scholarly journals Altered Growth Characteristics of Recombinant Respiratory Syncytial Viruses Which Do Not Produce NS2 Protein

1999 ◽  
Vol 73 (1) ◽  
pp. 466-473 ◽  
Author(s):  
Michael N. Teng ◽  
Peter L. Collins
Keyword(s):  
Reverse Genetics ◽  
Vaccine Development ◽  
Nonstructural Protein ◽  
Plaque Morphology ◽  
Wild Type ◽  
T7 Promoter ◽  
Virus Growth ◽  
Reading Frame ◽  
Syncytial Virus ◽  
Knockout Virus

ABSTRACT The second gene in the 3′-to-5′ gene order in respiratory syncytial virus (RSV) encodes the nonstructural protein NS2, for which there is no assigned function. To study the function of NS2, we have used a recently developed reverse genetics system to ablate expression of NS2 in recombinant RSV. A full-length cDNA copy of the antigenome of RSV A2 strain under the control of a T7 promoter was modified by introduction of tandem termination codons within the NS2 open reading frame (NS2stop) or by deletion of the entire NS2 gene (ΔNS2). The NS2 knockout antigenomic cDNAs were cotransfected with plasmids encoding the N, P, L, and M2-1 proteins of RSV, each controlled by the T7 promoter, into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Recombinant NS2stop and ΔNS2 RSVs were recovered and characterized. Both types of NS2 knockout virus displayed pinpoint plaque morphology and grew more slowly than wild-type RSV. The expression of monocistronic mRNAs for the five genes examined (NS1, NS2, N, F, and L) was unchanged in cells infected with either type of NS2 knockout virus, except that no NS2 mRNA was detected with the ΔNS2 virus. Synthesis of readthrough mRNAs was affected only for the ΔNS2 virus, where the NS1-NS2, NS2-N, and NS1-NS2-N mRNAs were replaced with the predicted novel NS1-N mRNA. Upon passage, the NS2stop virus stock rapidly developed revertants which expressed NS2 protein and grew with similar plaque morphology and kinetics wild-type RSV. Sequence analysis confirmed that the termination codons had reverted to sense, albeit not the wild-type assignments, and provided evidence consistent with biased hypermutation. No revertants were recovered from recombinant ΔNS2 RSV. These results show that the NS2 protein is not essential for RSV replication, although its presence greatly improves virus growth in cell culture. The attenuated phenotype of these mutant viruses, coupled with the expected genetic stability associated with gene deletions, suggests that the ΔNS2 RSV is a candidate for vaccine development.

scholarly journals Respiratory Syncytial Virus That Lacks Open Reading Frame 2 of the M2 Gene (M2-2) Has Altered Growth Characteristics and Is Attenuated in Rodents

2000 ◽  
Vol 74 (1) ◽  
pp. 74-82 ◽  
Author(s):  
Hong Jin ◽  
Xing Cheng ◽  
Helen Z. Y. Zhou ◽  
Shengqiang Li ◽  
Adam Seddiqui
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cdna Clone ◽  
Open Reading Frame ◽  
Plaque Morphology ◽  
Potential Candidate ◽  
Wild Type ◽  
Replication Process ◽  
Reading Frame ◽  
Syncytial Virus ◽  
In Cells

