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Biomedical subjects

M M Lai

Publications and source records attributed to M M Lai.

At least 127 records · Page 7Linked to original sources
PubMed1993

Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication.

All of the defective interfering (DI) RNAs of mouse hepatitis virus (MHV) contain both the 5' and 3' ends of the viral genomic RNA, which presumably include the cis sequences required for RNA replication. To define the replication signal of MHV RNA, we have used a vaccinia virus-T7 polymerase-transcribed MHV DI RNA to study the effects of sequence deletion on DI RNA replication. Following infection of susceptible cells with a recombinant vaccinia virus expressing T7 RNA polymerase, various cDNA clones derived from a DI RNA (DIssF) of the JHM strain of MHV, which is a 3.5-kb naturally occurring DI RNA, behind a T7 promoter were transfected. On superinfection with a helper MHV, the ability of various DI RNAs to replicate was determined. Serial deletions from the middle of the RNA toward both the 5' and 3' ends demonstrated that 859 nucleotides from the 5' end and 436 nucleotides from the 3' end of the MHV RNA genome were necessary for RNA replication. Surprisingly, an additional stretch of 135 nucleotides located at 3.1 to 3.3 kb from the 5' end of the genome was also required. This stretch is discontiguous from the 5'-end cis replication signal and is present in all of the naturally occurring DI RNAs studied so far. The requirement for a long stretch of 5'- and 3'-end sequences predicts that the subgenomic MHV mRNAs cannot replicate. The efficiency of RNA replication varied with different cDNA constructs, suggesting possible interaction between different regions of DI RNA. The identification of MHV RNA replication signals allowed the construction of an MHV DI-based expression vector, which can express foreign genes, such as the chloramphenicol acetyltransferase gene.

Animals↗
PubMed1993

Characterization of mouse hepatitis virus-specific cytotoxic T cells derived from the central nervous system of mice infected with the JHM strain.

The cytotoxic T lymphocyte (CTL) activity of spleen cells from BALB/c (H-2d) mice immunized with the neurotropic JHM strain of mouse hepatitis virus (JHMV) was stimulated in vitro for 7 days. CTL were tested for recognition of target cells infected with either JHMV or vaccinia virus recombinants expressing the four virus structural proteins. Only target cells infected with either JHMV or the vaccinia virus recombinant expressing the JHMV nucleocapsid protein were recognized. Cytotoxic T cell lines were established by limiting dilution from the brains of mice undergoing acute demyelinating encephalomyelitis after infection with JHMV. Twenty of the 22 lines recognized JHMV-infected but not uninfected syngeneic target cells, indicating that they are specific for JHMV. All T-cell lines except one were CD8+. The specificity of the CTL lines was examined by using target cells infected with vaccinia virus recombinants expressing the JHMV nucleocapsid, spike, membrane, and hemagglutinin-esterase structural proteins. Seventeen lines recognized target cells expressing the nucleocapsid protein. Three of the JHMV-specific T-cell lines were unable to recognize target cells expressing any of the JHMV structural proteins, indicating that they are specific for an epitope of a nonstructural protein(s) of JHMV. These data indicate that the nucleocapsid protein induces an immunodominant CTL response. However, no CTL activity specific for the nucleocapsid protein could be detected in either the spleens or cervical lymph nodes of mice 4, 5, 6, or 7 days after intracranial infection, suggesting that the CTL response to JHMV infection within the central nervous system may be induced or expanded locally.

Amino Acid Sequence↗
PubMed1993

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.

Animals↗
PubMed1993

Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen.

Hepatitis delta antigen (HDAg) consists of two protein species of 195 and 214 amino acids, respectively, which are identical in sequence except that the large HDAg has additional 19 amino acids at its C terminus and is prenylated. Previous studies have shown that the large HDAg and the surface antigen of hepatitis B virus (HBsAg) together can form empty hepatitis delta virus (HDV) particles. To understand the molecular mechanism of HDV virion morphogenesis, we investigated the possible direct protein-protein interaction between HDAg and HBsAg. We constructed recombinant baculoviruses expressing the major form of HBsAg and various mutant HDAgs and used these proteins for far-Western protein binding assays. We demonstrated that HBsAg interacted specifically with the large HDAg but not with the small HDAg. Using mutant HDAgs which have defective or aberrant prenylation, we showed that this interaction required isoprenylates on the cysteine residue of the C terminus of the large HDAg. Isoprenylation alone, without the remainder of the C-terminal amino acids of the large HDAg, was insufficient to mediate interaction with HBsAg. This study demonstrates a novel role of prenylates in HDV virion assembly.

