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CORD-19:e9900f49f3469114a7ad6b81b7692540cdf17817 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 0-60 Sentence denotes A New Cistron in the Murine Hepatitis Virus Replicase Gene ᰔ
T2 62-70 Sentence denotes Abstract
T3 71-263 Sentence denotes We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus.
T4 264-481 Sentence denotes MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33°C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5°C.
T5 482-737 Sentence denotes Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype.
T6 738-978 Sentence denotes This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31.
T7 979-1281 Sentence denotes The analysis of protein synthesis in virusinfected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12.
T8 1282-1374 Sentence denotes The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected.
T9 1375-1562 Sentence denotes Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature.
T10 1563-1743 Sentence denotes These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.
T11 1745-1839 Sentence denotes Coronaviruses are positive-stranded RNA viruses with genomes ranging in size from 27 to 32 kb.
T12 1840-1936 Sentence denotes Approximately twothirds of the genome encodes proteins that are involved in viral RNA synthesis.
T13 1937-2273 Sentence denotes The majority of these proteins are encoded in two 5Ј-proximal, overlapping open reading frames (ORFs), ORF1a and ORF1b, and are translated as polyproteins, pp1a and pp1ab, which are then processed by virus-encoded proteinases into 16 nonstructural proteins (nsp's): nsp1 to nsp11, encoded in ORF1a, and nsp12 to nsp16, encoded in ORF1b.
T14 2274-2554 Sentence denotes The translation products of ORF1a and ORF1ab, together with other viral proteins (41, 60) and, possibly, cellular proteins, assemble into replication-transcription complexes (RTCs) responsible for the synthesis of genome-sized RNA and a set of 3Ј-coterminal subgenome-sized mRNAs.
T15 2555-2744 Sentence denotes The subgenomesized mRNAs are produced by an unusual mechanism that involves discontinuous transcription during negative-strand RNA synthesis (for recent reviews, see references 39 and 43) .
T16 2745-2901 Sentence denotes The coronavirus RTCs accumulate at perinuclear regions and are associated with a network of endoplasmic reticulum-derived, modified membranes (16, 20, 28) .
T17 2902-3028 Sentence denotes The interconnected structures within this network are known as convoluted membranes (CMs) and double-membrane vesicles (DMVs).
T18 3029-3208 Sentence denotes Both structures are associated with viral replicase proteins and double-stranded RNA (16, 28, 53) , but it is not yet entirely clear if both are the sites of active RNA synthesis.
T19 3209-3434 Sentence denotes Hydrophobic transmembrane domains are present in nsp3, nsp4, and nsp6 and likely serve to anchor the nascent pp1a/pp1ab polyproteins to membranes during the first steps of RTC formation (1, 6, 16, 21, 26, (32) (33) (34) 54) .
T20 3435-3718 Sentence denotes A variety of studies suggest that most, if not all, coronavirus nsp's are recruited to the viral RTCs (16, 21, 35, 45) , and a complex and dynamic pattern of interactions between intermediate and mature RTC proteins is thought to play a role in the regulation of viral RNA synthesis.
T21 3719-4041 Sentence denotes Crystallographic analysis of individual and oligomeric complexes of coronavirus nsp's, as well as studies involving Saccharomyces cerevisiae two-hybrid, coimmunoprecipitation, cosedimentation, and cross-linking analysis (3, 47, 48, 55, 56) , provides insights into some of the structural aspects of these RTC interactions.
T22 4042-4530 Sentence denotes Many of the coronavirus nsp's have been shown, or are predicted, to have enzymatic functions, including papain-like cysteine proteinases (nsp3), ADP-ribose 1Љ-phosphatase (nsp3), 3C-like cysteine proteinase (nsp5), RNA-dependent RNA polymerase primase (RdRP-primase) (nsp8), RdRp (nsp12), 5Ј-to-3Ј helicase (nsp13), 3Ј-to-5Ј exonuclease (nsp14), N7-methyltransferase (nsp14), endoribonuclease (nsp15), and S-adenosylmethioninedependent 2Ј-O-methyltransferase (nsp16) (5, 22, 39, 44, 49) .
T23 4531-4674 Sentence denotes Clearly, some of these activities are common to many RNA viruses and are directly related to the biogenesis of the RTC and viral RNA synthesis.
T24 4675-4928 Sentence denotes However, others (e.g., the endoribo-nuclease of nsp15) are unique to coronaviruses or viruses closely related to them (23) and may be involved in unusual activities that are related to the size of the coronavirus genome or virus-host interactions (15) .
T25 4929-5300 Sentence denotes Also, the finding that 2Ј-Oribose-methylated RNA substrates are resistant to cleavage by the severe acute respiratory syndrome coronavirus (SARS-CoV) endoribonuclease indicates that there may be functional links between different nsp's: for example, the endonuclease activity of nsp15 and the S-adenosylmethionine-dependent 2Ј-O-methyltransferase activity of nsp16 (23) .
T26 5301-5436 Sentence denotes The nsp3 protein is the largest and, possibly, most complex of the proteins encoded in the murine hepatitis virus (MHV) replicase gene.
T27 5437-5596 Sentence denotes It is comprised of 2,005 amino acids (222 kDa) and is thought to contain as many as 15 domains that can be defined by structure, function, and phylogeny (31) .
T28 5597-6242 Sentence denotes These domains include amino-terminal ubiquitin-related (UB1) and hypervariable acidic (AC) domains, a papain-like cysteine proteinase domain (PLP1), an X domain with poly(ADP-ribose) binding activity (X or ADRP domain), a second ubiquitinrelated domain (UB2), a second papain-like cysteine proteinase domain (PLP2), group 2 specific nucleic acid binding (NAB) and marker (G2M) domains, a transmembrane domain (TMD) that includes at least 2 membrane-spanning helices, a putative metal-binding domain (ZF), and a carboxyl-terminal Y domain that contains three subdomains (Y1, Y2, and Y3) with characteristic patterns of conserved Cys/His residues.
T29 6243-6551 Sentence denotes The nsp3 protein is excised from its precursors, pp1a and pp1ab, by the concerted activity of the PLP1 and PLP2 domains, with PLP1 being responsible for the amino-terminal cleavage (cleavage site 2 [CS2]) and PLP2 being responsible for the carboxyl-terminal cleavage (cleavage site 3 [CS3]) (2, 12, 26, 59) .
T30 6552-6673 Sentence denotes The genetic analysis of functions associated with the coronavirus RTC has employed both classical and reverse approaches.
T31 6674-6954 Sentence denotes Reverse genetics has, for example, been used to introduce mutations at the active site of the coronavirus nsp14 exonuclease domain, which results in a failure to rescue recombinant virus (30) or the rescue of mutant viruses with significant growth and RNA synthesis defects (11) .
T32 6955-7243 Sentence denotes Similarly, mutant MHVs that are unable to liberate nsp1 from the nascent polyprotein (i.e., nsp1/nsp2 cleavage mutants) and mutant MHVs that lack extensive regions of the nsp1 or, indeed, the entire nsp2 coding region could be rescued but exhibited decreased or delayed replication (18) .
