P19.

Amino Acid Sequence↗
PubMed1995

A cis-acting viral protein is not required for the replication of a coronavirus defective-interfering RNA.

Mouse hepatitis virus (MHV), a coronavirus, generates defective-interfering (DI) RNAs of different sizes during passages at high multiplicities of infection. All MHV DI RNAs characterized so far contain an open reading frame (ORF) encoding a fused viral protein; in addition, DI RNAs with a long ORF have a competitive advantage over those with a shorter ORF. These findings suggest that DI RNA replication may require an ORF encoding a cis-acting viral protein. In this study, we used a naturally occurring DI RNA and inserted a 12-nucleotide (nt) amber-mutation linker at various positions to truncate the ORF. Most of the mutants replicated as well as the wild-type DI RNA, irrespective of the presence or absence and the length of the ORF in the RNA. Sequence analysis showed that all of the mutants retained the insertional mutations even after two viral passages in tissue culture, establishing that the mutant DI RNAs replicated. We have further introduced two 3-nucleotide substitutions of the first two AUG codons of the ORF, thus completely closing the ORF. This DI RNA replicated as well as the wild-type DI, but, after a single passage, the majority of the mutant RNAs was replaced by recombinant RNAs which contain a restored functional ORF. However, an additional insertion of a 12-nt amber-mutation linker downstream of the AUG substitutions prevented recombination, and the DI RNA still replicated. These data indicate that DI RNA replication does not require a DI-specific ORF encoding cis-acting viral proteins and that a 12-nucleotide insertion could prevent or delay the occurrence of RNA recombination, suggesting the importance of direct or indirect RNA alignment in homologous RNA recombination.

Animals↗
PubMed1995

Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase.

We have developed an expression vector system using a defective-interfering (DI) RNA of mouse hepatitis virus (MHV), a prototype coronavirus, to deliver and express a foreign gene in MHV-infected cells. This vector contains an MHV intergenic sequence to promote the expression of foreign genes. In this study, we used this vector to introduce a hemagglutinin-esterase (HE) protein, an optional MHV structural protein, into the MHV-infected cells. The engineered HE protein could be efficiently incorporated into the virion which did not synthesize its own HE protein, thus generating a pseudorecombinant virus that expresses an exogenous HE protein. The engineered HE protein could be made distinguishable from the native protein by attaching an 8-amino-acid peptide tag at the carboxyl-terminus. Both the engineered and native HE proteins from the HE-producing virus train could be incorporated into the virion, thus generating phenotypically mixed virus particles. We also showed that the HE-expressing DI RNA could be incorporated into viruses, and the engineered HE protein expressed in the infected cells for at least three serial virus passages. Furthermore, we have made two mutants, in which parts of the external domain of the HE protein have been deleted, to study the sequence requirements for the stable expression of HE and its incorporation into MHV virions. Although both of the mutant HE proteins could be expressed in the MHV-infected cells, they failed to be incorporated into virions, suggesting the importance of the extracellular domain of HE protein for its incorporation into virus particles. This vector system enabled the first successful incorporation of a selected coronaviral protein into virions and demonstrates its utility as an expression vector for studying the molecular biology of coronaviruses.

Animals↗
PubMed1995

Regulation of coronavirus RNA transcription is likely mediated by protein-RNA interactions.

Coronavirus mRNA transcription was thought to be regulated by the interaction between the leader RNA and the intergenic (IG) sequence, probably involving direct RNA-RNA interactions between complementary sequences. In this study, we found that a 9-nucleotide sequence immediately downstream of the leader RNA up-regulated mRNA transcription and that a particular strain of mouse hepatitis virus (MHV) lacking this 9-nucleotide transcribed subgenomic mRNA species containing unusually heterogeneous leader-fusion sites. These results suggest that the sequence complementarity between the leader and IG is not necessarily required for mRNA transcription. UV cross-linking experiments using cytoplasmic extracts of uninfected cells and the IG sequence showed that three different cellular proteins bound to IG of the template RNA. Deletion analyses and site-directed mutagenesis of IG further demonstrated a correlation between protein-binding and transcription efficiency, suggesting that these RNA-binding proteins are involved in the regulation of coronavirus mRNA transcription. We propose that coronavirus transcription is regulated by RNA-protein and protein-protein interactions.