Amino Acid Sequence↗
PubMed1993

RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication.

Hepatitis delta antigen (HDAg) is an RNA-binding protein with binding specificity for hepatitis delta virus (HDV) RNA (J. H. Lin, M. F. Chang, S. C. Baker, S. Govindarajan, and M. M. C. Lai, J. Virol. 64:4051-4058, 1990). By amino acid sequence homology search, we have identified within its RNA-binding domain two stretches of an arginine-rich motif (ARM), which is present in many prokaryotic and eukaryotic RNA-binding proteins. The first one is KERQDHRRRKA and the second is EDEKRERRIAG, and they are separated by 29 amino acids. Deletion of either one of these ARM sequences resulted in the total loss of the in vitro RNA-binding activity of HDAg. Thus, HDAg is different from other RNA-binding proteins in that it requires two ARM-like sequences for its RNA-binding activity. Replacement of the spacer sequence between the two ARMs with a shorter stretch of sequence also reduced RNA binding in vitro. Furthermore, site-specific mutations of the basic amino acid residues in both ARMs resulted in the total loss or reduction of RNA-binding activity. The biological significance of the RNA-binding activity was studied by examining the trans-activating activity of the RNA-binding mutants. The plasmids expressing HDAgs with various mutations in the RNA-binding motifs were cotransfected with a replication-defective HDV dimer cDNA construct into COS cells. It was found that all the HDAg mutants which had lost the in vitro RNA-binding activity also lost the ability to complement the defect of HDV RNA replication. We conclude that the trans-activating function of HDAg requires its binding to HDV RNA.

Amino Acid Sequence↗
PubMed1993

Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites.

Hepatitis delta virus (HDV) contains a circular RNA genome of 1.7 kb. HDV RNA replication is thought to proceed via a rolling-circle mechanism that is dependent on autocatalytic cleavage and ligation reactions. However, it has never been established that these ribozyme activities are indeed involved in HDV RNA replication. To investigate the possible biological significance of HDV RNA self-cleavage, we constructed several HDV dimer cDNAs containing single-base substitutions of the 3' nucleotide of the genomic and the antigenomic self-cleavage sites. These mutations were known to affect self-cleavage in vitro to various extents. The effects of these mutations on HDV RNA replication were examined in hepatic and nonhepatic cell lines. The results showed that all of the mutants which had lost the in vitro self-cleavage activity could not replicate. The only mutant which retained full cleavage activity replicated as efficiently as the wild-type RNA. Thus, this study established that self-cleavage activity is required for HDV RNA replication in cells. Interestingly, the level of HDV RNA detected in cells transfected with this replication-competent mutant and that detected in cells transfected with the wild-type construct were similar in COS-7 cells but vastly different in HepG2 and Huh-7 cells, suggesting that HDV RNA self-cleavage activity may be modulated by cell-specific factors. We also compared the effects of mutations when the primary transcripts of these constructs were of either genomic or antigenomic sense. In constructs which synthesize primary transcripts of genomic sense, all of the antigenomic self-cleavage mutants produced as much hepatitis delta antigen (HDAg) as did the wild-type construct, even in the absence of detectable HDV RNA replication, whereas the genomic self-cleavage mutants produced very little HDAg. These and other data suggest that (i) the primary HDV RNA transcripts of both genomic and antigenomic polarities must first be processed to serve as a template for HDV RNA transcription, (ii) efficient cleavage at the antigenomic self-cleavage site is not required for HDAg expression, and (iii) HDV RNA replication most likely occurs by a double-rolling-circle mechanism.

Animals↗
PubMed1992

Sequence and structure of the catalytic RNA of hepatitis delta virus genomic RNA.

