Screening bacterial effectors and human virus proteins in yeast to identify host factors driving tombusvirus RNA recombination: a role for autophagy and membrane phospholipid content.

Recombination in RNA viruses contributes to virus evolution and rapid emergence of new viral variants that helps evade host's antiviral strategies. Host factors play important but poorly characterized roles in viral RNA recombination. The authors expressed Legionella bacterium effector proteins and SARS-CoV-2 and human metapneumovirus (HMPV) proteins in yeast to test their effects on tomato bushy stunt virus (TBSV) RNA recombination. The identified 16 Legionella effectors, six SARS-CoV-2, and two HMPV proteins affecting TBSV recombination likely target shared host factors with TBSV. Among the targets of the effectors/viral proteins was the autophagy pathway. Inhibition of autophagy by expression of RavZ and LegA9 Legionella effectors reduced the production of TBSV recombinants in yeast and plants. Induction of autophagy by rapamycin, via nitrogen starvation of yeast or overexpression of ATG2 lipid transfer protein, led to enhanced viral RNA recombination. Using in vitro TBSV replicase assembly on giant unilamellar vesicles confirmed the critical role of phosphatidylethanolamine in RNA recombination. We suggest that the pro-recombination role of co-opted autophagy is to provide abundant phospholipids for viral replication organelle biogenesis. Overall, this work highlights the critical roles of membrane phospholipids and lipid context in the regulation of viral RNA recombination. We show that SARS-CoV-2 N and HMPV M2-1 proteins enhance TBSV RNA replication and recombination by protecting the viral RNAs from host Xrn1 5´-3´ exoribonuclease in yeast. Altogether, the novel strategy of using TBSV as a cellular system sensor might assist in the identification of novel functional targets of various viral and bacterial effectors in yeast.

Importance: Positive-strand (+)RNA viruses replicate in the cytosol of infected cells by exploiting cellular proteins and resources that frequently lead to diseases. Virus replication results in the generation of viral RNA recombinants that contribute to the emergence of new viral variants and adaptation to new hosts. The authors expressed Legionella bacterium effector proteins, SARS-CoV-2 and human metapneumovirus proteins in yeast to test their effects on tomato bushy stunt virus (TBSV) RNA recombination. This novel approach revealed that Legionella effectors and heterologous viral proteins target shared host factors with TBSV, including the autophagy pathway. In vitro approach revealed that the pro-recombination role of co-opted autophagy is to provide abundant phospholipids for viral replication. SARS-CoV-2 nucleocapsid protein and human metapneumovirus M2-1 protein are shown to enhance TBSV RNA replication and recombination by protecting the viral RNAs from host Xrn1 5´-3´ exoribonuclease in yeast. Thus, the TBSV/yeast system can be used as a cellular system sensor to find new functions of heterologous viral proteins.