Biochemical Characterization Of Cytoplasmic Polyhedrosis Virus Protein P44

Cypovirus (cytoplasmic polyhedrosis virus, CPV), which contains10genomic dsRNA segments, is one of the fifteen genera within the family Reoviridae, and is further classified into16types (cypovirus1-16) based on the electrophoretic migration profiles of their genome segments. CPV is the simplest virus of the family Reoviridae since it only has a single-layered capsid, and this characteristic makes cypovirus an ideal and simplified model for studying the mRNA/dsRNA synthesis and mRNA capping mechanisms of reoviruses and probably other dsRNA viruses. For this reason, extensive molecular and structural studies have been focused on CPVs in these years.For RNA viruses, viral RNA molecules (vRNAs) require proper secondary and tertiary structures or folding to form diverse cis-acting elements for their functions such as translation, replication, and encapsidation. For reoviruses, the interactions between5’-and3’-termini of vRNAs form a panhandle-like structure. But the panhandle should be disrupted when the RNA replication proceeds on the cyclized template.The formation and dissociation of RNA tertiary structures must be highly regulated for proper RNA functions in different processes. However, correct folding or unfolding of RNAs is always challenging, since RNAs can be easily trapped in incorrect intermediate structures that are locally stable in thermodynamics. In response, cells or viruses encode various RNA remodeling proteins, generally including ATP-dependent RNA helicases and ATP-independent RNA chaperones, to help overcome the thermodynamic barriers of kinetically trapped RNA molecules. For the viruses with vRNA5’-3’cyclization or panhandle, the nucleocapsid protein (N) of hantavirus and the core (capsid) protein of flavivirus are RNA chaperones, and hantavirus N has been found to be able to unwind the5’-3’panhandle structure in vitro. However, as the components of vRNA replication machinery, no capsid proteins of CPVs as well as other reoviruses has been found to be an RNA chaperone. The type5Helicoverpa armigera cypovirus (HaCPV-5) was initially isolated by our laboratory in2006from a mixture of HaCPVs, and can quickly cause lethal disease in Helicoverpa armigera that is one of the most serious agricultural pests in China. Thus far, the RNA genome of HaCPV-5has been completely sequenced. HaCPV-5RNA segment8contains a single open reading frame (ORF) that encodes a44-kDa protein, and further sequence analysis reveals that this44-kDa protein is the VP5capsid protein. In this study, we expressed HaCPV-5P44in a eukaryotic expression system and determined that this cypovirus P44contains the activities to hydrolyze NTP and bind to RNA processes. What’s more, P44also contains an RNA chaperone-like activity to ATP-independently destabilize RNA helix and accelerate strand annealing. Our further characterization of P44revealed that its helix destabilizing activity is RNA specific, lacks directionality, and could be inhibited by divalent metallic ions, such as Mg2+, Mn2+, Ca2+or Zn2+, to different extents. Moreover, we found that HaCPV-5P44could facilitate the transcription initiation of an alternative polymerase (reverse transcriptase) through a cypoviral panhandle-structured RNA template, suggesting the direct role of the RNA chaperone activity of P44in the initiation of cypoviral dsRNA replication.