Search PubMed⌕ Search

Biomedical subjects

M M Lai

Publications and source records attributed to M M Lai.

At least 109 records · Page 6Linked to original sources
PubMed1994

Identification of the cis-acting signal for minus-strand RNA synthesis of a murine coronavirus: implications for the role of minus-strand RNA in RNA replication and transcription.

Minus-strand RNA is the first RNA species made by plus-strand RNA viruses, such as mouse hepatitis virus (MHV), and serves as a template for subsequent RNA replication and transcription. The regulation of minus-strand RNA synthesis has been difficult to study because of the paucity of minus-strand RNA. We have optimized a ribonuclease (RNase) protection assay which enabled the detection of minus-strand RNA synthesis from nonreplicating RNAs, thus clearly separating minus-strand from plus-strand RNA synthesis. We used an MHV defective interfering (DI) RNA containing a chloramphenicol acetyltransferase gene as a reporter to determine the cis-acting signal for MHV minus-strand RNA synthesis. It was found that minus-strand RNAs existed in double-stranded RNA form in the cell. By using various deletion clones, we demonstrated that the cis-acting signal for minus-strand RNA synthesis resides in the 55 nucleotides from the 3' end plus poly(A) tail of the MHV genome. This is much shorter than the 436 nucleotides previously reported for the 3'-end replication signal. No specific upstream MHV sequence was required for the initiation of minus-strand RNA synthesis. This finding suggests that the requirement for minus-strand RNA synthesis is much less stringent than that for genomic and subgenomic plus-strand RNA synthesis and that some of the minus-strand RNAs made may not be functional since they may lack the recognition signals for RNA replication or transcription. We further showed that the DI clones which actively transcribed a subgenomic mRNA from an internal intergenic sequence synthesized much less minus-strand RNA than those clones which did not transcribe subgenomic mRNAs, indicating that minus-strand RNA synthesis was inhibited by transcription from an internal promoter of the same DI RNA. This result also suggests that the regulation of the quantities of subgenomic mRNAs is not at the point of minus-strand RNA synthesis but rather at plus-strand RNA synthesis. Furthermore, the finding that the leader sequence was not required for minus-strand RNA synthesis suggests that the leader RNA regulates mRNA transcription during plus-strand RNA synthesis.

Animals↗
PubMed1994

Expression of the hepatitis delta virus large and small antigens in transgenic mice.

Simultaneous infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) in humans is often associated with severe viral liver disease including fulminant hepatitis. Since HBV is thought to be noncytopathic to the hepatocyte, the enhanced disease severity observed during dual infection has been attributed to either simultaneous immune responses against the two viruses or direct cytotoxic effects of HDV products on the hepatocyte or both. To examine these alternate possibilities, we produced transgenic mice that express the small and large delta antigens (HDAg) in hepatocyte nuclei at levels equal to those observed during natural HDV infection. No biological or histopathological evidence of liver disease was detectable during 18 months of observation, suggesting that neither the large nor small form of HDAg is directly cytopathic to the hepatocyte in vivo.

Alanine Transaminase↗
PubMed1994

Isoprenylation masks a conformational epitope and enhances trans-dominant inhibitory function of the large hepatitis delta antigen.

Hepatitis delta antigen (HDAg) consists of two species, large (LHDAg) and small (SHDAg), which are identical in sequence except that the large form contains 19 extra amino acids at the C terminus. The large form is prenylated on the Cxxx motif. The small form can trans activate HDV RNA replication, while the large form inhibits it. To determine the molecular basis for their differential functions, we examined the effects of prenylation on the conformation and function of HDAg. We show that the presence of prenylates masks a conformational epitope which is present in SHDAg but hidden in wild-type LHDAg; this epitope becomes exposed in all of the nonprenylated mutant LHDAgs. Prenylation also plays a major role in conferring the trans-dominant negative inhibitory activity of LHDAg, since the loss of prenylation in LHDAg reduced its inhibitory activity. The primary amino acids of the C-terminal sequence also contributed to the maintenance of the HDAg protein conformation; a prenylated LHDAg mutant with a five-amino-acid deletion had an exposed C-terminal epitope. By examining LHDAg mutants which have deletions of various extents of C-terminal sequence, with or without the prenylation motif, we have further shown that all of the prenylated mutants have much higher levels of trans-dominant suppressor activities than do the corresponding nonprenylated mutants. Surprisingly, a few nonprenylated LHDAg mutants were able to trans activate HDV RNA replication, while all of the prenylated ones lost this function. These results suggest that isoprenylates cause the masking of a conformational epitope of HDAg and that conformational differences between the large and small HDAgs account for the differences in their trans-activating and trans-dominant inhibitory biological activities.

