Functional investigation of grass carp reovirus nonstructural protein NS80

Abstract

Background: Grass Carp Reovirus (GCRV), a highly virulent agent of aquatic animals, has an eleven segmented dsRNA genome encased in a multilayered capsid shell, which encodes twelve proteins including seven structural proteins (VP1-VP7), and five nonstructural proteins (NS80, NS38, NS31, NS26, and NS16). It has been suggested that the protein NS80 plays an important role in the viral replication cycle that is similar to that of its homologous protein μNS in the genus of Orthoreovirus.

Results: As a step to understanding the basis of the part played by NS80 in GCRV replication and particle assembly, we used the yeast two-hybrid (Y2H) system to identify NS80 interactions with proteins NS38, VP4, and VP6 as well as NS80 and NS38 self-interactions, while no interactions appeared in the four protein pairs NS38-VP4, NS38-VP6, VP4-VP4, and VP4-VP6. Bioinformatic analyses of NS80 with its corresponding proteins were performed with all currently available homologous protein sequences in ARVs (avian reoviruses) and MRVs (mammalian reoviruses) to predict further potential functional domains of NS80 that are related to VFLS (viral factory-like structures) formation and other roles in viral replication. Two conserved regions spanning from aa (amino acid) residues of 388 to 433, and 562 to 580 were discovered in this study. The second conserved region with corresponding conserved residues Tyr565, His569, Cys571, Asn573, and Glu576 located between the two coiled-coils regions (aa ~513-550 and aa ~615-690) in carboxyl-proximal terminus were supposed to be essential to form VFLS, so that aa residues ranging from 513 to 742 of NS80 was inferred to be the smallest region that is necessary for forming VFLS. The function of the first conserved region including Ala395, Gly419, Asp421, Pro422, Leu438, and Leu443 residues is unclear, but one-third of the amino-terminal region might be species specific, dominating interactions with other viral components.

Conclusions: Our results in this study together with those from previous investigations indicate the protein NS80 might play a central role in VFLS formation and viral components recruitment in GCRV particle assembly, similar to the μNS protein in ARVs and MRVs.

Figure 1

Yeast co-transformation and Y2H phenotype assessment of NS80, NS38, VP4, and VP6 interactions. Only yeast transformants that grew on both DDO and QDO plates and formed blue colonies could represent positive interaction. '+ or -' indicates 'growth or ungrowth'. 'B or W' corresponds to 'blue or white'.

Figure 2

Phylogenetic analysis based on sequence alignment of GCRV NS80 and its homologues. NS80 is highlighted in black and bootstrap values over 50% are shown. The scale bar 0.2 means 20% amino acids sequence substitution.

Figure 3

Alignment of GCRV NS80 with its homologous proteins. (A) Blastp analysis of GCRV NS80. GSRV (golden shiner reovirus), AGCRV (American grass carp reovirus), CSRV (chum salmon reovirus), SBRV (striped bass reovirus). ' × ' indicates incompatible alignment by accident. 'Red, pink, green, blue, and black' colours represents alignment scores of '≥200, 80-200, 50-80, 40-50, and <40', respectively. (B) Prediction of GCRV NS80 coiled-coil regions by Multicoil program. The x-axis and y-axis displays amino acid position and coiled-coil probabilities, respectively.

Figure 4

Schematic representation of GCRV NS80 conserved regions and potential functional domains. (A) NS80 conserved regions and potential functional domains. '+ (-) VFLS' indicated formation (not formation) of viral factory-like structures. (B) Multiple sequence alignment of NS80 and its homologues. Conserved residues were highlighted in black. 'X₄ or X₁₂ (painted in gray)' respectively showed four or twelve amino acid residues without any similarities.