Junín virus (JUNV), a member of the family Arenaviridae, is the etiological agent of Argentine hemorrhagic fever (AHF), a potentially deadly endemicepidemic disease affecting the population of the most fertile farming land of Argentina. Autophagy is a degradative process with a crucial antiviral role; however, several viruses subvert the pathway to their benefit. We determined the role of autophagy in JUNV-infected cells by analyzing LC3, a cytoplasmic protein (LC3-I) that becomes vesicle membrane associated (LC3-II) upon induction of autophagy. Cells overexpressing enhanced green fluorescent protein (EGFP)-LC3 and infected with JUNV showed an increased number of LC3 punctate structures, similar to those obtained after starvation or bafilomycin A1 treatment, which leads to autophagosome induction or accumulation, respectively. We also monitored the conversion of LC3-I to LC3-II, observing LC3-II levels in JUNV-infected cells similar to those observed in starved cells. Additionally, we kinetically studied the number of LC3 dots after JUNV infection and found that the virus activated the pathway as early as 2 h postinfection (p.i.), whereas the UV-inactivated virus did not induce the pathway. Cells subjected to starvation or pretreated with rapamycin, a pharmacological autophagy inductor, enhanced virus yield. Also, we assayed the replication capacity of JUNV in Atg5 knockout or Beclin 1 knockdown cells (both critical components of the autophagic pathway) and found a significant decrease in JUNV replication. Taken together, our results constitute the first study indicating that JUNV infection induces an autophagic response, which is functionally required by the virus for efficient propagation. IMPORTANCE Mammalian arenaviruses are zoonotic viruses that cause asymptomatic and persistent infections in their rodent hosts but may produce severe and lethal hemorrhagic fevers in humans. Currently, there are neither effective therapeutic options nor effective vaccines for viral hemorrhagic fevers caused by humanpathogenic arenaviruses, except the vaccine Candid no. 1 against Argentine hemorrhagic fever (AHF), licensed for human use in areas of endemicity in Argentina. Since arenaviruses remain a severe threat to global public health, more in-depth knowledge of their replication mechanisms would improve our ability to fight these viruses. Autophagy is a lysosomal degradative pathway involved in maintaining cellular homeostasis, representing powerful anti-infective machinery. We show, for the first time for a member of the family Arenaviridae, a proviral role of autophagy in JUNV infection, providing new knowledge in the field of host-virus interaction. Therefore, modulation of virus-induced autophagy could be used as a strategy to block arenavirus infections.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of non-enveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the G olgi c omplex (GC). In this work, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. Analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector G olgi-specific B FA resistance f actor 1 (GBF1), which activates the small GTPase A DP- r ibosylation f actor 1 (ARF1), is required for IBDV replication since inhibiting its activity by treatment with b re f eldin A (BFA) or G olgi c ide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative-mutant T31N over-expression hampered the IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnaviruses-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, being the lack of a transcriptionally active core the main differential feature. This structural trait, among others that resemble the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and have argued the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses. Here, we present original data showing the IBDV-induced GC reorganization and the crosstalk between IBDV and the Rab1b-GBF1-ARF1 mediated intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnaviruses-host cells and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.