Antigens, Viral↗
PubMed1994

Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription.

We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support subgenomic mRNA transcription. In this case, the leader sequences of the subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MHV leader RNA initiates in trans subgenomic mRNA transcription. Thus, the leader sequence can enhance subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of subgenomic mRNAs. The identification of an upstream cis-acting element for MHV subgenomic mRNA synthesis defines a novel sequence requirement for regulating mRNA synthesis in RNA viruses.

Animals↗
PubMed1994

Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis.

Mouse hepatitis virus (MHV), a coronavirus, utilizes a discontinuous transcription mechanism for subgenomic mRNA synthesis. Previous studies (C.-L. Liao and M. C. C. Lai, J. Virol. 68:4727-4737, 1994) have demonstrated that an upstream cis-acting leader sequence serves as a transcriptional enhancer, but the mechanism of transcriptional regulation is not clear. In this study, we constructed a series of defective interfering (DI) RNAs containing the chloramphenicol acetyltransferase (CAT) gene behind a differentially expressed transcription initiation (intergenic) sequence (for mRNA2-1). These DI RNAs had different copy numbers of the UCUAA pentanucleotide sequence at the 3' end of the leader. Transfection of these DI RNA constructs into cells infected with a helper MHV, which contains either two or three UCUAA copies at the 3' end of the leader, resulted in differential expression of CAT activities. We demonstrated that the copy number of UCUAA repeats in the leaders of both helper viral and DI RNAs affected the level of CAT activity, suggesting that MHV leader RNA could regulate both in trans and in cis the transcription of subgenomic mRNAs. The leader RNA of subgenomic mRNAs was derived from either the trans- or the cis-acting leader. Furthermore, insertion of a UA-rich sequence (UUUAUAAAC) immediately downstream of the leader in DI RNA, to match the sequence of helper viral RNA, enhanced the CAT activity by threefold, suggesting that this nine-nucleotide sequence is a cis-acting element. Interestingly, when the nine-nucleotide sequence was absent in DI RNA, the leaders of subgenomic mRNAs were exclusively derived from the helper virus. In contrast, when the nine-nucleotide sequence was present in DI RNA, the leaders were derived from both helper viral and DI RNAs. These results suggest that the nine-nucleotide sequence either is required for the leader RNA to initiate mRNA synthesis or, alternatively, serves as a transcription terminator for the leader RNA synthesis. However, when a constitutively expressed intergenic sequence (for mRNA7) was used, no difference in transcription efficiency was noted, regardless of the copy number of UCUAA in the DI RNA and helper virus. This study thus indicates that MHV subgenomic RNA transcription requires the interaction among the intergenic (promoter) sequence, a trans-acting leader, and a cis-acting leader sequence. A novel model of transcriptional regulation of coronavirus subgenomic mRNAs is presented.

Cell Line↗
PubMed1994

Pseudobulbar palsy associated with trismus.

A 60 year old patient presented with an acute pseudobulbar palsy associated with trismus. A computed tomography scan revealed low attenuation areas consistent with infarction affecting the genu of the internal capsules bilaterally. Trismus has not previously been described as the presenting feature of a pseudobulbar palsy.

Brain↗
PubMed1993

Regulation of prostatic acid phosphatase expression and secretion by androgen in LNCaP human prostate carcinoma cells.

