Mechanisms of Exosome-Autophagy Interplay in Host Response to the Cytoplasmic RNA Virus (Rift Valley Fever Virus) and Development of Promising Countermeasure Approaches

Abstract

Rift Valley fever virus (RVFV) is from the Bunyaviridae family, genus Phlebovirus, and causes Rift Valley Fever (RVF) disease for which no licensed vaccines or effective therapies are available. RVF is an important emerging zoonotic disease that poses serious threat to livestock and human life, and its causative agent, RVFV, is classified as a pathogen of highest concern (Category A) by NIAID. While RVFV is primarily transmitted by mosquitoes, human cases can also be acquired through contact with blood or organs of an infected animal. RVF disease causes abortion storms in ruminants that can reach 100%, and in serious human cases it can lead to encephalitis, hemorrhagic fever, hepatic disease, ocular disease, and mortality. Case fatality rates from RVFV infection can reach as high as 30% in non-endemic regions. RVFV is an enveloped negative sense single-stranded RNA virus with a diameter between 80-120 nm, and the RVFV genome consists of three segments that are named based on their relative sizes: Large (L), Medium (M), and Small (S). Autophagy is an intracellular pathway that allows for the degradation of cytoplasmic organelles during cellular stress, and is also an important host response mechanism during several pathogenic infections. The role of autophagy during viral infections is complex. In some cases, it slows the progression of the infection, whereas, in some other cases the virus uses the autophagy system to enhance its replication. In a previous publication, we have demonstrated that exosomes released from cells infected with RVFV (designated as EXi-RVFV) protect the cells that receive them against RVFV infection through activation of autophagy. The results showed that EXi-RVFV activate the viral RNA sensor RIG-I to cause IFN-B activation and that IFN-B in turn activates autophagy. Thus, treatment of naïve U937 monocytes with EXi-RVFV leads to a significant (2 fold) induction of autophagy as compared to untreated cells or cells treated with equivalent amounts of exosomes purified from uninfected cells (EXu). We have also deciphered the molecular mechanisms through which IFN-B activation leads to activation of autophagy in the recipient cells. Our result show that the signaling proteins AKT, mTOR, and ERK1/2 are involved in the observed autophagy activation, as well as the core autophagy proteins Beclin1 and ATG5. Furthermore, we have analyzed the type of autophagy involved, to determine whether the LC3-Associated Phagocytosis (LAP) or the canonical autophagy pathway becomes engaged. Based on both protein marker analysis and electron microscopy studies, we have demonstrated that the canonical pathway and not LAP autophagy is induced by EXi-RVFV treatment of naïve U937 cells. These results provide insights into the host response mechanisms that can be utilized towards development of novel countermeasures against RVFV in the future.