T33 7244-7452 Sentence denotes In contrast to these attenuated phenotypes, mutation of the active site of the Human coronavirus strain 229E (HCoV-229E) nsp3 ADRP domain has no significant effect on virus RNA synthesis or virus titer (36) .
T34 7453-7602 Sentence denotes The analysis of classical temperature-sensitive (ts) mutants is also a powerful tool for investigating the complexities of coronavirus RNA synthesis.
T35 7603-7858 Sentence denotes We have recently reported a genetic and functional analysis of a panel of MHV ts mutants that are unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature, i.e., mutants with an RNA-negative phenotype.
T36 7859-8103 Sentence denotes One of the main conclusions of this analysis is that mutations in the nsp12, nsp14, and nsp16 proteins define distinct cistrons (cistrons II, IV, and VI) while mutations in the nsp4, nsp5, and nsp10 proteins define a single cistron (cistron I).
T37 8104-8416 Sentence denotes On the basis of these results, we have predicted that most of the ORF1a gene products (more specifically, nsp4 to nsp10) function as a polyprotein before cleavage into individual polypeptides, or they are as-sembled into the RTC and are then cleaved, with a gain of function expressed in individual polypeptides.
T38 8417-8561 Sentence denotes In contrast, the ORF1b-encoded nsp's (nsp12 to nsp16) are diffusible and assemble and function in viral RNA synthesis after cleavage from pp1ab.
T39 8562-8816 Sentence denotes We have also suggested that replicase polyprotein processing may have an important role in regulating the different functions associated with the coronavirus RTC: for example, minus-and plus-strand RNA synthesis and the aging of the active complex (40) .
T40 8817-8956 Sentence denotes The model that we have presented is consistent with our understanding of the organization and expression of the coronavirus replicase gene.
T41 8957-9008 Sentence denotes However, there are still many unanswered questions.
T42 9009-9158 Sentence denotes For example, it has not been shown that, as we predict, the nsp6, nsp7, nsp8, and nsp9 proteins function in cis, together with nsp4, nsp5, and nsp10.
T43 9159-9320 Sentence denotes To do this, it will be necessary to find MHV RNA-negative ts mutants with causal mutations in the relevant proteins and to complement them with existing mutants.
T44 9321-9470 Sentence denotes Similarly, our model leaves open the question of whether or not other ORF1a-encoded proteins will be able to complement defects in cistron I mutants.
T45 9471-9677 Sentence denotes As mentioned above, it is known that particular domains of nsp1 and nsp3 are not essential for virus replication, but it is, of course, still possible that these proteins do have functions in RNA synthesis.
T46 9678-9850 Sentence denotes Finally, the classical genetic approach can be further exploited to provide mechanistic insights into the functional inactivation of nsp's at the nonpermissive temperature.
T47 9851-10019 Sentence denotes For example, a recent biochemical analysis of the MHV RNAnegative mutant LA ts6 has suggested that nsp10 acts as a regulatory cofactor of nsp5 proteinase activity (9) .
T48 10020-10236 Sentence denotes It would be interesting to investigate the polyprotein processing pattern of additional mutants in order to establish whether similar interactions exist between other proteins encoded in the MHV replicase gene locus.
T49 10237-10255 Sentence denotes Cells and viruses.
T50 10256-10524 Sentence denotes 17Cl-1 (17 clone 1) mouse fibroblast cells (46) and HeLa-D98OR (27) cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin, 100 g/ml streptomycin, and 5% tryptose phosphate broth (TPB).
T51 10525-10763 Sentence denotes Monkey kidney (CV-1) cells and baby hamster kidney (BHK-21) cells were obtained from the European Collection of Cell Cultures (ECACC) and cultured in minimal essential medium (MEM) supplemented with HEPES (25 mM), 5% FCS, and antibiotics.
T52 10764-10838 Sentence denotes MHV strain A59 (MHV-A59) was originally obtained from the laboratory of L.
T53 10839-10907 Sentence denotes Sturman, Wadsworth Center for Laboratories and Research, Albany, NY.
T54 10908-11235 Sentence denotes The characterized ts mutant viruses used in this study have been described previously (40) and were derived from the original mutant isolates after plaque purification and propagation in 17Cl-1 cells cultured at 33°C in low-pH MEM (pH 6.6 to 6.8) containing 5% FCS, 100 U/ml penicillin, 100 g/ml streptomycin, and 5% TPB (38) .
T55 11236-11376 Sentence denotes Revertant viruses were picked from plaques of mutants whose titers were determined at 39.5°C and were plaque purified three times at 39.5°C.
T56 11377-11636 Sentence denotes Virus stocks were obtained by using virus from a single plaque (ϳ10 7 PFU) to infect ϳ4 ϫ 10 6 cells to yield 6 ml of stock virus, which was then used to infect ϳ1 ϫ 10 7 cells at 33°C for 16 h to yield passage 2 virus stocks with a titer of ϳ5 ϫ 10 8 PFU/ml.
T57 11637-11766 Sentence denotes Vaccinia virus (WR strain) and vaccinia virus recombinants were propagated, titrated, and purified as described previously (50) .
T58 11767-11799 Sentence denotes Isolation of Bristol ts mutants.
T59 11800-11999 Sentence denotes 17Cl-1 cells were infected with MHV-A59 at a multiplicity of infection (MOI) of 5 PFU/cell in Hanks' balanced salt solution (HBSS) containing 50 g/ml DEAE-dextran and 0.2% bovine serum albumin (BSA).
T60 12000-12196 Sentence denotes After 30 min at 33°C, the inoculum was removed and the cells were incubated in DMEM containing 10% FCS, 100 U/ml penicillin, 100 g/ml streptomycin, and 150 g/ml of 5-fluorouracil at 33°C for 16 h.
T61 12197-12290 Sentence denotes This concentration of pyrimidine analogue was determined to inhibit virus replication by 80%.
T62 12291-12363 Sentence denotes The mutagenized virus stock (ϳ2 ϫ 10 6 PFU/ml) was stored at minus 80°C.
T63 12364-12537 Sentence denotes The mutagenized virus was diluted to 1.5 PFU/ml, and 200-l aliquots were incubated with ϳ10 4 17Cl- Characterization of ts mutant stocks for titer and efficiency of plating.
T64 12538-12712 Sentence denotes 17Cl-1 cells in six-well plates were infected in duplicate at room temperature with 0.5 ml of 10-fold dilutions of virus in HBSS containing 50 g/ml DEAE-dextran and 0.2% BSA.
T65 12713-12796 Sentence denotes The inoculum was removed after 30 min, and the cells were washed with 2 ml of DMEM.
T66 12797-13033 Sentence denotes Monolayers were overlaid with MEM containing 5% FCS, 100 U/ml of penicillin, 100 g/ml streptomycin, and 0.1% Gelrite gellan gum (Sigma-Aldrich, Poole, Dorset, United Kingdom) and incubated at the appropriate temperature with 7.5% CO 2 .