The expression and the secretion of human prostatic acid phosphatase (PAcP), a differentiation antigen which is the major acid phosphatase in prostate epithelial cells, are thought to be regulated by an androgen. We investigated this regulatory mechanism at the post-transcriptional level in LNCaP human prostate carcinoma cells using a cDNA clone for the secretory form of PAcP. 5 alpha-Dihydrotestosterone (DHT, an active form of endogenous androgen) stimulated the secretion of PAcP from cells grown in a steroid-reduced medium and in a defined serum-free medium, respectively. The secreted PAcP activity was increased following a DHT dose in a dose-dependent fashion at concentrations of up to 1 microM. Further, the stimulation of PAcP secretion occurred following the period of exposure to DHT. During a 5-day treatment period, with 10 nM of DHT in the steroid-reduced medium, the secretion of PAcP was stimulated approximately 150% over that from control cells. Nevertheless, PAcP was secreted from cells grown in the absence of added DHT. First, the androgen dependency of PAcP expression was examined. The expression and the secretion of PAcP were observed in cells that were grown in a defined serum-free medium and grown in a steroid-reduced medium, in the absence of DHT. The increased secretion by DHT was further demonstrated to be in part due to an increase in PAcP mRNA level, as evidenced by Northern blot analysis. PAcP mRNA levels were elevated approximately 2-fold and corresponded to an increase of approximately 2.5-fold in the secreted level of newly synthesized 35S-PAcP. Then, the effect of DHT on the secretory process was investigated. Results of pulse-chase labeling experiments indicated that the secretory rate of PAcP was stimulated by about 50% on average by DHT. In conclusion, our data demonstrated that, in LNCaP cells, the expression and the secretion of PAcP regulated by androgen are apparently hormone-responsive processes. Further, DHT stimulation of PAcP secretion operates within at least two levels: increased PAcP mRNA and stimulation of the secretory pathway.

Acid Phosphatase↗
PubMed1993

The detection and characterization of multiple hemagglutinin-esterase (HE)-defective viruses in the mouse brain during subacute demyelination induced by mouse hepatitis virus.

It has previously been shown that passive immunization with antibodies specific for the hemagglutinin-esterase (HE) protein of mouse hepatitis virus (MHV) prevents acute lethal encephalitis, resulting in a subacute and chronic demyelination in mice infected with JHM(2), an isolate of the neurotropic JHM strain of MHV. To determine possible genetic changes occurring during infection, viruses were isolated from the brain of infected mice at various time points after infection and examined for the patterns of their structural proteins. The results showed that the sizes and expression levels of the viral spike, membrane, and nucleocapsid proteins were constant among 161 virus isolates throughout the infection. In contrast, most of the viruses isolated later in infection did not synthesize HE protein. This finding suggests that the HE gene expression is extremely variable and is preferentially lost during prolonged viral infections. In contrast, when viruses were passaged in tissue culture, no significant accumulation of HE protein-defective mutants was observed, suggesting that the accumulation of HE protein-defective mutants in infected animals was most likely the result of the positive selection for these mutants during the subacute and chronic infection. The genetic defects of HE gene in these mutants were characterized by cloning, sequencing, and in vitro translation of HE genes. Most of the mutations in the HE protein-defective mutants consisted of deletions of various lengths at different sites within the HE-coding region, resulting in the change of the reading frame and early termination. However, most of the truncated HE proteins were not detected in the infected cells. Since viruses from different mice exhibited different types of defects, the HE mutations probably occurred de novo in the brain. These results demonstrated the exceptionally rapid selection of HE-defective mutants during viral infection of mice and suggest that their selection may be related to the neuropathogenicity or persistence of MHV.

Animals↗
PubMed1993

A unique conformation at the carboxyl terminus of the small hepatitis delta antigen revealed by a specific monoclonal antibody.

