Andrias davidianus Ranavirus (ADRV) Genome Replicate Efficiently by Engaging Cellular Mismatch Repair Protein MSH2

Abstract

As nucleocytoplasmic large DNA viruses, replication of ranaviruses (genus Ranavirus, family Iridoviridae) involves a series of viral and host proteins. We have described that the replication and transcription machinery of Andrias davidianus ranavirus (ADRV) which was isolated from the Chinese giant salamander contained host factors. Here, a new host factor, the MutS homolog 2 (MSH2), was proved as an important protein that participated in ADRV infection. Expression of MSH2 was stable during ADRV infection in cultured cells and it localized at the cytoplasmic viral factories and colocalized with virus nascent DNA, indicating its possible role in virus genome replication. Investigation of the viral proteins that interacted with MSH2 by co-immunoprecipitation showed that A. davidianus MSH2 can interact with ADRV-35L (possible components associated with virus transcription), ADRV-47L (virus DNA polymerase), and ADRV-98R. Further knockdown MSH2 expression by RNAi significantly reduced the late gene expression of ADRV. Additionally, MSH2 knockout by CRISPR/Cas9 significantly reduced viral titers, genome replication, and late gene transcription of ADRV. Thus, the current study proved that ADRV can engage cellular MSH2 for its efficient genome replication and late gene transcription, which provided new information for understanding the roles of host factors in ranavirus replication and transcription.

Chinese Giant Salamander Iridovirus 025L Is a Viral Essential Gene

“…Viral genomes were extracted according to the Viral Genome Extraction Kit (OMEGA). CGSIV MCP gene primers were designed to evaluate CGSIV DNA replication [ 1 ], and the pMD18T−MCP plasmid (constructed and preserved in this experiment) was used as the standard. CGSIV MCP−F and CGSIV MCP−R were used as upstream and downstream primers, and the absolute quantitative PCR was performed on an ABI 7300 fluorescence qPCR instrument using Takara SYBR ® Premix Ex Taq™ II.…”

“…Ranaviruses (family Iridoviridae, Alphairidovirinae, genus Ranavirus) are large, double−stranded DNA viruses that infect a variety of ectothermic vertebrates, such as amphibians, reptiles, and fish [ 1 , 2 , 3 , 4 ]. The Chinese giant salamander is the largest amphibian in the world and has great ecological and commercial value.…”

Siniperca chuatsi Rhabdovirus (SCRV)-Induced Key Pathways and Major Antiviral Genes in Fish Cells

“…Each qPCR mixture contained 1 μL of cDNA, 5 μL of SYBR Premix (2×), 1 μL of primers (0.5 μL for each primer), and 3 μL of ultrapure water. The qPCR conditions were conducted as described previously [15]. The β-actin gene of S. chuatsi was used as an internal control.…”

DNAJA4 Promotes the Replication of the Chinese Giant Salamander Iridovirus

“…Viral genomes were extracted using the Viral Genome Extraction Kit (omega, Norcross, USA). Using the pMD18T-MCP plasmid as the standard, CGSIV MCP gene primers were designed to evaluate CGSIV DNA replication [22]. qMCP-F/R was used as upstream and downstream primers, and the copy number of viral genomic DNA was determined by absolute quantitative PCR on an ABI 7300 PCR instrument using Takara SYBR ® Premix Ex Taq™ II.…”

“…However, host factors have not revealed the mechanism involved in infection replication. At present, some host factors, such as IRF, Uba2, and MSH2, are involved in virus replication in the studies of ranavirus [21][22][23], but the role of DNAJA4 in ranavirus infection has not been reported. In addition, only the effects of CGSIV MCP on CGSIV infection have been reported [20], and no studies have found that host factors are involved in the replication of CGSIV infection.…”