T67 13034-13109 Sentence denotes Infected cells were incubated at 33°C for 3 days or 37°C/39.5°C for 2 days.
T68 13110-13270 Sentence denotes Cells were fixed with 5% formaldehyde in phosphate-buffered saline (PBS) and stained with a solution of 0.2% toluidine blue, 0.2% azure blue, and 1% boric acid.
T69 13271-13371 Sentence denotes The efficiency of plating (EOP) was determined by dividing the titer at 39.5°C by the titer at 33°C.
T70 13372-13392 Sentence denotes Viral RNA synthesis.
T71 13393-13666 Sentence denotes Viral RNA synthesis was measured by [ 3 H]uridine incorporation into acid-precipitable material in the presence of 20 g/ml actinomycin D. [5-3 H]uridine (Ͼ1.0 TBq/mmol; Amersham Radiochemicals, Chalfont, United Kingdom) was added to the medium at 1.85 MBq/ml or 7.4 MBq/ml.
T72 13667-13904 Sentence denotes After incubation, the radioactive medium was removed and the cells were lysed with 5% lithium dodecyl sulfate and 200 g/ml proteinase K in LEH buffer (0.1 M LiCl, 0.001 M EDTA, 0.01 M HEPES [pH 6.6]) at 2 ϫ 10 5 to 5 ϫ 10 5 cells per ml.
T73 13905-14019 Sentence denotes DNA was sheared by repeated passage of the lysate through a 27-gauge needle attached to a 1-ml tuberculin syringe.
T74 14020-14325 Sentence denotes Duplicate or triplicate samples of 5 ϫ 10 4 cells were precipitated with trichloroacetic acid, the precipitates were collected on glass fiber filters (Fisher Scientific, Leicestershire, United Kingdom) and dried under a heat lamp, and the radioactivity was determined by liquid scintillation spectroscopy.
T75 14326-14344 Sentence denotes Northern blotting.
T76 14345-14619 Sentence denotes RNA for Northern blotting was obtained by infecting ϳ10 7 17Cl-1 cells with virus at an MOI of 10, incubating the cells for 8 h at 33°C or 6 h at 39.5°C, and isolating total RNA using the Trizol reagent (Invitrogen, Paisley, United Kingdom) as described by the manufacturer.
T77 14620-14741 Sentence denotes The poly(A)-containing RNA was isolated using oligo(dT) 25 Dynabeads (Dynal, Oslo, Norway) as previously described (52) .
T78 14742-14957 Sentence denotes The RNA was then incubated with formamide (50%) and formaldehyde (2.2 M) at 70°C for 10 min and electrophoresed in a 1% agarose gel containing 20 mM MOPS (3-N-morpholinopropanesulfonic acid) and 600 mM formaldehyde.
T79 14958-15234 Sentence denotes After electrophoresis, the gel was soaked in 0.05 N NaOH, neutralized, and equilibrated with 20ϫ SSC (3 M NaCl, 0.3 M sodium citrate, 1 mM EDTA) before being vacuum blotted onto a nylon membrane (Optitran BA-S 83; Schleicher and Schuell, Sigma-Aldrich, Poole, United Kingdom).
T80 15235-15478 Sentence denotes The RNA was cross-linked to the membrane using UV light, and MHV RNAs were detected by hybridization with a 466-bp, ␣-32 P-random-prime-labeled PCR product corresponding to sequences in the nucleocapsid protein ORF of the MHV-A59 genome (10) .
T81 15479-15504 Sentence denotes Complementation analysis.
T82 15505-15679 Sentence denotes Complementation analysis was done by infecting 17Cl-1 cells in 35-mm dishes either singly with one ts mutant or doubly with two ts mutants to give a total MOI of 20 PFU/cell.
T83 15680-15807 Sentence denotes After incubation at room temperature for 30 min, the inoculum was removed and the monolayers were washed three times with HBSS.
T84 15808-15948 Sentence denotes The cells were then incubated with low-pH MEM containing 5% FCS, 100 U/ml penicillin, 100 g/ml streptomycin, and 5% TPB prewarmed to 39.5°C.
T85 15949-15997 Sentence denotes Infected cells were incubated at 39.5°C for 8 h.
T86 15998-16082 Sentence denotes Supernatants were harvested and clarified by centrifugation at 10,000 rpm for 5 min.
T87 16083-16138 Sentence denotes The virus titer was determined by plaque assay at 33°C.
T88 16139-16385 Sentence denotes The complementation indices (CI) were calculated as described previously (40) , and a CI of Ͼ2 between mutant pairs was consistent with complementation, i.e., Ϯ4-fold difference above background, while a CI of Ͻ2 was negative for complementation.
T89 16386-16408 Sentence denotes RT-PCR and sequencing.
T90 16409-16666 Sentence denotes Viral RNA for reverse transcription-PCR (RT-PCR) and sequencing was obtained by infecting ϳ10 7 17Cl-1 cells with virus at an MOI of 10, incubating the cells for 8 h at 33°C, and isolating total RNA using the Trizol reagent as described by the manufacturer.
T91 16667-17043 Sentence denotes The replicase gene coding region (ORF1a and ORF1b) of MHV-Brts31 and MHV-Br31R1 virus (nucleotides [nt] 211 to 21751) was amplified and sequenced using a set of 121 oligonucleotides (P1 to P121) that are complementary to sequences spaced at approximately 350-nucleotide intervals along the positive-and negative-strand copies of the viral RNA (sequences available on request).
T92 17044-17216 Sentence denotes A part of the nsp3 protein coding region of MHV-Brtsc31 and MHV-Br31cR1 (nt 3704 to 4517) was amplified and sequenced using six oligonucleotides of the oligonucleotide set.
T93 17217-17287 Sentence denotes RT-PCR of viral RNA and sequencing were done using standard protocols.
T94 17288-17354 Sentence denotes Sequencing analysis was done by MWG Eurofins (Ebersberg, Germany).
T95 17355-17460 Sentence denotes Computer-assisted analysis of sequence data was done using the Lasergene biocomputing software (DNAStar).
T96 17461-17630 Sentence denotes Recombinant MHV-inf-1 and MHV-Brtsc31 were derived from vaccinia virus vMHV-inf-1, which contains a cloned, full-length MHV-A59 cDNA (GenBank accession number AY700211).
T97 17631-17712 Sentence denotes Mutagenesis was done using the reverse genetics system described previously (7) .
T98 17713-17889 Sentence denotes Briefly, two rounds of vaccinia virus-mediated homologous recombination were done using the Escherichia coli guanine-phosphoribosyltransferase (GPT) gene as a selection marker.
T99 17890-18175 Sentence denotes First, part of the nsp3 protein coding region within the vMHV-inf-1 cDNA was replaced by the GPT gene using homologous recombination with plasmid pGPT-nsp3(N). pGPT-nsp3(N) encodes the GPT gene flanked on its left by MHV-A59 nt 3751 to 4161 and on its right by MHV-A59 nt 5044 to 5460.