Two forms of the hepatitis delta antigen (HDAg), a small (24 kDa) and a large (27 kDa) one, have different functions in the hepatitis delta virus (HDV) replication cycle. The small HDAg trans-activates RNA replication, while the large one inhibits RNA replication. The lack of the trans-acting activity in the large HDAg, even though it contains the complete sequence of small HDAg, suggests that the large HDAg lacks a certain functional conformation. To test such a possibility, monoclonal antibodies (MAbs) were generated from mice immunized with recombinant baculovirus-expressed small HDAg. As expected, most of the MAbs recognized both small and large HDAg. In addition, one MAb (9E4) was obtained which recognized only the small HDAg, but not the large one, in Western blot and immunoprecipitation analysis, suggesting that it recognized an epitope unique to small HDAg. However, MAb 9E4 detected both forms of HDAg in virus-infected cells by immunofluorescence and reacted with TrpE-large HDAg fusion proteins expressed in Escherichia coli, suggesting that this MAb recognizes a conformation-dependent epitope which is not present in the native large HDAg molecule but is detectable in LHDAg when its conformation is altered. The 9E4 epitope was mapped within a region of 32 amino acids at the carboxyl-terminus of small HDAg, indicating that this region contains a unique conformation not present in the native molecule of large HDAg. Since this is the only structure identified that is unique to small HDAg, the C-terminal region may contain the domain associated with the biological activities unique to the small HDAg.

Animals↗
PubMed1993

Generation and selection of coronavirus defective interfering RNA with large open reading frame by RNA recombination and possible editing.

All of the coronavirus defective interfering (DI) RNAs analyzed thus far contain an open reading frame (ORF) from which DI RNA-specific protein(s) are translated, although the function of the DI-specific protein and the significance of the ORF are not known. A complete cDNA clone of a mouse hepatitis virus (MHV) DI RNA, NE-1, containing a single nucleotide deletion in the 5' region of the ORF was obtained and analyzed. Due to this single nucleotide deletion, a DI-specific protein of 7.5-kDa was made from NE-1, in contrast to the 88-kDa protein made from the wild-type DI RNA. NE-1 RNA was efficiently replicated after transfection into MHV-infected cells. However, after one passage of NE-1 RNA-containing virus, the 88-kDa wild-type protein was synthesized, indicating that the large ORF was restored during NE-1 DI RNA replication. Sequence analysis of NE-1 DI RNA from infected cells demonstrated that in approximately half of the DI RNA population, the ORF was restored by RNA recombination between NE-1 DI RNA and helper virus genomic sequence. The sequences of other DI RNAs contained an additional nontemplated A at the five-A sequence nine nucleotides upstream of the deletion site, resulting in a stretch of six consecutive As. In these "edited"-type DI RNAs, the original nucleotide deletion was maintained and no RNA recombination was observed. This "editing" produced an ORF of the same size as the wild-type DI RNA. We conclude that the DI RNA with a large ORF has a selective advantage. There was no significant difference in replication efficiency among these RNAs when they replicated alone. However, cotransfection of two DI RNA species and time course experiments suggested that homologous interference and other mechanism(s) during the early stage of virus multiplication are responsible for the accumulation of DI RNAs containing the large ORF.

Amino Acid Sequence↗
PubMed1993

Natural evolution of coronavirus defective-interfering RNA involves RNA recombination.

Defective-interfering (DI) RNAs of RNA viruses, in general, are generated and continue to evolve in size during serial undiluted passages of viruses. This evolution was thought to occur by independent generation of DI RNAs during virus passages and subsequent selection of new DI RNAs under new cellular conditions. Here we demonstrate that recombination between the old DI RNA and the helper viral RNA can be one of the mechanisms for natural DI RNA evolution. A mouse hepatitis virus (MHV) DI RNA, DIssE RNA, was transcribed in vitro and transfected into a mouse cell line infected with a different MHV strain (A59), which is distinguishable from the original natural helper MHV (JHM). During subsequent serial undiluted passages of the harvested virus, several novel DI RNA species were generated, while the original DIssE RNA disappeared by passage 11. cDNA cloning and sequence analysis of one of these novel DI RNAs, designated DI-2, revealed that it is composed of four discontinuous regions of the genomic sequence and is different from the structure of the original DIssE RNA. Sequence comparison among DI-2, DIssE, and helper MHV-A59 RNAs showed that DI-2 sequence is similar to DIssE in the first and second regions, but similar to the helper A59 virus in the third and last regions. Thus, this DI RNA was generated by RNA recombination between the original DIssE RNA and the helper viral RNA. These results indicate that recombination between DI RNA and helper virus RNA can be involved in the natural evolution of DI RNAs.

Animals↗
PubMed1993

A spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry.

