Identification of Cryptic Promoter Activity in cDNA Sequences Corresponding to PRRSV 5' Untranslated Region and Transcription Regulatory Sequences.

Jayeshbhai Chaudhari,Hiep L X Vu,The Nhu Nguyen

doi:10.3390/v14020400

Abstract

To investigate the role of PRRSV nonstructural proteins (nsps) in viral RNA replication and transcription, we generated a cDNA clone of PRRSV strain NCV1 carrying the nanoluciferase (nluc) gene under the control of the transcription regulatory sequence 6 (TRS6) designated as pNCV1-Nluc. Cells transfected with the pNCV1-Nluc DNA plasmid produced an infectious virus and high levels of luciferase activity. Interestingly, cells transfected with mutant pNCV1-Nluc constructs carrying deletions in nsp7 or nsp9 regions also exhibited luciferase activity, although no infectious virus was produced. Further investigation revealed that the cDNA sequences corresponding to the PRRSV 5′ untranslated region (UTR) and TRS, when cloned upstream of the reporter gene nluc, were able to drive the expression of the reporter genes in the transfected cells. Luciferase signals from cells transfected with a reporter plasmid carrying PRRSV 5′ UTR or TRS sequences upstream of nluc were in the range of 6- to 10-fold higher compared to cells transfected with an empty plasmid carrying nluc only. The results suggest that PRRSV 5′ UTR and TRS-B in their cDNA forms possess cryptic eukaryotic promoter activity.

Highlights

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a disease of swine characterized by reproductive failure in pregnant sows and respiratory diseases in young pigs
As a part of our effort to develop a new generation of PRRSV vaccine, we generated an infectious cDNA clone of a type-2 PRRSV strain, designated as NCV1, by cloning the full-length viral cDNA genome into a bacterial plasmid, immediately downstream of the human cytomegalovirus promoter (Figure 1a)
To facilitate real-time monitoring of viral infection, the reporter gene nluc was inserted into the pNCV1 cDNA genome as an additional open reading frames (ORFs) between ORF7 and 30 untranslated region (UTR), under the control of transcription regulatory sequence 6 (TRS6) (Figure 1a)

Summary

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a disease of swine characterized by reproductive failure in pregnant sows and respiratory diseases in young pigs. ORF1a and ORF1b are translated to produce two large polyproteins, pp1a and pp1b, respectively, which are cleaved by virally encoded proteases to generate 13–16 nonstructural proteins (nsps) [4]. The nsps are responsible for the replication and transcription of the viral RNA genome, as well as the modulation of the host immune responses (reviewed in [6]). Of these nsps, nsp is the most immunogenic protein. A single mutation within the catalytic domain of nsp abolishes its polymerase activity [12]

Objectives

Methods

Results

Discussion

Conclusion