ABSTRACT The M2 gene of respiratory syncytial virus (RSV) encodes two putative proteins: M2-1 and M2-2; both are believed to be involved in the RNA transcription or replication process. To understand the function of the M2-2 protein in virus replication, we deleted the majority of the M2-2 open reading frame from an infectious cDNA clone derived from the human RSV A2 strain. Transfection of HEp-2 cells with the cDNA clone containing the M2-2 deletion, together with plasmids that encoded the RSV N, P, and L proteins, produced a recombinant RSV that lacked the M2-2 protein (rA2ΔM2-2). Recombinant virus rA2ΔM2-2 was recovered and characterized. The levels of viral mRNA expression for 10 RSV genes examined were unchanged in cells infected with rA2ΔM2-2, except that a shorter M2 mRNA was detected. However, the ratio of viral genomic or antigenomic RNA to mRNA was reduced in rA2ΔM2-2-infected cells. By use of an antibody directed against the bacterially expressed M2-2 protein, the putative M2-2 protein was detected in cells infected with wild-type RSV but not in cells infected with rA2ΔM2-2. rA2ΔM2-2 displayed a small-plaque morphology and grew much more slowly than wild-type RSV in HEp-2 cells. In infected Vero cells, rA2ΔM2-2 exhibited very large syncytium formation compared to that of wild-type recombinant RSV. rA2ΔM2-2 appeared to be a host range mutant, since it replicated poorly in HEp-2, HeLa, and MRC5 cells but replicated efficiently in Vero and LLC-MK2 cells. Replication of rA2ΔM2-2 in the upper and lower respiratory tracts of mice and cotton rats was highly restricted. Despite its attenuated replication in rodents, rA2ΔM2-2 was able to provide protection against challenge with wild-type RSV A2. The genotype and phenotype of the M2-2 deletion mutant were stably maintained after extensive in vitro passages. The attenuated phenotype of rA2ΔM2-2 suggested that rA2ΔM2-2 may be a potential candidate for use as a live attenuated vaccine.

scholarly journals Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice

2007 ◽  
Vol 81 (17) ◽  
pp. 9490-9501 ◽  
Author(s):  
Christine D. Krempl ◽  
Anna Wnekowicz ◽  
Elaine W. Lamirande ◽  
Giw Nayebagha ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Virulent Strain ◽  
Consensus Sequence ◽  
Cytoplasmic Tail ◽  
Syncytial Virus

ABSTRACT Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (ΔG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the ΔG mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

scholarly journals Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

2015 ◽  
Vol 89 (9) ◽  
pp. 4907-4917 ◽  
Author(s):  
Anna M. Mielech ◽  
Xufang Deng ◽  
Yafang Chen ◽  
Eveline Kindler ◽  
Dorthea L. Wheeler ◽  
...  
Keyword(s):  
Viral Replication ◽  
Protease Activity ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Viral Pathogenesis ◽  
Wild Type ◽  
Viral Protease ◽  
Novel Approach ◽  
Protease Stability

ABSTRACTUbiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation.IMPORTANCEIntroducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

scholarly journals Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Benjamin Brennan ◽  
Veronica V. Rezelj ◽  
Richard M. Elliott
Keyword(s):  
Virus Infection ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Transcription Termination ◽  
Severe Disease ◽  
Open Reading Frame ◽  
Coding Region ◽  
Reading Frame ◽  
Green Fluorescent

ABSTRACT SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3′ rapid amplification of cDNA ends (RACE), we mapped the 3′ end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3′ end of the N mRNA terminates upstream of a 5′-GCCAGCC-3′ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5′-GCCAGCC-3′ motif present in the virus genomic S RNA.

scholarly journals Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Satoshi Komoto ◽  
Yuta Kanai ◽  
Saori Fukuda ◽  
Masanori Kugita ◽  
Takahiro Kawagishi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Growth Curves ◽  
Single Step ◽  
Replication Cycle ◽  
Nonstructural Proteins ◽  
Virus Growth ◽  
Severe Diarrhea

ABSTRACT The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

scholarly journals The Full-Length Protein Encoded by Human Cytomegalovirus Gene UL117 Is Required for the Proper Maturation of Viral Replication Compartments

2008 ◽  
Vol 82 (7) ◽  
pp. 3452-3465 ◽  
Author(s):  
Zhikang Qian ◽  
Baoqin Xuan ◽  
Te Tee Hong ◽  
Dong Yu
Keyword(s):  
Human Cytomegalovirus ◽  
Large Scale ◽  
Full Length ◽  
Wild Type ◽  
Virus Growth ◽  
Reading Frame ◽  
Early Proteins ◽  
Immediate Early Proteins ◽  
Novel Transcripts ◽  
Related Proteins