Animals↗
PubMed1995

Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed.

Previous studies suggested that coronavirus RNA transcription involves interaction between leader RNA and the intergenic (IG) sequences, probably via protein-RNA interactions (X. M. Zhang, C.-L. Liao, and M. M. C. Lai, J. Virol., 68:4738-4746, 1994; X. M. Zhang and M. M. C. Lai, J. Virol., 68:6626-6633, 1994). To determine whether cellular proteins are involved in this process, we performed UV cross-linking experiments using cytoplasmic extracts of uninfected cells and the IG (promoter) sequence between genes 6 and 7 (IG7) and the 5' untranslational region of mouse hepatitis virus genomic RNA. We demonstrated that three different cellular proteins (p70, p48, and p35/38) bound to the promoter sequence of the template RNA. Deletion analyses of the template RNA mapped the binding site of p35/38 at the consensus transcription initiation signal. In contrast, the binding of p70 and p48 was less specific. p35/38 is the same protein as the one previously identified to bind to the complementary strand of the leader RNA; its binding affinity to the leader was approximately 15 times stronger than that to IG7. Site-directed mutagenesis of the IG sequence revealed that mutations in the consensus sequence of IG7 (UCUAAUCUAAAC to UCGAAAC and GCUAAAG), which resulted in reduced subgenomic mRNA transcription, also caused correspondingly reduced levels of p35/38 binding. These results demonstrated that the extent of protein binding to the IG sequences correlated with the amounts of subgenomic mRNAs transcribed from the IG site. These studies suggest that these RNA-binding proteins are involved in coronavirus RNA transcription and may represent transcription factors.

Base Sequence↗
PubMed1995

Transgenic mice support replication of hepatitis delta virus RNA in multiple tissues, particularly in skeletal muscle.

Hepatitis delta virus (HDV) is hepatotropic and frequently causes fulminant hepatitis in both human and nonhuman primate hosts. To understand the molecular basis of HDV tissue tropism and the mechanism of pathogenesis, transgenic mice in which replication-competent HDV dimeric RNA is expressed under the control of either liver-specific or universal transcriptional promoters were developed. The expressed RNA replicated efficiently in the liver and several tissues of nonhepatic origin. Surprisingly, maximal replication of HDV RNA occurred in skeletal muscle and was almost 100-fold greater than in the liver. These findings suggest that the hepatotropism of HDV is most likely a receptor-mediated restriction and that muscle-specific factors may facilitate HDV RNA replication. No evidence of cytopathology was apparent in most of the tissues examined, including the liver, supporting the contention that hepatocellular disease is not mediated by direct cytopathological effects associated with HDV RNA replication and gene expression. However, mild muscle atrophy in some of the transgenic mice was noted. Delta antigen was detected in the nuclei of myocytes. Only the small form, not the large form, of delta antigen was detected, suggesting that the RNA editing event which causes the conversion of delta antigen did not occur in transgenic mice. Furthermore, the 0.8-kb antigenomic RNA species, which is postulated to be the mRNA for delta antigen, was not detected in mice. The preferential replication of HDV RNA in skeletal muscle suggests that HDV RNA replication can be facilitated by certain muscle-specific factors.

Actins↗
PubMed1995

Replication of hepatitis delta virus RNA in mice after intramuscular injection of plasmid DNA.

To establish a readily manipulable small-animal system for the study of human hepatitis delta virus (HDV) replication in vivo, plasmid DNAs containing head-to-tail cDNA dimers of HDV were inoculated intramuscularly into mice. Genomic-sense HDV RNA was detected in the injected muscle within 1 week and increased to substantial levels by week 7 postinjection. The intramuscular accumulation of HDV RNA was determined to be the direct result of viral RNA replication by three lines of evidence: (i) injected tissues also accumulated antigenomic-sense HDV RNA, (ii) plasmid DNA that synthesized primary transcripts of antigenomic sense also led to the accumulation of genomic-sense HDV RNA, and (iii) injection of a cDNA dimer defective in antigenomic RNA cleavage failed to produce detectable HDV RNA in muscle. Immunohistochemical analysis of injected muscle demonstrated the presence and nuclear localization of hepatitis delta antigen in myocytes. Finally, sera from DNA-injected mice contained antibodies specific for delta antigen, indicating the induction of an immunological response to the intracellularly expressed antigen. These findings demonstrated the ability of HDV RNA to replicate in skeletal muscle and provide a useful system for the study of HDV replication, delta antigen processing, and its presentation to the immune system in vivo. Furthermore, this system offers an efficiently replicating RNA as a potential vehicle for in vivo gene transfer.