T100 18176-18397 Sentence denotes Second, the GPT gene within the recombinant vaccinia virus vMHV-nsp3GPT cDNA was replaced by a mutated nsp3 protein coding region using homologous recombination with plasmid pnsp3-M475I (MHV-A59 nt 4129 to 4131; ATG3ATA).
T101 18398-18559 Sentence denotes The plasmid pnsp3-M475I was produced by overlap PCR using mutagenic primers (details are available from the authors upon request) and vMHV-inf-1 DNA as template.
T102 18560-18705 Sentence denotes The resulting PCR product, which encompasses MHV-A59 nt 3751 to 5460, was then cloned into pCR-Blunt II-TOPO (Invitrogen) to produce pnsp3-M475I.
T103 18706-18825 Sentence denotes The identities of plasmids and recombinant vaccinia viruses were confirmed by sequence analysis of the mutated regions.
T104 18826-18921 Sentence denotes Further cloning details, plasmid maps, and sequences are available from the authors on request.
T105 18922-19209 Sentence denotes Recombinant coronaviruses, MHV-inf-1 and MHV-Brtsc31, were rescued from cloned cDNA using purified, EagI-cleaved vaccinia virus DNA as a template for the transcription of recombinant, full-length MHV genomic RNA, which was electroporated into BHK-MHV-N cells as described previously (7).
T106 19210-19338 Sentence denotes Following electroporation, the transfected BHK-MHV-N cells were mixed with a 4-fold excess of 17Cl-1 cells and cultured at 33°C.
T107 19339-19478 Sentence denotes At days 1 and 2 postelectroporation, tissue culture supernatants were taken and recombinant coronaviruses were plaque purified three times.
T108 19479-19639 Sentence denotes Virus stocks were obtained by using virus from a single plaque to infect 17Cl-1 cells to yield passage 1 virus stocks with a titer greater than 1 ϫ 10 8 PFU/ml.
T109 19640-19768 Sentence denotes The identities of recombinant MHV-A59 and recombinant mutant viruses were confirmed by sequence analysis of the mutated regions.
T110 19769-19843 Sentence denotes Viral protein synthesis and immunoprecipitation of MHV replicase products.
T111 19844-20021 Sentence denotes 17Cl-1 cells (ϳ10 6 cells) in 60-mm-diameter cell culture dishes were either mock infected or infected with viruses at an MOI of 20.0 and incubated at 33°C in serum-free medium.
T112 20022-20131 Sentence denotes After 1 h, the medium was replaced with DMEM supplemented with 5% FCS and containing 5 g/ml of actinomycin D.
T113 20132-20275 Sentence denotes At 4.0 h postinfection, the medium was replaced with medium lacking methionine and cysteine and supplemented with 2% dialyzed fetal calf serum.
T114 20276-20378 Sentence denotes At 4.5 h postinfection, 35 S-trans label (100 Ci/ml) was added and one dish was transferred to 39.5°C.
T115 20379-20550 Sentence denotes At 6.5 h postinfection, cells were washed with PBS and then lysed in 250 l of lysis buffer A (4% SDS, 3% dithiothreitol [DTT], 40% glycerol, and 0.065 M Tris-HCl, pH 6.8).
T116 20551-20618 Sentence denotes Cell lysates were immunoprecipitated as described previously (42) .
T117 20619-20902 Sentence denotes Briefly, radiolabeled cell lysates (1 ϫ 10 5 cell equivalents) were diluted in 1 ml RIPA buffer (0.5% Triton X-100, 0.1% SDS, 300 mM NaCl, 4 mM EDTA, and 50 mM Tris-HCl, pH 7.4) and immunoprecipitated with 2 l of nsp-specific antibodies and protein A Sepharose beads (GE Healthcare).
T118 20903-21157 Sentence denotes Protein-bead conjugates were washed four times in RIPA buffer, and the proteins were eluted from beads by incubation with 2ϫ sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue, 0.125 M Tris HCl, pH 6.8) at 37°C for 30 min.
T119 21158-21282 Sentence denotes Protein products were separated by SDS-PAGE in 5 to 12.5% gradient polyacrylamide gels and were analyzed by autoradiography.
T120 21283-21368 Sentence denotes The 14 C molecular mass marker (GE Healthcare) was used as a molecular mass standard.
T121 21369-21382 Sentence denotes TEM analysis.
T122 21383-21461 Sentence denotes 17Cl-1 cells were infected with virus at an MOI of 20.0 and incubated at 33°C.
T123 21462-21534 Sentence denotes At 3.5 h postinfection, one set of infected cells was shifted to 39.5°C.
T124 21535-21706 Sentence denotes At 5.5 h postinfection, the cells were washed two times with PBS, scraped from the dish, and pelleted in a microcentrifuge tube at 1,000 ϫ g for 5 min at room temperature.
T125 21707-21873 Sentence denotes PBS was replaced with 0.1 M cacodylate-buffered 4% glutaraldehyde before processing for transmission electron microscopy (TEM) analysis as previously described (16) .
T126 21874-22049 Sentence denotes Briefly, cells fixed in glutaraldehyde were rinsed two times for 10 min each in 0.1 M cacodylate buffer and postfixed in 0.1 M cacodylate-buffered 1% osmium tetroxide for 1 h.
T127 22050-22153 Sentence denotes After two rinses in buffer, the cells were serially dehydrated in increasing concentrations of ethanol.
T128 22154-22280 Sentence denotes After the dehydration step, the ethanol was replaced with 100% propylene oxide (transition fluid) by 3 changes of 10 min each.
T129 22281-22405 Sentence denotes The cells were then immersed in a 1:1 solution of resin-propylene oxide and left on a rotator overnight at room temperature.
T130 22406-22603 Sentence denotes Next day, the solution was replaced with a 3:1 solution of resinpropylene oxide for 7 to 8 h, after which the solution was changed to 100% resin and left on a rotator overnight at room temperature.
T131 22604-22722 Sentence denotes The samples were then embedded in a freshly prepared 100% Embed 812 resin (Electron Microscopy Sciences) for 7 to 8 h.
T132 22723-22775 Sentence denotes The molds were then placed in a 56°C oven overnight.
T133 22776-22975 Sentence denotes Sections 0.5 m thick were stained with 1% toluidine blue, and selected areas were thin sectioned (80 nm), mounted on copper grids, and stained with 5% uranyl acetate and Reynolds' lead citrate (37) .
T134 22976-23044 Sentence denotes The grids were examined and photographed on a Hitachi H600 at 75 kV.
T135 23045-23117 Sentence denotes Negatives were scanned into digital files using a Microtek i800 scanner.
T136 23118-23156 Sentence denotes Isolation and phenotype of MHV-Brts31.
T137 23157-23404 Sentence denotes MHV-Brts31 was isolated as described in Materials and Methods and identified as a putative RNA-negative, ts mutant due to its inability to cause cytopathic effects in 17Cl-1 cells that were infected and maintained at the nonpermissive temperature.
