Arboviruses represent a group of emerging pathogens of both medical and agricultural importance for which there are few therapies. RVFV is a particularly important member of this group that causes disease both in humans and livestock, and is considered a Category A pathogen due to its high pathogenesis and potential for geographical spread. Here, we identified AMPK as a novel antiviral factor that restricts RVFV infection independent of the type I IFN system. This restriction is dependent on the canonical upstream activator LKB1. Furthermore, we found that AMPK is activated by RVFV infection, and this activation restricts infection at the level of RNA replication likely by reducing fatty acid biosynthesis, an essential process in RVFV infection. We extended these studies by demonstrating that additional arboviruses, known to require lipid biosynthesis, were also restricted by this pathway. Since treatment with drugs that activate AMPK restricted infection, this could represent a novel therapeutic strategy toward the control of many RNA viruses.
AMPK is a central regulator of cellular energy that regulates a number of cellular pathways that could influence viral replication, including protein and lipid biosynthesis 
. AMPK activation inhibits protein translation through two major downstream pathways. First, AMPK activation inhibits translation initiation by inhibiting mTORC1 activity. Second, AMPK inhibits translation elongation through inactivation of eEF2. We explored these two targets as potentially regulating RVFV infection. However, we found RVFV was insensitive to treatment with the mTORC1 inhibitor, Rapamycin, regardless of AMPK status. Furthermore, eEF2 phosphorylation induced by drugs that alter the energy status of the cell was not affected in the absence of AMPK, indicating additional upstream regulators are contributing to eEF2 activity. Therefore, we explored lipid biosynthesis as a potential target for AMPK-dependent anti-viral activity.
AMPK controls fatty acid metabolism through ACC, and may be the only physiologically relevant kinase that controls ACC activity 
. This is consistent with our findings that ACC phosphorylation was exquisitely dependent on AMPK, in contrast to eEF2, which was phosphorylated during energy depletion even in the absence of AMPK. ACC is the enzyme responsible for the conversion of acetyl-CoA to malonyl-CoA 
. Malonyl-CoA production impacts lipid metabolism in at least three ways. Malonyl-CoA is a substrate driving de novo
palmitate production, and is also important in converting simple essential fatty acids into more complex polyunsaturated fatty acids that can be used to build triglycerides and other cellular lipids 
. Finally, malonyl-CoA inhibits transport of fatty acids to the mitochondria, thus inhibiting fatty acid oxidation 
. In addition to its role in fatty acid metabolism, AMPK is also an important regulator of HMG-CoA reductase (HMGCR), the rate limiting enzyme in the synthesis of isoprenoids and sterols, including cholesterol. Cholesterol is known to contribute to infection of multiple viruses, and therefore could also be targeted in AMPK-mediated virus restriction.
Since we found that fatty acid biosynthesis was required for RVFV infection, and changes to AMPK expression and activation status led to global changes in cellular lipid levels, we hypothesized that inhibiting fatty acid synthesis downstream of ACC was responsible for AMPK-mediated restriction of RVFV. This was supported by our finding that we could bypass the requirement for malonyl-CoA production by introducing exogenous palmitate. Since the addition of palmitate rescued RVFV overcoming the restriction mediated by AMPK activation (), the ability of AMPK to inhibit fatty acid biosynthesis is likely the most important determinant of AMPK-mediated RVFV restriction. Palmitate is a substrate for the biosynthesis of a number of lipid moieties that could contribute to RVFV infection. Palmitate undergoes chain elongation and additional modifications in the ER to produce saturated fatty acids as well as triglycerides, phospholipids, and cholesterol esters 
. It is also a substrate for sphingolipid biosynthesis in the Golgi. Sphingolipids become incorporated into cellular membranes and participate in signaling events that could contribute to RVFV infection. Finally, palmitate addition is a form of post-translational modification of some proteins 
There are several stages during the course of RVFV infection where cellular lipids are utilized. Many RNA viruses induce the formation of novel membranous structures derived from various organelles within the cell to support the viral replication complex 
. Notably, formation of these structures is often dependent on de novo
fatty acid synthesis 
. While RVFV-induced membrane alterations have not been well characterized, a related Bunyavirus, Bunyamwera virus, was reported to induce Golgi-derived tubular structures with globular heads in association with the viral replication complex, suggesting that other Bunyaviruses could likewise induce membrane changes 
. In addition to RNA replication, enveloped viruses bud from cellular membranes, thereby incorporating those lipids into the viral particle 
. RVFV assembly occurs on Golgi membranes, with virus particles ultimately budding into the Golgi for transport and release at the plasma membrane 
. Cellular lipids derived from de novo
palmitate production downstream of ACC could contribute to each of these steps, although our findings that viral RNA synthesis is inhibited by AMPK suggests that RNA replication is a key target.
