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

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.

Keywords: NSP6; reverse genetics; rotavirus; viral replication.

FIG 1

Generation of the rSA11-delNSP6 virus incapable of expressing the NSP6 protein. (A) Schematic presentation of the plasmids encoding segment 11 (NSP5/6 genes), used for rescue of the wild-type (wt) rSA11 and mutant rSA11-delNSP6 viruses (pT7-NSP5SA11 and pT7-NSP5SA11-delNSP6, respectively). To generate plasmid pT7-NSP5SA11-delNSP6, the start codon and three AUG codons in the NSP6 ORF of SA11 were disrupted by replacing them with ACG codons. The arrowheads indicate the ACG mutation sites. UTR, untranslated region; aa, amino acid. (B) PAGE of viral genomic dsRNAs extracted from the native SA11, rSA11, and rSA11-delNSP6 viruses. Lane 1, dsRNAs from the native SA11; lanes 2 and 3, dsRNAs from the rescued rSA11 (lane 2) and rSA11-delNSP6 (lane 3) viruses. The numbers on the left indicate the order of the genomic dsRNA segments of the native SA11. (C) Rescued rSA11 and rSA11-delNSP6 contain a signature mutation in their VP4 genes as a gene marker. Nucleotide substitutions (T to C and A to C at nucleotide positions 1365 and 1368, respectively) were introduced to eliminate a unique PstI site in the VP4 gene (15). (D) The VP4 gene RT-PCR products were digested with PstI, followed by separation in a 1.2% agarose gel. Native SA11 (lanes 1 and 2), rSA11 (lanes 3 and 4), and rSA11-delNSP6 (lanes 5 and 6) are shown. The 2,362-bp fragments (lanes 1, 3, and 5) were digested with PstI (lanes 2, 4, and 6). M, 1-kb ladder markers. (E) Expression of the NSP6 protein and other RVA proteins in MA104 cells infected with rSA11 or rSA11-delNSP6. Whole-cell lysates of infected cells were analyzed by immunoblotting using anti-NSP6 antiserum, anti-NSP5 antiserum, anti-VP6 antiserum, or anti-β-actin monoclonal antibody. The anti-β-actin antibody was used as a loading control. Mock (lane 1), rSA11 (lane 2), and rSA11-delNSP6 (lane 3) virus infection is shown.

FIG 2

Infectivity of the rSA11-delNSP6 virus lacking NSP6 expression. (A) Single-step growth curves of rSA11 and rSA11-delNSP6. MA104 cells were infected with rSA11 or rSA11-delNSP6 at an MOI of 5 and then incubated for various times. Virus titers in the cultures were determined by plaque assay. The data shown are the mean viral titers and standard deviations (SDs) from three independent cell cultures. (B) Plaque formation by rSA11 and rSA11-delNSP6. rSA11 or rSA11-delNSP6 was directly plated onto CV-1 cells for plaque formation. The experiment was repeated three times with similar results, and representative results are shown.

FIG 3

Generation of recombinant SA11-based monoreassortant viruses having a KU-derived segment 11 capable or incapable of NSP6 expression. (A) Schematic presentation of the plasmids encoding KU segment 11 (NSP5/6 genes) for rescue of the wild-type rSA11-s11KU and mutant rSA11-s11KU-delNSP6 viruses (pT7/NSP5KU and pT7/NSP5KU-delNSP6, respectively). To generate plasmid pT7/NSP5KU-delNSP6, the start codon and five AUG codons in the NSP6 ORF of KU were disrupted by replacing them with ACG codons. The arrowheads indicate the ACG mutation sites. (B) PAGE of viral dsRNAs extracted from rSA11, rSA11-s11KU, rSA11-s11KU-delNSP6, and native KU. Lane 1, dsRNAs from rSA11; lanes 2 and 3, rSA11-s11KU (lane 2) and rSA11-s11KU-delNSP6 (lane 3); lane 4, native KU. The numbers on the left indicate the order of the genomic dsRNA segments of rSA11. (C) Expression of the NSP6 protein and other RVA proteins in MA104 cells infected with rSA11-s11KU or rSA11-s11KU-delNSP6. Whole-cell lysates of infected cells were analyzed by immunoblotting using anti-NSP6 antiserum, anti-NSP5 antiserum, anti-VP6 antiserum, or anti-β-actin monoclonal antibody. Shown are mock (lane 1), rSA11-s11KU (lane 2), and rSA11-s11KU-delNSP6 (lane 3) infection.

FIG 4

Infectivity of the rSA11-s11KU-delNSP6 monoreassortant virus lacking NSP6 expression. (A) Single-step growth curves of rSA11-s11KU and rSA11-s11KU-delNSP6. MA104 cells were infected with rSA11-s11KU or rSA11-s11KU-delNSP6 at an MOI of 5 and then incubated for various times. Virus titers in the cultures were determined by plaque assay. The data shown are the mean viral titers and SDs for three independent cell cultures. (B) Plaque formation by rSA11-s11KU and rSA11-s11KU-delNSP6. rSA11-s11KU or rSA11-s11KU-delNSP6 was directly plated onto CV-1 cells for plaque formation. The experiment was repeated three times with similar results, and representative results are shown.

FIG 5

Single-step growth curves of the NSP6-deficient viruses in IFN-α/β-deficient Vero E6 cells. Vero E6 cells were infected with rSA11 or rSA11-delNSP6 (A) or with rSA11-s11KU or rSA11-s11KU-delNSP6 (B) at an MOI of 5 and then incubated for various times. The virus titers in the cultures were determined by plaque assay. The data shown are the mean viral titers and SDs from three independent cell cultures.