T138 23405-23528 Sentence denotes To analyze the phenotype of MHV-Brts31 in more detail, we determined first the EOP of a high-titer (passage 2) virus stock.
T139 23529-23671 Sentence denotes At 33°C, the stock had a titer of 1 ϫ 10 8 PFU/ml, and at 39.5°C, the stock had a titer of 1.5 ϫ 10 3 PFU/ml, which is an EOP of 1.5 ϫ 10 Ϫ5 .
T140 23672-23724 Sentence denotes This value is indicative of a single point mutation.
T141 23725-23854 Sentence denotes Of the plaques that formed at the nonpermissive temperature, all that we tested were no longer ts and are, therefore, revertants.
T142 23855-23905 Sentence denotes One of these revertants was designated MHV-Br31R1.
T143 23906-24073 Sentence denotes The plaque size and morphology of MHV-Brts31 and MHV-Br31R1 at 33°C and of MHV-Br31R1 at 39.5°C were identical to those of the parental MHV-A59 (Fig. 1A, left panel) .
T144 24074-24121 Sentence denotes MHV-Brts31 also failed to form plaques at 37°C.
T145 24122-24193 Sentence denotes Next, we next asked whether or not MHV-Brts31 is an RNAnegative mutant.
T146 24194-24407 Sentence denotes First, we determined the amounts of viral RNA synthesized in cells infected with MHV-A59, MHV-Brts31, or MHV-Br31R1 and maintained at the permissive and nonpermissive temperatures of 33°C and 39.5°C, respectively.
T147 24408-24683 Sentence denotes Figure 1B shows that at 33°C, all three viruses produce genome-sized and subgenome-sized virus RNA in the expected ratios, although the Northern blot suggests that MHV-Brts31 may synthesize slightly less viral RNA than does MHV-A59 or MHV-Br31R at the permissive temperature.
T148 24684-24784 Sentence denotes At the nonpermissive temperature of 39.5°C, MHV-Brts31 did not synthesize a detectable level of RNA.
T149 24785-24891 Sentence denotes In contrast, MHV-Br31R1 synthesizes amounts of virus RNA that are comparable to those of parental MHV-A59.
T150 24892-24956 Sentence denotes These results confirm that MHV-Brts31 is an RNA-negative mutant.
T151 24957-25223 Sentence denotes In order to look more closely at the RNA synthesis phenotype of MHV-Brts31 and MHV-Br31R1, we next analyzed the kinetics of viral RNA synthesis in cells infected with MHV-A59, MHV-Brts31, or MHV-Br31R1 and maintained at the permissive and nonpermissive temperatures.
T152 25224-25253 Sentence denotes Figure 2A higher temperature.
T153 25254-25613 Sentence denotes We have also confirmed that the kinetics and levels of virus RNA synthesis in MHV-Brts31-infected cells maintained at the nonpermissive temperature cannot be distinguished from those of mock-infected cells and that cells infected with MHV-Br31R1 and MHV-A59 are indistinguishable in terms of virus RNA synthesis at the permissive temperature (data not shown).
T154 25614-25725 Sentence denotes These results show that MHV-Brts31 has near-normal levels of total RNA synthesis at the permissive temperature.
T155 25726-25798 Sentence denotes They also suggest that MHV-Br31R1 is most likely to be a true revertant.
T156 25799-26161 Sentence denotes To show definitively whether or not the kinetics and levels of viral RNA synthesis in MHV-Brts31-infected cells at the permissive temperature are identical to those of MHV-A59-infected cells will require a more detailed analysis, possibly using different multiplicities of infection and a more accurate measure of RNA levels, e.g., quantitative RT-PCR (qRT-PCR).
T157 26162-26425 Sentence denotes A major advantage of using ts mutants is that they can be analyzed by temperature shift protocols, i.e., by allowing them to gain function at the permissive temperature, shifting them to the nonpermissive temperature, and then determining if the function is lost.
T158 26426-26571 Sentence denotes Thus, we determined the effect of temperature shift on the levels of total viral RNA synthesis in MHV-Brts31and MHV-Br31R1-infected 17Cl-1 cells.
T159 26572-26757 Sentence denotes To do this, infections were initiated at 33°C and infected cells were incubated until RNA synthesis reached about 20% of the maximum rate, which is approximately 4 to 5 h postinfection.
T160 26758-26860 Sentence denotes At this time, one set of cells were pulse-labeled with [ 3 H]uridine in the presence of actinomycin D.
T161 26861-27013 Sentence denotes Another set of virus-infected cells were shifted to 39.5°C and pulse-labeled for 30 min at 0 and 30 min after shifting to the nonpermissive temperature.
T162 27014-27155 Sentence denotes The cells were pulse-labeled with [ 3 H]uridine in the presence of actinomycin D and in the presence or absence of 100 g/ml of cycloheximide.
T163 27156-27260 Sentence denotes It should be noted that at 5 h postinfection the virus RTC produces mainly (Ͼ90%) plus-strand RNA (38) .
T164 27261-27295 Sentence denotes The results are shown in Fig. 2B .
T165 27296-27440 Sentence denotes The pulse-labeling prior to temperature shift shows again that MHV-Brts31-and MHV-BR31R1-infected cells have comparable levels of RNA synthesis.
T166 27441-27557 Sentence denotes After temperature shift, total RNA synthesis increased in MHV-Br31R1-infected cells in the absence of cycloheximide.
T167 27558-27660 Sentence denotes This reflects the amplification of virus RTCs and the increased metabolic rate at higher temperatures.
T168 27661-27773 Sentence denotes In the presence of cycloheximide, the activity fails to increase, but after shift, significant activity remains.
T169 27774-27917 Sentence denotes We conclude that the MHV-Br31R1 replicase-transcriptase activity formed before temperature shift is relatively resistant to higher temperature.
T170 27918-28079 Sentence denotes In MHV-Brts31-infected cells, there is also significant RNA synthesis activity after temperature shift, irrespective of the presence or absence of cycloheximide.
T171 28080-28255 Sentence denotes We interpret this to mean that for MHV-Brts31, preexisting complexes are also relatively resistant and continue to synthesize plus-strand RNA at the nonpermissive temperature.
T172 28256-28421 Sentence denotes To decide whether or not RTCs in MHV-Brts31-infected cells are able to synthesize minus-strand RNA at the nonpermissive temperature will require further experiments.
T173 28422-28447 Sentence denotes Complementation analysis.
T174 28448-28579 Sentence denotes Previous work by Sawicki et al. (40) has divided a large panel of RNA-negative, ts mutants of MHV into four complementation groups.
T175 28580-28714 Sentence denotes We crossed MHV-Brts31 with at least one virus from each of these known complementation groups using a classical complementation assay.
T176 28715-28749 Sentence denotes The results are shown in Table 1 .
T177 28750-28917 Sentence denotes Basically, MHV-Brts31 was able to complement all of the mutant viruses tested from each complementation group, with complementation indices (CI) of between 28 and 638.