Human hepatitis delta virus (HDV) RNA has been shown to contain a self-catalyzed cleavage activity. The sequence requirement for its catalytic activity appears to be different from that of other known ribozymes. In this paper, we define the minimum contiguous sequence and secondary structure of the HDV genomic RNA required for the catalytic activity. By using nested-set deletion mutants, we have determined that the essential sequence for the catalytic activity is contained within no more than 85 nucleotides of HDV RNA. These results are in close agreement with the previous determinations and confirmed the relative insignificance of the sequence at the 5' side of the cleavage site. The smallest catalytic RNA, representing HDV genomic RNA nucleotide positions 683 to 770, was used as the basis for studying the secondary structure requirements for catalytic activity. Analysis of the RNA structure, using RNase V1, nuclease S1 and diethylpyrocarbonate treatments showed that this RNA contains at least two stem-and-loop structures. Other larger HDV RNA subfragments containing the catalytic activity also have a very similar secondary structure. By performing site-specific mutagenesis studies, it was shown that one of the stem-and-loop structures could be deleted to half of its original size without affecting the catalytic activity. In addition, the other stem-and-loop contained a six base-pair helix, and the structure, rather than the sequence, of this helix was required for the catalytic activity. However, the structure of a portion of the stem-and-loop remains uncertain. We also report that this RNA can be divided into two separate molecules, which alone did not have cleavage activity but, when mixed, one of the RNAs could be cleaved in trans. This study thus reveals some features of the secondary structure of the HDV genomic RNA involved in self-catalyzed cleavage. A model of this RNA structure is presented.

Base Sequence↗
PubMed1992

Sequence analysis of the spike protein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity.

Mouse hepatitis virus (MHV), a coronavirus, causes encephalitis and demyelination in susceptible rodents. Previous investigations have shown that the MHV spike (S) protein is a critical determinant of viral tropism and pathogenicity in mice and rats. To understand the molecular basis of MHV neuropathogenesis, we studied the spike protein gene sequences of several neutralization-resistant variants of the JHM strain of MHV, which were selected with monoclonal antibodies (MAbs) specific for the S protein. We found that variant 2.2-V-1, which was selected with MAb J.2.2 and primarily caused demyelination, had a single point mutation at nucleotide (NT) 3340, as compared to the parental JHM virus, which predominantly caused encephalitis. This site was in the S2 subunit of the S protein. In contrast, variant 7.2-V-1, which was selected with MAb J.7.2 and primarily caused encephalitis, had two point mutations at NT 1766 and 1950, which were in the S1 subunit. Finally, the double mutant 2.2/7.2-V-2, which was selected with both MAbs J.2.2 and J.7.2, and was attenuated with respect to both virulence and the ability to cause demyelination, had a deletion spanning from NT 1523 to 1624 in the S1 and a point mutation at NT 3340 in the S2. We conclude that at least two regions of the S protein contribute to neuropathogenicity of MHV. We have also isolated a partial revertant of 2.2-V-1, which was partially resistant to MAb J.2.2 but retained the same neuropathogenicity as the variant 2.2-V-1. This revertant retained the mutation at NT 3340, but had a second-site mutation at NT 1994, further confirming that NT 3340 contributed to the pathogenic phenotype of MHV. By comparing these results with MHV variants isolated in other laboratories, which had mutations in other sites on the S gene and yet retained the demyelinating ability, we suggest that the ability of JHM viruses to induce demyelination is determined by the interaction of multiple sites on the S gene, rather than the characteristics of a single, unique site. Our study also revealed the possible presence of microheterogeneity of S gene sequence, particularly in the S1 region, in these viruses. The sequence microheterogeneity may also contribute to the differences in their biological properties.

Animals↗
PubMed1992

Evolution of hepatitis delta virus RNA during chronic infection.

The complete RNA sequences of hepatitis delta virus (HDV) isolated at three different time points from a chronic delta hepatitis patient were determined. These time points represented three different periods of clinical flare-ups. The sequence analysis showed that these three different HDV isolates evolved at a rate ranging from 3.0 x 10(-2) to 3.0 x 10(-3) substitutions/nucleotide/year, depending on the period of infection. The evolution rates appeared to correlate with the changes of clinical pictures of hepatitis, i.e., the more drastic the change in the symptom of hepatitis was, the more nucleotide changes were detected. Except during the transition from the acute phase to chronic phase of delta hepatitis, when there was a much larger number of changes in HDV RNA sequence, the overall evolution rate of HDV RNA in the chronic phase appeared to be similar to those of other RNA viruses. Sequence relationship of these HDV RNAs suggested that acute exacerbations in chronic delta hepatitis were associated with the evolution of the persistently infected HDV, rather than resulting from new viral infections. However, some of the mutations were not cumulative, suggesting that HDV isolated at a later time was not directly evolved from the immediately previous one. Thus, HDV at any time point was a mixture of viruses with slight sequence variations, and a specific HDV RNA species was selected from this virus population under different environments. These findings indicate that HDV RNA is heterogeneous and evolves at a fast rate. The evolution rates in different parts of HDV RNA also varied. The evolution rate of HDV RNA determined here was higher than the ones determined previously from partial RNA sequences of two Japanese HDV isolates.

Adult↗
PubMed1992

Coronavirus mRNA synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences.

