scholarly journals Induction of a Cellular DNA Damage Response by Porcine Circovirus Type 2 Facilitates Viral Replication and Mediates Apoptotic Responses

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Li Wei ◽  
Shanshan Zhu ◽  
Jing Wang ◽  
Rong Quan ◽  
Xu Yan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Viral Replication ◽  
Dna Damage Response ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Damage Response ◽  
Cellular Dna

scholarly journals Differential Requirements of the C Terminus of Nbs1 in Suppressing Adenovirus DNA Replication and Promoting Concatemer Formation

2008 ◽  
Vol 82 (17) ◽  
pp. 8362-8372 ◽  
Author(s):  
Seema S. Lakdawala ◽  
Rachel A. Schwartz ◽  
Kevin Ferenchak ◽  
Christian T. Carson ◽  
Brian P. McSharry ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Viral Replication ◽  
Dna Damage Response ◽  
Viral Genomes ◽  
Mrn Complex ◽  
E4 Region ◽  
C Terminus ◽  
Damage Response ◽  
Cellular Dna

ABSTRACT Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.

scholarly journals Harnessing the Genetic Plasticity of Porcine Circovirus Type 2 to Target Suicidal Replication

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1676
Author(s):  
Agm Rakibuzzaman ◽  
Pablo Piñeyro ◽  
Angela Pillatzki ◽  
Sheela Ramamoorthy
Keyword(s):  
Viral Replication ◽  
Body Weight Loss ◽  
Neutralizing Antibody ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Mutation Rates ◽  
Porcine Circovirus ◽  
Emerging Viruses ◽  
Weanling Piglets

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.

scholarly journals Porcine MKRN1 Modulates the Replication and Pathogenesis of Porcine Circovirus Type 2 by Inducing Capsid Protein Ubiquitination and Degradation

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Tongtong Wang ◽  
Qian Du ◽  
Xingchen Wu ◽  
Yingying Niu ◽  
Lijuan Guan ◽  
...  
Keyword(s):  
Viral Replication ◽  
Capsid Protein ◽  
Virus Replication ◽  
Ring Finger ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Lysine Residues

ABSTRACT Porcine circovirus type 2 (PCV2) capsid protein (Cap) is a unique structure protein that plays pivotal roles in the process of viral replication and pathogenesis. Herein, we characterized a putative porcine Makorin RING finger protein 1 (pMKRN1) variant, an N-terminal-truncated variant of putative full-size porcine MKRN1 which has a unique expression pattern resulting from the porcine mkrn1 gene and which interacts with PCV2 Cap. A domain mapping assay showed that the C terminus of pMKRN1 and fragments (amino acids 108 to 198) of Cap are required for this interaction. PCV2 transiently upregulated pMKRN1 in PK-15 cells, but persistent viral infection downregulated pMKRN1 in major pathological tissues of PCV2-infected piglets. Overexpression of pMKRN1 significantly inhibited the generation of progeny PCV2 via ubiquitination and degradation of Cap, whereas knockout of pMKRN1 blocked Cap degradation and promoted progeny virus replication. pMKRN1 specifically targeted PCV2 Cap lysine residues 164, 179, and 191 to induce polyubiquitination and subsequent degradation. Mutation of either of the three lysine residues in the Cap protein or mutation of the histidine at residue 243 within the RING finger domain of pMKRN1 abrogated the E3 ligase activity of pMKRN1, rendering cells incapable of inducing Cap ubiquitination and degradation. Consistent with this finding, a Cap ubiquitination-deficient PCV2 strain showed enhanced virus replication and produced severe histological lesions in the lung and lymph node tissues compared with wild-type PCV2. Taken together, the results presented here suggest that PCV2 downregulates the pMKRN1 variant to avoid pMKRN1-mediated Cap ubiquitination and degradation, thus promoting viral replication and pathogenesis in its targeted tissues. IMPORTANCE Porcine circovirus type 2 is the pathogen to which pigs are the most susceptible, causing immense economic losses in the global swine industry, but whether host cells have developed some strategies to prevent viral replication is still unclear. Here, we found that porcine MKRN1 (pMKRN1) was upregulated in the early stage of PCV2 infection and mediated the polyubiquitination and degradation of Cap protein to block PCV2 replication, yet persistent PCV2 infection downregulated pMKRN1 levels to avoid degradation, promoting viral replication and pathogenesis in its targeted tissues. These data present new insight into the molecular mechanisms underlying the antiviral effects of pMKRN1 E3 ligase during PCV2 infection and also suggest potential new control measures for PCV2 outbreaks.

scholarly journals Characterization of a Previously Unidentified Viral Protein in Porcine Circovirus Type 2-Infected Cells and Its Role in Virus-Induced Apoptosis

2005 ◽  
Vol 79 (13) ◽  
pp. 8262-8274 ◽  
Author(s):  
Jue Liu ◽  
Isabelle Chen ◽  
Jimmy Kwang
Keyword(s):  
Viral Replication ◽  
Caspase 3 ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Caspase 8 ◽  
Infected Cells ◽  
Apoptotic Activity ◽  
Induced Apoptosis

ABSTRACT Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs. In this study, transcription and translation of a novel viral gene (termed ORF3 here) was detected during productive infection of PCV2 in PK15 cells. The results of infection with ORF3-deficient PCV2 by site-directed mutagenesis indicated that the protein is not essential for viral replication. To investigate the underlying mechanism of cell death caused by replication of PCV2, apoptosis characterized by chromosomal condensation and fragmentation, formation of apoptotic bodies, and significant increase in hypodiploids were detected in infected cells. We further demonstrated that PCV2-induced apoptosis required the activation of caspase-8 but not caspase-9. The activation of caspase-8 results in the activation of caspase-3 as shown by an increase in the cleavage of the caspase substrate in the infected cells. To determine whether ORF3 protein could trigger apoptosis, ORF3 as well as ORF1 and ORF2 genes were transiently expressed in PK15 and Cos-7 cells for apoptotic activity assay. Transfection of cells with the ORF3 alone induced apoptosis using a pathway similar to that described in the context of viral infection. This is further confirmed by a significant decrease in apoptotic activity of infected cells in the absence of the ORF3 expression, suggesting that the protein plays a major role in the induction of virus-induced apoptosis. Altogether, these results indicate that ORF3 is a novel PCV2 protein that is not essential for viral replication in cultured cells but is involved in PCV2-induced apoptosis by activating caspase-8 and caspase-3 pathways.

scholarly journals Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Reshma Nazeer ◽  
Fadi S. I. Qashqari ◽  
Abeer S. Albalawi ◽  
Ann Liza Piberger ◽  
Maria Teresa Tilotta ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Viral Replication ◽  
Signaling Pathways ◽  
Dna Damage Response ◽  
Replication Fork ◽  
Dependent Manner ◽  
Damage Response ◽  
Adenovirus E1b ◽  
Cellular Dna

ABSTRACT Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication. IMPORTANCE Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.

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