T178 28918-29194 Sentence denotes This contrasts with a CI of 1.8 obtained for a cross involving mutants in the same complementation group (MHV-Albts6, CG1, versus MHV-Albts16, CG1) and a CI of 240 for a cross involving mutants from different complementation groups (MHV-Albts16, CG1, versus MHV-Albts22, CG2).
T179 29195-29385 Sentence denotes These results indicate that MHV-Brts31 forms the first member of a new complementation group and defines a new MHV replicase gene cistron, which we wish to provisionally designate cistron 0.
T180 29386-29464 Sentence denotes Identification of the mutation responsible for the ts phenotype of MHV-Brts31.
T181 29465-29572 Sentence denotes Thiel et al. have shown that MHV replicase gene products suffice for subgenome-length mRNA synthesis (51) .
T182 29573-29821 Sentence denotes We reasoned, therefore, that MHV-Brts31 would have a mutation somewhere in the replicase gene, and to identify this mutation, we sequenced the entire replicase genes of both MHV-Brts31 and MHV-Br31R1 (data not shown; sequence available on request).
T183 29822-30046 Sentence denotes This comparative analysis identified a single coding mutation, G to A at nucleotide 4131 (codon 1307 in ORF1a, amino acid 1307 in pp1a), which resulted in a conservative replacement of methionine-475 with isoleucine in nsp3.
T184 30047-30255 Sentence denotes The methionine residue at position 475 in the MHV nsp3 is conserved among group 2a coronaviruses (e.g., MHV, bovine coronavirus [BCoV], and human coronavirus OC43) but is not conserved in other coronaviruses.
T185 30256-30378 Sentence denotes Both MHV-A59 and the revertant MHV-Br31R1 have a methionine at this position, showing that MHV-Br31R1 is a true revertant.
T186 30379-30505 Sentence denotes The numbering system used was that assigned to our infectious clone of the MHV-A59 genome (GenBank accession number AY700211).
T187 30506-30839 Sentence denotes In order to confirm that G 4131 to A is the mutation responsible for the ts phenotype of MHV-Brts31, we used a vaccinia virus-based reverse genetics system to produce a recombinant virus, MHV-Brtsc31, that has a single G-to-A nucleotide exchange at position 4131 compared to the parental recombinant MHV-A59, which we call MHV-inf-1.
T188 30840-30946 Sentence denotes MHV-Brtsc31 was rescued and plaque purified three times at 33°C, and a passage 2 virus stock was obtained.
T189 30947-31066 Sentence denotes Sequence analysis of RNA from this stock of virus confirmed the presence of the G 4131 -to-A mutation (data not shown).
T190 31067-31221 Sentence denotes At 33°C, the MHV-Brtsc31 stock had a titer of 6 ϫ 10 8 PFU/ml, and at 39.5°C, the stock had a titer of 1.0 ϫ 10 4 PFU/ml, which is an EOP of 1.7 ϫ 10 Ϫ5 .
T191 31222-31295 Sentence denotes MHV-Brtsc31 also failed to form plaques at 37°C (Fig. 1A, right panel) .
T192 31296-31458 Sentence denotes One of the plaques that formed in cells infected with MHV-Brtsc31 and maintained at the nonpermissive temperature of 39.5°C was picked and designated MHV-Br31cR1.
T193 31459-31639 Sentence denotes The plaque size and morphology of MHV-Brtsc31 and MHV-Br31cR1 at 33°C and those of MHV-Br31cR1 at 39.5°C were identical to those of the parental MHV-inf-1 (Fig. 1A, right panel) .
T194 31640-31801 Sentence denotes Sequence analysis of RNA isolated from 17Cl-1 cells infected with a passage 2 stock of MHV-Br31cR1 showed that it was, indeed, a true revertant (data not shown).
T195 31802-32005 Sentence denotes Next, we determined the amounts of viral RNA synthesized in cells infected with MHV-inf-1 and MHV-Brtsc31 and maintained at the permissive and nonpermissive temperatures of 33°C and 39.5°C, respectively.
T196 32006-32122 Sentence denotes Figure 1C shows that at 33°C both viruses produce genome-sized and subgenome-sized virus RNA in the expected ratios.
T197 32123-32224 Sentence denotes At the nonpermissive temperature of 39.5°C, MHV-Brtsc31 did not synthesize a detectable level of RNA.
T198 32225-32325 Sentence denotes Finally, we repeated the same complementation analysis described above using MHV-Brtsc31 (Table 1) .
T199 32326-32498 Sentence denotes Again, the ts mutant virus was able to complement all of the mutant viruses tested from each complementation group, with complementation indices (CI) of between 27 and 717.
T200 32499-32738 Sentence denotes Taken together, these results confirm that a single G 4131 -to-A mutation is responsible for the ts phenotype of MHV-Brts31, conclusively locate cistron 0 in ORF1a, and show that nsp3 has, at least, one essential role in MHV RNA synthesis.
T201 32739-33026 Sentence denotes To determine if proteolytic processing of the replicase polyprotein(s) is affected in MHV-Brtsc31-and MHV-Br31cR1-infected cells at the nonpermissive temperature, we did temperature shift experiments with metabolic labeling and immunoprecipitation using nspspecific antibodies (Fig. 3) .
T202 33027-33162 Sentence denotes As described in Materials and Methods, 17Cl-1 cells were infected with either MHV-Brtsc31 or MHV-Br31cR1 at 33°C and incubated for 4 h.
T203 33163-33268 Sentence denotes At this time, one set of virus-infected cells was shifted to 39.5°C and a duplicate set was left at 33°C.
T204 33269-33375 Sentence denotes All the virus-infected cells were then radiolabeled with 35 S-trans label from 4.5 to 6.5 h postinfection.
T205 33376-33553 Sentence denotes At the end of the radiolabeling, whole-cell lysates were prepared, the viral nsp's were immunoprecipitated with nspspecific antisera, and the products were analyzed by SDS-PAGE.
T206 33554-33949 Sentence denotes Our first experiment (Fig. 4) showed that, in MHV-Brtsc31infected cells incubated at the nonpermissive temperature, compared to the permissive temperature, there is a major disruption of replicase polyprotein processing with respect to the processing of the pp1a p150 intermediate to its products, nsp4, nsp5 and nsp8 (compare boxed areas in Fig. 4A , lanes 3 to 5, with those in lanes 8 to 10).
T207 33950-34076 Sentence denotes This disruption of p150 processing was not seen in cells infected with MHV-Br31cR1 (Fig. 4B, lanes 3 to 5 and lanes 8 to 10) .
T208 34077-34275 Sentence denotes In contrast, there was no obvious impairment of the processing of nsp1, nsp2, and nsp3 in the MHV-Brtsc31-infected cells at the nonpermissive temperature (Fig. 4A, lanes 1 and 2 and lanes 6 and 7) .