The mRNA synthesis of mouse hepatitis virus (MHV) has been proposed to be the result of interaction between the leader RNA and the intergenic sites. Previously, we have identified a transcription initiation site (for mRNA 2-1), which is more efficiently transcribed by viruses containing two copies of UCUAA sequence in the leader RNA than by those with three copies. In this study, we have identified several sites which are regulated in the opposite way, namely, they are efficiently transcribed by the leader RNA with three UCUAA copies but not by those with two copies. These sites were characterized by primer extension and amplification by polymerase chain reaction. One of these sites is in the gene 3 region of a recombinant virus between A59 and JHM strains of MHV. Another is in the gene 2 region of MHV-1 strain. Both of these sites have a sequence similar to but different from the consensus transcription initiation signal (UCUAAUCUAUC and UUUAAUCUU, as opposed to UCUAAAC). These two novel intergenic sequences are not present in the genome of the JHM strain, consistent with the absence of these mRNAs in the JHM-infected cells. The discovery of this type of transcription initiation site provides additional evidence for the importance of the leader RNA in the transcription initiation of MHV mRNAs.

Base Sequence↗
PubMed1992

Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modifications between the large and small forms.

Hepatitis delta virus (HDV) encodes only one protein, the hepatitis delta antigen (HDAg). Two forms of HDAg, a large (27 kDa) and a small (24 kDa) one, participate in the various steps of HDV replication. To further understand the properties of HDAg, we have constructed recombinant baculoviruses and expressed both forms of the HDAg in insect cells. The gene encoding HDAg was placed under the control of the polyhedrin promoter of Autographa Californica nuclear polyhedrosis virus (AcNPV) by homologous recombination. When Spodoptera frugiperda (Sf9) cells were infected with the recombinant viruses, both the small HDAg and the large HDAg were expressed at high levels. The HDAgs produced by the recombinants were similar in size and antigenic properties to those of the proteins produced in mammalian hepatoma cell lines. It was also localized exclusively in the nuclei. In addition, both proteins bound to HDV RNA in an in vitro assay. No difference in the RNA-binding affinity was noted between the two forms of HDAg, suggesting that the trans-dominant inhibitory activity of the large HDAg on HDV replication is not due to its competition with the small HDAg for RNA binding. Two RNA-protein complexes could be detected, suggesting either that there are at least two binding sites on the HDV RNA or that HDAg binds to HDV RNA in two multimeric forms. We have further shown that both the large and the small HDAgs are phosphoproteins, with the former having an approximately sixfold higher level of phosphorylation. Finally, it was demonstrated that the large HDAg was isoprenylated, while the small one was not. These differences in post-translational modifications are the first differences in biochemical properties demonstrated between the two forms and may explain the differential effects of the large and small HDAgs on HDV RNA replication and virus packaging.

Animals↗
PubMed1992

RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur between replicating MHV RNAs and RNA fragments which do not replicate, suggesting the potential of RNA recombination for genetic engineering.

Base Sequence↗
PubMed1992

Mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors.

The cellular receptor for the murine coronavirus mouse hepatitis virus (MHV) has been identified as a member of the murine carcinoembryonic antigen (CEA) family (R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). However, the receptor protein was not detected in some of the susceptible mouse tissues. We therefore examined whether other types of MHV receptor might exist. By polymerase chain reaction with the conserved sequences of murine CEA gene family members (mmCGM) as primers, we detected two CEA-encoding RNAs in the mouse liver. One of them (1.3 kb) encodes mmCGM1, which has previously been identified as the receptor for MHV, and the other one (0.8 kb) was shown to encode another member of mouse CGM, mmCGM2. The sequence analysis showed that mmCGM2 lacks 564 nucleotides in the middle of the gene compared with mmCGM1. These two CEA transcripts are probably derived from the same gene by an alternative splicing mechanism. Expression of either of these cDNA clones in COS-7 cells rendered these cells susceptible to MHV infection, suggesting that not only mmCGM1 but also mmCGM2 serves as a receptor for MHV. The mmCGM2 was the major CEA species in the mouse brain, which is a main target organ for the neurotropic strains of MHV. Very little mmCGM1 was detected in the mouse brain or in cells of the susceptible mouse astrocytoma cell line DBT. This result indicates that MHV may utilize different CEA molecules as the major receptor in the mouse brain and in the liver. This is a first identification of multiple receptors for a single virus. The presence of different receptors in different tissues may explain the target cell specificity of certain MHVs.

Base Sequence↗