Previous studies have shown that some mouse strains are resistant to mouse hepatitis virus (MHV) infection despite the presence of functional viral receptors (K. Yokomori and M. M. C. Lai, J. Virol. 66, 6931-6938, 1992). To determine the molecular requirement for MHV infection, several cell lines derived from both susceptible and resistant mouse strains were tested for their ability to support infection by two different MHV strains, JHM and A59. Most of the cell lines tested, including ones from susceptible mouse strains, exhibited selective resistance to JHM, but were susceptible to A59, suggesting that there is an additional cellular factor(s) discriminating JHM from A59 infection. Both RNA and protein syntheses of JHM were inhibited in the resistant cells; however, transfection of JHM genomic RNA into these cells led to the production of infectious virus, suggesting that the restriction step(s) is during an early stage of viral replication cycle. The mRNA for the MHV receptor (the murine homolog of the carcinoembryonic antigen) is expressed in all cell lines, and expression in COS cells of the receptor isolated from the resistant murine cell lines rendered the COS cells susceptible to both A59 and JHM infections. Furthermore, the transfection of additional MHV receptors into the resistant cells did not overcome the resistance to JHM virus infection. These results suggested that the viral receptor is functional; nevertheless, the JHM infection is restricted at an early step of infection in these cells. The study of the growth properties of the various recombinant viruses between A59 and JHM revealed that one of the viral genes determining viral replication in these cell lines is the S protein gene; thus, the second factor required for viral infection may interact directly or indirectly with the S protein at an early step of infection. Taken together, these studies suggest that expression of a functional viral receptor is not sufficient to establish MHV infection, and that an additional factor(s) is required for an early step of viral infection, possibly during virus entry.

Animals↗
PubMed1993

Endogenous promoters can direct the transcription of hepatitis delta virus RNA from a recircularized cDNA template.

Transcription and replication of hepatitis delta virus (HDV) RNA is thought to be performed by host RNA polymerase II. The mechanism which enables polymerase II to use RNA as a template is unclear. However, since extensive intramolecular complementarity allows HDV RNA to form a rod-shaped structure, it is possible that the mostly double-stranded HDV RNA may resemble double-stranded DNA in structure, and can thus be used by RNA polymerase II as a template. To investigate this possibility, we examined whether the cDNA counterpart of HDV RNA contains a promoter and thus can drive the transcription and replication of HDV RNA. Circularized monomers of HDV cDNA, when transfected into various cell lines, were found to generate both monomeric and dimeric forms of HDV RNA and hepatitis delta antigen at levels comparable to those generated with HDV cDNA multimers under the control of a SV40 late promoter, suggesting that HDV cDNA contains endogenous promoters. Using chloramphenicol acetyltransferase and human growth hormone as reporter genes, the specific promoter activity for the synthesis of antigenomic HDV RNA was localized to a 29-nucleotide region (nucleotides 1650-1679), although an additional 224-nucleotide upstream region was also necessary for maximum activity. Similarly, promoter activity for the synthesis of genomic RNA was localized to a 160-nucleotide region around position 1679 that overlapped with the antigenomic promoter region. Since these regions are in a highly conserved double-stranded region of HDV RNA, they may represent RNA promoters recognized by RNA polymerase II. This result also suggests a convenient method, using circularized monomer HDV cDNA, to study HDV RNA replication.

Base Sequence↗
PubMed1993

Cell fusion studies identified multiple cellular factors involved in mouse hepatitis virus entry.