T209 34276-34485 Sentence denotes Also, there was no temperature-sensitive effect on nsp1, nsp2, and nsp3 processing in MHV-Br31cR1-infected cells at either permissive or nonpermissive temperatures (Fig. 4B, lanes 1 and 2 and lanes 6 and 7) .
T210 34486-34748 Sentence denotes These results indicate that, at the nonpermissive temperature, the replacement of methionine-475 with isoleucine in nsp3 of MHV-Brtsc31 is affecting nsp5-mediated processing of pp1a and, more specifically, the processing of p150 to generate nsp4, nsp5, and nsp8.
T211 34749-34918 Sentence denotes These results were unexpected and the first demonstration of a relationship be- tween nsp3 function and pp1a processing by the 3CLpro cysteine protease activity of nsp5.
T212 34919-35107 Sentence denotes In addition to mediating the proteolytic processing of the pp1a polypeptide, nsp5 also mediates the processing of the ORF1b-encoded region of pp1ab, i.e., the production of nsp12 to nsp16.
T213 35108-35324 Sentence denotes To see if the replacement of methionine-475 with isoleucine in nsp3 of MHV-Brtsc31 has any effect on pp1ab processing, we repeated the experiment described above using an antiserum specific for nsp12, the viral RdRp.
T214 35325-35607 Sentence denotes The results shown in Fig. 5 lead us to conclude that the processing of nsp12 is also impaired at the nonpermissive temperature compared to the permissive temperature in cells infected with MHV-Brtsc31 but not in MHV-Br31cR1-infected cells (Fig. 5 , lanes 2 and 4 and lanes 6 and 8).
T215 35608-35788 Sentence denotes As a control, we once again demonstrated the impaired processing of nsp5 in MHV-Brtsc31-infected cells at the nonpermissive temperature (Fig. 5, lanes 1 and 3 and lanes 5 and 7) .
T216 35789-35931 Sentence denotes TEM analysis of MHV-infected cells reveals that DMVs can be visualized as dark-ringed vesicles in the cytoplasm adjacent to the nucleus (16) .
T217 35932-36198 Sentence denotes In cells infected with MHV-Br31R1, the typical accumulation of DMV structures (indicated by arrows) was evident after incubation at the permissive (Fig. 6A) and nonpermissive (Fig. 6B ) temperatures, with an increased number of DMVs in the cells incubated at 39.5°C.
T218 36199-36534 Sentence denotes In contrast, we found that although the number of DMVs in MHV-Brts31infected cells incubated at the permissive temperature (Fig. 6C ) was similar to the number found in revertant virus-infected cells, the number of DMVs in MHV-Brts31-infected cells that had been shifted to the nonpermissive temperature was reduced ( Fig. 6D and F) .
T219 36535-36774 Sentence denotes We also noted a small number of aberrant vesicles at the nonpermissive temperature (indicated by asterisks in Fig. 6D and F) that stained with uranyl acetate and Reynolds' lead citrate but do not have the typical doublemembrane morphology.
T220 36775-36855 Sentence denotes Further work will be necessary to determine if these are possibly residual DMVs.
T221 36856-37109 Sentence denotes Taken together, these results indicate that the replacement of methionine-475 with isoleucine in nsp3 of MHV-Brts31 impairs both 3CLpro-mediated processing of replicase polyproteins and the assembly or stability of DMVs at the nonpermissive temperature.
T222 37111-37201 Sentence denotes Previously, we have identified four complementation groups of MHV RNA-negative ts mutants.
T223 37202-37495 Sentence denotes Three of these groups, groups II, IV, and VI, were defined by ts viruses with mutations in the ORF1b-encoded proteins nsp12, nsp14, and nsp16, respectively, while the fourth group, group I, was defined by ts viruses with mutations in three of the ORF1a-encoded proteins, nsp4, nsp5, and nsp10.
T224 37496-37712 Sentence denotes This led us to the hypothesis that each of the five MHV ORF1b cleavage products (nsp12 to nsp16) may represent distinct cistrons but that the majority of the ORF1a cleavage products will function as a single cistron.
T225 37713-37826 Sentence denotes More specifically, it was proposed that cistron I would encompass the regions of ORF1a that encode nsp4 to nsp10.
T226 37827-37948 Sentence denotes The complementation analysis of MHV-Brts31 described here identifies a new cistron (cistron 0) in the MHV replicase gene.
T227 37949-38127 Sentence denotes Furthermore, the identification of the mutation responsible for the ts phenotype of MHV-Brts31 (G 4131 to A) shows that cistron 0 encompasses, at least, the region encoding nsp3.
T228 38128-38228 Sentence denotes For a number of reasons, we suggest that cistron 0 will not extend beyond the coding region of nsp3.
T229 38229-38444 Sentence denotes First, as mentioned above, extensive deletion of sequences encoding the carboxyl region of nsp1 or, indeed, the entire coding region of nsp2 has an attenuating effect but is not lethal for MHV replication (13, 18) .
T230 38445-38890 Sentence denotes Second, although it has been shown that an RNA stemloop in the bovine coronavirus (BCoV) 5Ј proximal coding region of nsp1 is essential for RNA replication (4) (which would explain the lethality of deleting the entire nsp1 coding region), the majority of evidence suggests that the coronavirus nsp1 protein primarily has a role in the virus-host interaction (8, 24, 25, 29) or regulatory, rather than essential, functions in RNA synthesis (19) .
T231 38891-39218 Sentence denotes Although the possibility that other ORF1a-or ORF1b-encoded nsp's (i.e., nsp6, nsp7, nsp8, nsp9, nsp11, nsp13, or nsp15) could fall into the same complementation group as MHV-Brts31 cannot be ruled out, it would, in our opinion, be difficult to reconcile this with our current view of MHV replicase gene expression and function.
T232 39219-39395 Sentence denotes Our analysis of replicase polyprotein processing and DMV structures in MHV-Brtsc31-infected cells at the nonpermissive temperature allows us to reach two important conclusions.
T233 39396-39605 Sentence denotes First, the ts lesion of MHV-Brtsc31 is affecting nsp5-mediated processing of pp1a and pp1ab, specifically, the processing of p150 to generate nsp4, nsp5, and nsp8 and the processing of pp1ab to generate nsp12.
T234 39606-39714 Sentence denotes However, there is no effect on the processing of the replicase polyproteins to produce nsp1, nsp2, and nsp3.
T235 39715-39759 Sentence denotes We interpret this to mean one of two things.
T236 39760-40020 Sentence denotes Either there is an interaction between nsp3 and another component of the RTC, which is necessary to facilitate nsp5-mediated polyprotein processing, or the normal structure or function of nsp3 is needed to provide pp1a/pp1ab substrates upon which nsp5 can act.
T237 40021-40283 Sentence denotes In this respect, the precedent for protein-protein interactions within the coronavirus RTC that result in a modification of nsp5 activity has been given by Donaldson et al. (9) , who have suggested that nsp10 may act as a cofactor for 3CLpro-mediated processing.
