In:
Journal of Virology, American Society for Microbiology, Vol. 95, No. 14 ( 2021-06-24)
Abstract:
Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in humans and livestock and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana . We first developed a mini-replicon system expressing an RSV genomic RNA3 enhanced green fluorescent protein (eGFP) reporter [MR3 (−)eGFP ], a nucleocapsid (NP), and a codon usage-optimized RNA-dependent RNA polymerase (RdRp opt ). Using this mini-replicon system, we determined that RSV NP and RdRp opt are indispensable for the eGFP expression from MR3 (−)eGFP . The expression of eGFP from MR3 (−)eGFP can be significantly enhanced in the presence of four viral suppressors of RNA silencing (VSRs), NSs, and P19-HcPro-γb. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana . However, we found that the RSV NS3 coding sequence acts as a cis element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that the mini-replicon system described here will allow studies of the RSV replication, transcription, cell-to-cell movement, and host machinery underpinning RSV infection in plants. IMPORTANCE Plant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into three genera: Orthotospovirus, Tenuivirus , and Emaravirus . Reverse-genetics systems have been established for members of the genera Orthotospovirus and Emaravirus . However, there is still no reverse-genetics system available for Tenuivirus . Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damage to the rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana . This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plants.
Type of Medium:
Online Resource
ISSN:
0022-538X
,
1098-5514
DOI:
10.1128/JVI.00589-21
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2021
detail.hit.zdb_id:
1495529-5
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