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Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines.

SUBMITTER: Suhardiman M

PROVIDER: S-EPMC7114486 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

Suhardiman Maman M Kramyu Jarin J Narkpuk Jaraspim J Jongkaewwattana Anan A Wanasen Nanchaya N

Virus research 20141006

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover ...[more]

PMID: 25300804

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Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. Severe interstitial pneumonia is the typical pathological characteristic of PRRSV-infected swine. Accumulating evidence has suggested that hypoxia-inducible factor 1α (HIF-1α) plays vital roles in the development of inflammation and the viral life cycle. However, the role and the underlying mechanism of HIF-1α in PRRSV infection remain elusive. Here, we found that PRRSV infection elevated HIF-1α expression. Furthermore, overexpression of HIF-1α increased PRRSV replication, whereas knockdown of HIF-1α inhibited PRRSV infection. Our further mechanistic analysis revealed that PRRSV-encoded nonstructural protein 1β (nsp1β) promoted HIF-1α transcription via its N-terminal nuclease activity and degraded the polyubiquitin chain of HIF-1α via its C-terminal deubiquitylation (DUB) enzyme activity, collectively stabilizing HIF-1α. Meanwhile, nsp1β interacted with both HIF-1α and von Hippel-Lindau tumor suppressor (pVHL) to form a ternary complex, which may have hindered pVHL-mediated ubiquitination degradation of HIF-1α by impairing the interaction between HIF-1α and pVHL. Interestingly, pVHL also stabilized nsp1β via K63-linked ubiquitination, forming a positive feedback loop to stabilize HIF-1α. Taken together, these results indicate that PRRSV infection stabilizes HIF-1α to facilitate viral proliferation and that viral nsp1β plays a vital role in enhancing the expression and stabilization of HIF-1α. The regulation of HIF-1α may have great therapeutic potential for the development of novel drugs against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has devastated the swine industry worldwide for over 30 years and shows no signs of slowing down. In this study, we found that PRRSV infection elevated hypoxia-inducible factor 1α (HIF-1α) expression. In addition, overexpressed HIF-1α contributed to PRRSV replication, whereas knockdown of HIF-1α reduced PRRSV growth. The PRRSV-encoded nonstructural protein 1β (nsp1β) exerted a stabilizing effect on HIF-1α through its nuclease protease and papain-like cysteine protease enzymatic domains. PRRSV nsp1β also interacted with von Hippel-Lindau tumor suppressor (pVHL) and HIF-1α, whereby nsp1β impaired the interaction between HIF-1α and pVHL. This work deepens our understanding of the molecular mechanisms involved in PRRSV infection and provides new insights for the development of HIF-1α-based anti-PRRSV therapies.

Dataset Information

Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

Publications

Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

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