ABSTRACT Previously, two large-scale mutagenic analyses showed that mutations in the human cytomegalovirus (HCMV) gene UL117 resulted in a defect in virus growth in fibroblasts. Early transcriptional analyses have revealed several mRNAs from the UL119-UL115 region; however, specific transcripts encoding UL117-related proteins have not been identified. In this study, we identified two novel transcripts arising from the UL117 gene locus, and we reported that the UL117 open reading frame encoded the full-length protein pUL117 (45 kDa) and the shorter isoform pUL117.5 (35 kDa) as the result of translation initiation at alternative in-frame ATGs. Both proteins were expressed with early kinetics, but pUL117 accumulated at a lower abundance relative to that of pUL117.5. During HCMV infection, both proteins localized predominantly to the nucleus, and the major fraction of pUL117 localized in viral nuclear replication compartments. We constructed mutant HCMV viruses in which the entire UL117 coding sequence was deleted or the expression of pUL117 was specifically abrogated. The growth of mutant viruses was significantly attenuated, indicating that pUL117 was required for efficient virus infection in fibroblasts. Cells infected with the pUL117-deficient mutant virus accumulated representative viral immediate-early proteins and early proteins normally. In the absence of pUL117, the accumulation of replicating viral DNA was reduced by no more than twofold at early times and was indistinguishable from that of the wild type at 72 h postinfection. Strikingly, there was a 12- to 24-h delay in the development of nuclear replication compartments and a marked delay in the expression of late viral proteins. We conclude that pUL117 acts to promote the development of nuclear replication compartments to facilitate viral growth.

scholarly journals Attenuation of Zika Virus by Passage in Human HeLa Cells

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 93 ◽  
Author(s):  
Li ◽  
Collins ◽  
Widen ◽  
Davis ◽  
Kaiser ◽  
...  
Keyword(s):  
Hela Cell ◽  
Hela Cells ◽  
Zika Virus ◽  
Infected Animal ◽  
Vaccine Development ◽  
Nonstructural Protein ◽  
A549 Cells ◽  
Wild Type ◽  
Nonstructural Protein 1

Zika virus (ZIKV) is a mosquito-borne Flavivirus. Previous studies have shown that mosquito-transmitted flaviviruses, including yellow fever, Japanese encephalitis, and West Nile viruses, could be attenuated by serial passaging in human HeLa cells.  Therefore, it was hypothesized that wild-type ZIKV would also be attenuated after HeLa cell passaging. A human isolate from the recent ZIKV epidemic was subjected to serial HeLa cell passaging, resulting in attenuated in vitro replication in both Vero and A549 cells. Additionally, infection of AG129 mice with 10 plaque forming units (pfu) of wild-type ZIKV led to viremia and mortality at 12 days, whereas infection with 103 pfu of HeLa-passage 6 (P6) ZIKV led to lower viremia, significant delay in mortality (median survival: 23 days), and increased cytokine and chemokine responses.  Genomic sequencing of HeLa-passaged virus identified two amino acid substitutions as early as HeLa-P3: pre-membrane E87K and nonstructural protein 1 R103K. Furthermore, both substitutions were present in virus harvested from HeLa-P6-infected animal tissue. Together, these data show that, similarly to other mosquito-borne flaviviruses, ZIKV is attenuated following passaging in HeLa cells. This strategy can be used to improve understanding of substitutions that contribute to attenuation of ZIKV and be applied to vaccine development across multiple platforms.

scholarly journals Basic Residues of the Helix Six Domain of Influenza Virus M1 Involved in Nuclear Translocation of M1 Can Be Replaced by PTAP and YPDL Late Assembly Domain Motifs

2003 ◽  
Vol 77 (12) ◽  
pp. 7078-7092 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Subrata Barman ◽  
Tae Yong Yang ◽  
Debi P. Nayak
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Nuclear Translocation ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Plaque Morphology ◽  
Wild Type ◽  
Wild Type Phenotype

ABSTRACT Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (ΔYRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, ΔYRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.

scholarly journals Vaccinia virus kelch protein A55 is a 64 kDa intracellular factor that affects virus-induced cytopathic effect and the outcome of infection in a murine intradermal model