We have previously reported that certain murine cell lines are susceptible to mouse hepatitis virus (MHV) A59 strain infection but resistant to JHM strain, despite the expression of the viral receptor for both strains. This restriction on viral infection has been shown to occur at the virus entry level (Yokomori et al., Virology 196, 45-56, 1993). To study whether JHM resistance of these cell lines is due to a defective cellular factor necessary for JHM virus entry, or due to the presence of an inhibitor, hybrid cells were obtained by polyethylene glycol (PEG)-mediated fusion between two resistant cell lines, i.e., a Balb/c mouse-derived cell line, BC10 cells, which are resistant to JHM infection but express a viral receptor, and the simian cell line COS cells, which are resistant to JHM because of the absence of the viral receptor. JHM could replicate in the hybrid BC10-COS cells, indicating that the restriction of viral infection in BC10 cells could be complemented by COS cells. This result indicates that the resistance of BC10 cells to JHM infection is due to the defectiveness of a cellular factor rather than the presence of an inhibitor. JHM virus-binding and penetration into BC10 cells appeared to be normal. However, JHM internalization into BC10 cells by PEG-induced virus-cell fusion did not lead to viral replication, suggesting that the restriction of JHM infection in BC10 cells is at the level of viral uncoating. This restriction could be complemented by PEG-mediated fusion with other murine cell lines which have the virion-uncoating activity, but not by cell lines which lack this activity. Furthermore, the viral resistance of most of other murine cell lines, which express MHV receptors, could not be overcome by fusion with COS cells, suggesting that different murine cell lines are defective in different viral entry functions. Therefore, we conclude that JHM viral entry process requires multiple cellular factors secondary to the viral receptor.

Adsorption↗
PubMed1993

The Coronaviridae now comprises two genera, coronavirus and torovirus: report of the Coronaviridae Study Group.

At the April 1992, mid-term meeting of the International Committee on Taxonomy of Viruses (ICTV) a proposal from the Coronaviridae Study Group (CSG) to include the torovirus genus in the Coronaviridae was accepted. Following another proposal, the arterivirus genus was removed from the Togaviridae but not assigned to another family. The arteriviruses have some features in common with the Coronaviridae but also have major differences. After much debate, culminating in September 1992, it was decided that the CSG would not recommend inclusion of arterivirus in the Coronaviridae. It was agreed that (a) the nomenclature used for coronavirus genes, mRNAs and polypeptides (Cavanagh et al., 1990) should be used for toroviruses, (b) that the small (about 100 amino acids) membrane-associated protein, which is distinct from the integral membrane glycoprotein M, associated with virions of infectious bronchitis (Liu & Inglis, 1991) and transmissible gastroenteritis (Godet et al., 1992) coronaviruses would be referred to by the acronym sM (lower case 's') and (c) that 'pol' (polymerase) should be used as a working term for gene 1, which comprises open reading frames (ORFs) 1a and 1b in both genera of the Coronaviridae.

Arterivirus↗
PubMed1993

Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus.

The murine coronavirus mouse hepatitis virus gene 1 is expressed as a polyprotein, which is cleaved into multiple proteins posttranslationally. One of the proteins is p28, which represents the amino-terminal portion of the polyprotein and is presumably generated by the activity of an autoproteinase domain of the polyprotein (S. C. Baker, C. K. Shieh, L. H. Soe, M.-F. Chang, D. M. Vannier, and M. M. C. Lai, J. Virol. 63:3693-3699, 1989). In this study, the boundaries and the critical amino acid residues of this putative proteinase domain were characterized by deletion analysis and site-directed mutagenesis. Proteinase activity was monitored by examining the generation of p28 during in vitro translation in rabbit reticulocyte lysates. Deletion analysis defined the proteinase domain to be within the sequences encoded from the 3.6- to 4.4-kb region from the 5' end of the genome. A 0.7-kb region between the substrate (p28) and proteinase domain could be deleted without affecting the proteolytic cleavage. However, a larger deletion (1.6 kb) resulted in the loss of proteinase activity, suggesting the importance of spacing sequences between proteinase and substrate. Computer-assisted analysis of the amino acid sequence of the proteinase domain identified potential catalytic cysteine and histidine residues in a stretch of sequence distantly related to papain-like cysteine proteinases. The role of these putative catalytic residues in the proteinase activity was studied by site-specific mutagenesis. Mutations of Cys-1137 or His-1288 led to a complete loss of proteinase activity, implicating these residues as essential for the catalytic activity. In contrast, most mutations of His-1317 or Cys-1172 had no or only minor effects on proteinase activity. This study establishes that mouse hepatitis virus gene 1 encodes a proteinase domain, in the region from 3.6 to 4.4 kb from the 5' end of the genome, which resembles members of the papain family of cysteine proteinases and that this proteinase domain is responsible for the cleavage of the N-terminal peptide.

Amino Acid Sequence↗