PRRSV utilizes MALT1-regulated autophagy flux to switch virus spread and reserve.

ABSTRACT

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major swine pathogen, which can survive host antiviral immunity with various mechanisms. PRRSV infection induces macroautophagy/autophagy, facilitating virus replication. MALT1, a central immune regulator, was manipulated by PRRSV to optimize viral infection at different stages of the virus cycle. In this study, the key role of MALT1 in autophagy regulation during PRRSV infection was characterized, enlightening the role of autophagy flux in favor of virus spread and persistent infection. PRRSV-induced autophagy was confirmed to facilitate virus proliferation. Furthermore, autophagic fusion was dynamically regulated during PRRSV infection. Importantly, PRRSV-induced MALT1 facilitated autophagosome-lysosome fusion and autolysosome formation, thus contributing to autophagy flux and virus proliferation. Mechanically, MALT1 regulated autophagy via mediating MTOR-ULK1 and -TFEB signaling and affecting lysosomal homeostasis. MALT1 inhibition by inhibitor Mi-2 or RNAi induced lysosomal membrane permeabilization (LMP), leading to the block of autophagic fusion. Further, MALT1 overexpression alleviated PRRSV-induced LMP via inhibiting ROS generation. In addition, blocking autophagy flux suppressed virus release significantly, indicating that MALT1-maintained complete autophagy flux during PRRSV infection favors successful virus spread and its proliferation. In contrast, autophagosome accumulation upon MALT1 inhibition promoted PRRSV reserve for future virus proliferation once the autophagy flux recovers. Taken together, for the first time, these findings elucidate that MALT1 was utilized by PRRSV to regulate host autophagy flux, to determine the fate of virus for either proliferation or reserve.Abbreviations 3-MA 3-methyladenine; BafA1 bafilomycin A1; BFP/mBFP monomeric blue fluorescent protein; CQ chloroquine; DMSO dimethyl sulfoxide; dsRNA double-stranded RNA; GFP green fluorescent protein; hpi hours post infection; IFA indirect immunofluorescence assay; LAMP1 lysosomal associated membrane protein 1; LGALS3 galectin 3; LLOMe L-leucyl-L-leucine-methyl ester; LMP lysosomal membrane permeabilization; mAb monoclonal antibody; MALT1 MALT1 paracaspase; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; MOI multiplicity of infection; MTOR mechanistic target of rapamycin kinase; NFKB/NF-κB nuclear factor kappa B; nsp nonstructural protein; ORF open reading frame; pAb polyclonal antibody; PRRSV porcine reproductive and respiratory syndrome virus; PRRSV-N PRRSV nucleocapsid protein; Rapa rapamycin; RFP red fluorescent protein; ROS reactive oxygen species; SBI SBI-0206965; siRNA small interfering RNA; SQSTM1/p62 sequestosome 1; TCID50 50% tissue culture infective dose; TFEB transcription factor EB; ULK1 unc-51 like autophagy activating kinase 1.