Animals↗
PubMed1995

The molecular biology of hepatitis delta virus.

Hepatitis delta virus (HDV) contains a circular, viroid-like RNA genome, the only animal viral RNA of its kind. It possesses a ribozyme activity, which can autocatalytically cleave and ligate itself. The ribozyme has a unique structural requirement different from other known ribozymes. HDV RNA undergoes RNA-dependent RNA replication via a double rolling circle mechanism, which is probably mediated by cellular RNA polymerase II, utilizing modified cellular transcription machineries. HDV RNA encodes a single protein, hepatitis delta antigen, which is a nuclear, RNA-binding phosphoprotein and required for viral RNA replication. During replication, HDV RNA undergoes a specific RNA editing event to extend its open reading frame and produce a longer, isoprenylated delta antigen, which suppresses RNA replication and initiates viral particle assembly. Ribozyme, cell-mediated RNA-dependent RNA replication, and RNA editing are some of the unique properties and unresolved issues of the molecular biology of HDV.

Animals↗
PubMed1995

Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression.

The hemagglutinin-esterase (HE) protein of mouse hepatitis virus (MHV) is an optional envelope protein present in only some MHV isolates. Its expression is regulated by the copy number of a UCUAA pentanucleotide sequence present in the leader sequence of the viral genomic RNA. The functional significance of this viral protein so far is not clear. In this report, we compared the neuropathogenicity of two MHV isolates, JHM(2) and JHM(3), which express different amounts of HE protein. Intracerebral inoculation of these two viruses into C57BL/6 mice showed that JHM(2), which expresses an abundant amount of HE protein, was more neurovirulent than JHM(3), which expresses very little HE. Histopathology showed that early in infection, JHM(2) infected primarily neurons, while JHM(3) infected mainly glial cells. JHM(3) eventually infected neurons and caused a delayed death relative to JHM(2)-infected mice, suggesting that the progression of JHM(3) infection in the central nervous system was slower than JHM(2). In vitro infection of JHM(3) in primary mixed glial cell cultures of astrocyte-enriched cultures yielded higher virus titers than JHM(2), mimicking the preferential growth of JHM(3) in glial cells in vivo. These findings suggest that the reduced neuropathogenicity of JHM(3) may correlate with its preferential growth in glial cells. Sequence analysis showed that the S genes of these two viruses are identical, thus ruling out the S gene as the cause of the difference in neuropathogenicity between these two viruses. We conclude that the HE protein contributes to viral neuropathogenicity by influencing either the rate of virus spread, viral cell tropism or both.

Acetylesterase↗
PubMed1995

Molecular biologic and pathogenetic analysis of hepatitis delta virus.

Hepatitis delta virus (HDV) consists of an envelope derived from hepatitis B virus (HBV) and a nucleocapsid consisting of hepatitis delta antigen and a circular RNA genome. Both the delta antigen and the RNA possess many unique properties which play crucial roles in the life cycle of HDV. They may also contribute indirectly to the mechanism of viral pathogenesis.

Antigens, Viral↗
PubMed1994

Isoprenylation of large hepatitis delta antigen is necessary but not sufficient for hepatitis delta virus assembly.

