Mutagenesis analysis of porcine reproductive and respiratory syndrome virus nonstructural protein 7

Abstract

Nonstructural protein 7 (nsp7), which is flanked by nsp6 and nsp8, is one of the most conserved nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein (nsp)-specific antibodies are produced in high titers in response to virus replication, especially against nsp1a, nsp1b, nsp2, and nsp7. However, many regional aspects of nsp7 are still veiled, such as its impact on viral replication and virulence or the immunological mechanism between virus and host. Based on the structure of the predicted nsp7 domain, we have constructed a series of large mutations and deletions. We ultimately demonstrated all mutations (nsp7, nsp7α/nspβ) and the majority of substitutions of nsp7 affected the PRRSV replicative cycle in some ways and were fatal for viral recovery, which indicates that these are significant to structure or function of the nsp7. What's more, the mutant vOKXH-nsp7 (F40A) indeed caused some of the variation compared with the parental virus vOKXH-GD, which shortens the amount of time needed to reach its highest viral titer, and decreases the concentration of the highest viral titer, obstructing viral mRNA and protein synthesis. Consequently, these valuable results possibly provide the first direct evidence that the nsp7 is really a critical protein domain for the RNA synthesis and the translation of viral protein of PRRSV.

Origin, Genetic Diversity, and Evolutionary Dynamics of Novel Porcine Circovirus 3

“…Predicting the Structure of PCV3a and PCV3b : The tertiary structure models of PCV3a and PCV3b ORF2 protein were built using the I‐TASSER online tool60 (https://zhanglab.ccmb.med.umich.edu) based on the structure of PCV2. PCV3a (Strain: KX778720 PCV3‐US/MO2015) and PCV3b (Strain:MF589105 PCV3/CN/Guangdong‐SG1/2016) sequences were used for the comparative analysis of epitopes and viral structure.…”

Characterization and utility of phages bearing peptides with affinity to porcine reproductive and respiratory syndrome virus nsp7 protein

“…A previous study demonstrated that nsp7 reacted strongly with pig immune sera, and that specific antibodies were detected as early as 14 days pi and sustained up to 202 days pi (Brown et al, 2009). We recently found that nsp7 was an important protein for viral RNA synthesis and protein translation (Zhang et al, 2013). These results suggest that nsp7 may have important diagnostic and antiviral roles.…”

“…However, among these three proteins, nsp7 was considered to be the most appropriate for diagnostic development for several reasons: it can be expressed as a soluble recombinant protein in bacterial culture, the region coded by nsp7 is more homologous among different strains within a genotype, and its antibody response could last up to 202 days post-infection (pi). In addition, we previously demonstrated that nsp7 was a critical protein domain for viral RNA synthesis and translation in PRRSV (Zhang et al, 2013). These results suggest that PRRSV nsp7 may be a potential target for diagnostic and antiviral development.…”

“…PRRSV nsp7 is the largest subunit in the nsp5-nsp8 region, and is flanked by nsp6 and nsp8. Studies of equine arteritis virus suggest that nsp7 is cleaved internally by the nsp4 protease to generate two small subunits, nsp7␣ and nsp7␤ (Shi et al, 2011;Zhang et al, 2013). A previous study demonstrated that nsp7 reacted strongly with pig immune sera, and that specific antibodies were detected as early as 14 days pi and sustained up to 202 days pi (Brown et al, 2009).…”

“…PRRSV XH-GD (GenBank accession no. EU624117) and the infectious PRRSV cDNA clone pOKXH-GD (Zhang et al, 2013) were stored in our laboratory.…”