T238 40284-40463 Sentence denotes Second, the decrease in the number of DMV structures in MHV-Brts31-infected cells at the nonpermissive temperature indicates that the assembly or stability of the RTC is impaired.
T239 40464-40705 Sentence denotes A quantitative analysis of DMV numbers in cells infected with MHV-Brts31, together with experiments to determine the turnover rate of DMV structures in MHV-A59-infected cells, will be needed to determine the precise defect in DMV biogenesis.
T240 40706-40978 Sentence denotes However, our data suggest that preformed RTCs are able to continue plus-strand RNA synthesis at the nonpermissive temperature, and it seems more likely that the failure to correctly process the replicase polyproteins would affect assembly rather than stability of the RTC.
T241 40979-41172 Sentence denotes On the other hand, an earlier analysis of cells infected with the RNA-negative, ts mutant MHV-Albts6 (6) has shown that DMV formation and replicase polyprotein processing are not always linked.
T242 41173-41290 Sentence denotes In this case, DMV formation was severely impaired even though replicase polyprotein processing appeared to be normal.
T243 41291-41536 Sentence denotes The finding that both MHV-Brts31 and MHV-Albts6 fail to accumulate DMVs at the nonpermissive temperature is consistent with the idea that DMVs either are required for coronavirus RNA synthesis or are generated as a result of viral RNA synthesis.
T244 41537-41801 Sentence denotes In the light of these conclusions, the obvious challenge is to identify the specific function or functions of nsp3 that are impaired at the nonpermissive temperature in MHV-Brts31/ MHV-Brtsc31-infected cells and to relate this to the phenotype of the mutant virus.
T245 41802-41989 Sentence denotes In this context, it is important to note that the data do not allow us to discern whether the phenotypic changes that we observe are directly or indirectly related to the causal mutation.
T246 41990-42202 Sentence denotes The M475I mutation, which is located, essentially, at the junction of the PLP1 and X domains of nsp3, could lead to multiple effects that may then be separately related to the different phenotypic manifestations.
T247 42203-42454 Sentence denotes Indeed, Graham and Denison (17) have shown that recombinant MHV mutants with changes in the nsp1-to-nsp3 protein coding region may have a variety of different phenotypes with respect to replicase polyprotein processing, replication, and RNA synthesis.
T248 42455-42549 Sentence denotes In general, we can offer three possible ways in which the M475I mutation may exert its effect.
T249 42550-42693 Sentence denotes First, it is possible that one or more of the enzymatic activities associated with nsp3 are rendered inactive at the nonpermissive temperature.
T250 42694-42765 Sentence denotes For a number of reasons, we consider this the least likely explanation.
T251 42766-42902 Sentence denotes For example, we consider it unlikely that inactivation of PLP1 activity is responsible for the phenotype of MHV-Brts31 (or MHV-Brtsc31).
T252 42903-43105 Sentence denotes Our data do not provide any evidence for a defect in nsp1, nsp2, and nsp3 processing, and previous studies have shown that MHV PLP1 activity is not essential for virus replication in cell culture (17) .
T253 43106-43250 Sentence denotes Nevertheless, mutants of this sort are highly impaired on recovery and we cannot rule out that the inactivation of PLP1 may somehow be involved.
T254 43251-43476 Sentence denotes Similarly, although it has been shown that the HCoV-229E PLP2 activity is essential for replication (58), our results do not indicate any defect in this activity at the nonpermissive temperature in MHV-Brtsc31-infected cells.
T255 43477-43613 Sentence denotes A second type of function associated with nsp3 is the ADP-ribose 1Љ-phosphatase and poly(ADP-ribose) binding activities of the X domain.
T256 43614-43898 Sentence denotes Again, we consider it unlikely that inactivation of one or both of these activities can account for the phenotype of MHV-Brts31 because it has been shown previously that mutation of the active site of the HCoV-229E nsp3 X domain has no significant effect on virus RNA synthesis (36) .
T257 43899-44040 Sentence denotes The inactivation of nsp3-associated deubiquitination activity (57) and other, as-yet-unidentified enzymatic activities remains a possibility.
T258 44041-44194 Sentence denotes A second and perhaps more likely explanation is that, at the nonpermissive temperature, interactions between nsp3 and cellular membranes are compromised.
T259 44195-44332 Sentence denotes Although there is still some uncertainty, the current model of MHV nsp3 topology predicts at least 2 transmembrane helices (26, 31, 32) .
T260 44333-44463 Sentence denotes This would place the bulk of nsp3 on the same membrane face occupied by nsp5 and the nsp12 to nsp16 proteins processed from pp1ab.
T261 44464-44646 Sentence denotes Kanjanahaluethai et al. (26) recently showed that removing the nsp3 transmembrane domain abolished PLP2 activity, presumably by preventing the interaction of PLP2 with its substrate.
T262 44647-44866 Sentence denotes If the MHV-Brts31/MHV-Brtsc31 nsp3 protein is no longer stably inserted into the cellular membrane at the nonpermissive temperature, then this could compromise the integrity of the whole replicase-transcriptase complex.
T263 44867-45050 Sentence denotes The same might be true if the replacement of methionine-475 with isoleucine in nsp3 had a global effect upon the structure of nsp3, or even the pp1a and pp1ab polyproteins as a whole.
T264 45051-45459 Sentence denotes Our current view of nsp3 as a large protein with a functional and structural domain architecture might argue against a global effect (31) , and the position of this substitution, which is essentially predicted to be in a flexible region separating conserved functional domains, would be more consistent with the idea that intramolecular nsp3 domain interactions are affected at the nonpermissive temperature.
T265 45460-45620 Sentence denotes The third possibility is that, at the nonpermissive temperature, nsp3 protein-protein, or perhaps nsp3 protein-RNA, interactions within the RTC are compromised.
T266 45621-45734 Sentence denotes This could be manifested as an inability to assemble new complexes or as the assembly of a nonfunctional complex.
T267 45735-45943 Sentence denotes Putative metal-binding domains are located in the PLP, ZF, and Y domains of the MHV nsp3, and they may represent zinc-finger motifs that are involved in the recognition of other protein components of the RTC.
T268 45944-46018 Sentence denotes The MHV nsp3 protein also contains a putative nucleic acid-binding domain.
T269 46019-46295 Sentence denotes Neuman et al. (31) have shown that the corresponding SARS-coronavirus NAB domain of nsp3 has both single-stranded nucleic acid-binding activity and doublestranded unwinding activity, and they suggest that these activities are consistent with a nucleic acid chaperone function.
T270 46296-46519 Sentence denotes It is easy to see how the disruption of MHV nsp3 protein-protein or nsp3 protein-RNA interactions could prevent the assembly of a functional RTC and result in the ts phenotype that we observe for MHV-Brts31 and MHV-Brtsc31.
T271 46520-46744 Sentence denotes Work is under way to investigate some of these possibilities, and we are confident that RNA-negative ts mutants such as MHV-Brts31 will prove to be valuable tools for unraveling the complexities of coronavirus RNA synthesis.