Hepatitis delta virus (HDV) encodes two proteins, the small hepatitis delta antigen (SHDAg) and large hepatitis delta antigen (LHDAg). Both proteins are identical except for the presence of additional 19 amino acids at the C terminus of LHDAg. While SHDAg is required for HDV RNA replication, LHDAg inhibits replication and is required together with hepatitis B surface antigen for the assembly of HDV. The C-terminal last 4 amino acids of LHDAg (Cys-Arg-Pro-Gln) is an isoprenylation motif. It has previously been shown that the mutation of the Cys inhibited the assembly of HDV. In order to discern whether this effect is due to change of amino acid residue or abolition of isoprenylation, we constructed several LHDAg mutants of the terminal three amino acid residues and tested their abilities to be packaged with HBsAg by cotransfection experiments. We also made GST-fusion proteins of these mutants and tested their abilities to be isoprenylated in rabbit reticulocyte lysate system. We found that some, but not all, of the substitutions of the amino acid residues other than the Cys also inhibited isoprenylation and that the status of isoprenylation of these mutant proteins correlated well with their abilities to be packaged with HBsAg into virions. This result indicates that isoprenylation, rather than the primary amino acid sequence, is required for LHDAg packaging. Furthermore, we found that the attachment of an isoprenylation motif to SHDAg did not enable it to be packaged with HBsAg and that the deletions of any 5 amino acids in the last 15 amino acids (amino acids 196 to 210) unique to the LHDAg abolished the packaging ability. In contrast, the deletion of 33 amino acids (amino acids 163 to 195) upstream of the last C-terminal 19 amino acids of LHDAg did not interfere with its packaging ability. Therefore, we conclude that the 15 amino acids upstream of the isoprenylation site of LHDAg are also essential for HDV assembly, and a large portion of the alleged C-terminal Pro/Gly-rich region (amino acids 146 to 195) is not required for the assembly process.

Amino Acid Sequence↗
PubMed1994

Mouse hepatitis virus receptors: more than a single carcinoembryonic antigen.

Mouse hepatitis virus (MHV), a murine coronavirus, has been shown to utilize carcinoembryonic antigen (CEA) as the receptor. We have demonstrated that MHV can utilize a different isoform of CEA, which is an alternatively spliced gene product that is expressed in different tissues, as a receptor. Furthermore, the CEA molecules from a resistant mouse strain (SJL) have different sequences and yet serve as functional viral receptors. Thus, MHV can use more than a single type of CEA molecule as the receptor. We have also shown that some mouse cell lines express functional CEA molecules and yet are resistant to infections by certain MHV strains. Biochemical studies of the infected cells indicate that MHV infections in these cell lines are blocked at the steps of virus entry. We conclude that MHV entry requires additional cellular factors other than CEA, the viral receptor. The significance of viral receptors and the additional cellular factors in regulating viral tropism is discussed.

Animals↗
PubMed1994

Unusual heterogeneity of leader-mRNA fusion in a murine coronavirus: implications for the mechanism of RNA transcription and recombination.

Coronavirus mRNA transcription was thought to be regulated by the interaction between the leader RNA and the intergenic sequence (IS), probably involving direct RNA-RNA interactions between complementary sequences. In this study, we found that a particular strain of mouse hepatitis virus, JHM2c, which has a deletion of a 9-nucleotide (nt) sequence (UUUAUAAAC) immediately downstream of the leader RNA, transcribed subgenomic mRNA species containing a whole array of heterogeneous leader fusion sites. Using a transfected defective interfering RNA which contains an IS and a reporter (chloramphenicol acetyltransferase) gene and JHM2c as a helper virus, we demonstrated that subgenomic mRNAs transcribed from the defective interfering RNAs were extremely heterogeneous. The leader-mRNA fusion sites in this virus can be grouped into five types. In type I, the leader is fused with the consensus IS of the template RNA at a site within the UCUAA repeats, consistent with the classical model of discontinuous transcription. In type II, the leader is fused with the consensus IS as in type I, but the leader of mRNA contains some nucleotide substitutions within the UCUAA repeats. In type III, the leader is fused with mRNAs at a site either upstream or downstream of the consensus IS. The sequences around the fusion sites bear little or no homology to the leader. As a result, mRNAs contain sequences complementary to the template sequences upstream of the IS or have sequence deletions downstream of the IS. In type IV, the leader is fused to the IS at the 9-nt sequence immediately downstream of the UCUAA repeats. In type V, the leader-mRNA fusion site contains a duplication of a portion of the leader sequence or an insertion of nontemplated sequences which are not present in either leader or template RNA. These patterns of leader-mRNA fusion resemble the aberrant homologous recombination frequently seen in other RNA viruses. The degree of heterogeneity of leader fusion sites is dependent on the sequences of both the leader RNA and IS. These results suggest that leader-mRNA fusion in coronavirus transcription does not require direct RNA-RNA interaction between complementary sequences. A modified model of RNA transcription and recombination based on protein-RNA and protein-protein interactions is proposed. This study also provides a paradigm for aberrant homologous recombination.

Animals↗