2006 ◽  
Vol 87 (6) ◽  
pp. 1521-1529 ◽  
Author(s):  
Philippa M. Beard ◽  
Graham C. Froggatt ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Cytopathic Effect ◽  
Plaque Morphology ◽  
Wild Type ◽  
Intracellular Protein ◽  
Reading Frame ◽  
Btb Domain ◽  
Broad Complex ◽  
Vacv Strain

The vaccinia virus (VACV) protein A55 is a BTB/kelch protein with a broad-complex, tramtrack and bric-a-brac (BTB) domain in the N-terminal region and five kelch repeats in the C-terminal half. The BTB/kelch subgroup of the kelch superfamily of proteins has been associated with a wide variety of functions including regulation of the cytoskeleton. VACV contains three genes predicted to encode BTB/kelch proteins: A55R, F3L and C2L. The A55R gene product has been identified as an intracellular protein of 64 kDa that is expressed late in infection. A VACV strain lacking 93.6 % of the A55R open reading frame (vΔA55) was constructed and found to have an unaltered growth rate in vivo but a different plaque morphology and cytopathic effect, as well as reduced development of VACV-induced Ca2+-independent cell/extracellular matrix adhesion. In a murine intradermal model of VACV infection, a virus lacking the A55R gene induced larger lesions than wild-type and revertant control viruses.

scholarly journals The nsp3 Macrodomain Promotes Virulence in Mice with Coronavirus-Induced Encephalitis

2014 ◽  
Vol 89 (3) ◽  
pp. 1523-1536 ◽  
Author(s):  
Anthony R. Fehr ◽  
Jeremiah Athmer ◽  
Rudragouda Channappanavar ◽  
Judith M. Phillips ◽  
David K. Meyerholz ◽  
...  
Keyword(s):  
Immune System ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Disease Onset ◽  
Viral Pathogenesis ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Structural Domain ◽  
Type Virus ◽  
Wild Type

ABSTRACTAll coronaviruses encode a macrodomain containing ADP-ribose-1″-phosphatase (ADRP) activity within the N terminus of nonstructural protein 3 (nsp3). Previous work showed that mouse hepatitis virus strain A59 (MHV-A59) with a mutated catalytic site (N1348A) replicated similarly to wild-type virus but was unable to cause acute hepatitis in mice. To determine whether this attenuated phenotype is applicable to multiple disease models, we mutated the catalytic residue in the JHM strain of MHV (JHMV), which causes acute and chronic encephalomyelitis, using a newly developed bacterial artificial chromosome (BAC)-based MHV reverse genetics system. Infection of mice with the macrodomain catalytic point mutant virus (N1347A) resulted in reductions in lethality, weight loss, viral titers, proinflammatory cytokine and chemokine expression, and immune cell infiltration in the brain compared to mice infected with wild-type virus. Specifically, macrophages were most affected, with approximately 2.5-fold fewer macrophages at day 5 postinfection in N1347A-infected brains. Tumor necrosis factor (TNF) and interferon (IFN) signaling were not required for effective host control of mutant virus as all N1347A virus-infected mice survived the infection. However, the adaptive immune system was required for protection since N1347A virus was able to cause lethal encephalitis in RAG1−/−(recombination activation gene 1 knockout) mice although disease onset was modestly delayed. Overall, these results indicate that the BAC-based MHV reverse genetics system will be useful for studies of JHMV and expand upon previous studies, showing that the macrodomain is critical for the ability of coronaviruses to evade the immune system and promote viral pathogenesis.IMPORTANCECoronaviruses are an important cause of human and veterinary diseases worldwide. Viral processes that are conserved across a family are likely to be good targets for the development of antiviral therapeutics and vaccines. The macrodomain is a ubiquitous structural domain and is also conserved among all coronaviruses. The coronavirus macrodomain has ADP-ribose-1″-phosphatase activity; however, its function during infection remains unclear as does the reason that coronaviruses have maintained this enzymatic activity throughout evolution. For MHV, this domain has now been shown to promote multiple types of disease, including hepatitis and encephalitis. These data indicate that this domain is vital for the virus to replicate and cause disease. Understanding the mechanism used by this enzyme to promote viral pathogenesis will open up novel avenues for therapies and may give further insight into the role of macrodomain proteins in the host cell since these proteins are found in all living organisms.

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