In addition to RVFV, we found that three additional viruses including the Togavirus SINV, the Flavivirus KUNV, and the Rhabdovirus VSV are restricted by AMPK and LKB1 (). Importantly, this group includes members of the three major families of arboviruses that contribute to human disease. Members of the Togavirus family including Semliki Forest virus and Rubella virus have been described to induce characteristic modified endosomal and lysosomal structures termed cytopathic vacuoles that support the viral replication complex 
. Furthermore, a number of Flaviviruses have been shown to have important lipid dependencies. KUNV, a strain of West Nile virus, has been described as forming two distinct membrane structures that include double membrane spherical vesicles that are the sites of viral replication, as well as arrays of convoluted membranes that are the sites of viral polyprotein processing 
. Moreover, both fatty acid synthesis and oxidation have been shown to be essential for another Flavivirus, Dengue virus (DENV). Infection is characterized by virally-induced increases in cellular fatty acid synthesis and a redistribution of the enzyme fatty acid synthase to sites of DENV replication 
. Free fatty acids are also derived through autophagosomal processing of triglycerides, and exogenous addition of the fatty acid oleate was able to rescue DENV infection when autophagy is inhibited 
. Furthermore, induction of ER-derived lipid droplet formation is necessary for DENV particle formation 
. Therefore DENV and perhaps many other viruses require complex and unique interactions with cellular lipid metabolism through both synthesis and degradation pathways. In addition, Hepatitis C Virus (HCV), a distantly related Flavivirus, induces formation of a membranous web derived from intracellular vesicles, whose formation requires fatty acid synthesis for replication 
. Interestingly, AMPK has been implicated to play a role in HCV infections. AMPK-activating drugs inhibited the replication of HCV replicons concomitant with a decrease in cellular lipid levels, while knock down of the upstream activator LKB1 led to increased replication, 
, consistent with our findings with RVFV, KUNV, SINV, and VSV. Importantly, KUNV could be partially rescued from AMPK-mediated restriction by the addition of the fatty acid palmitate. Thus, AMPK may restrict multiple families of viruses through this mechanism. Since all positive strand RNA viruses are thought to induce membrane modifications for viral RNA replication, and include a large number of medically significant groups (e.g., Picornaviruses, and Coronaviruses) 
, it will be important to determine the full scope of viruses restricted by AMPK as well as the mechanism of restriction.
Since many disparate viruses are restricted by AMPK, it is interesting to speculate how AMPK could be activated in response to these viral infections. We have found that both live virus and UV-inactivated replication incompetent RVFV is capable of activating AMPK via LKB1. This suggests that the energy sensing pathway is responsible for this activation yet we were unable to detect global changes in cellular energy levels during the period in infection when AMPK becomes phosphorylated. Thus, we hypothesize that RVFV infection induces a localized drop in cellular energy to activate AMPK. Since this is independent of viral replication and can restrict a large panel of disparate viruses that have the commonality of entering cells via endocytic routes and fusing within these compartments, we postulate that a local energy drop may occur during these steps. Since endocytosis is a highly energetic process usurped by many viruses, it is possible that increased levels could themselves could provide the trigger for this rapidly inducible antiviral response. We have previously reported that receptor-mediated endocytosis, employed by many viruses including KUNV, SINV and VSV for entry is intact in AMPK deficient cells 
. Therefore at least some routes of endocytic entry used by viruses are unaffected by AMPK, and may provide a trigger for activation rather than a point of restriction. This would allow broad activation of AMPK by many types of viruses internalized by such routes and provide a rapid response to restrict virus infection by inhibiting fatty acid synthesis.
Since AMPK activators are currently in the clinic to treat metabolic disorders such as type II diabetes 
, and restrict RVFV and KUNV replication in cell culture, they may prove to be useful antiviral therapeutics. Several AMPK activating drugs have been shown to reduce morbidity and mortality during lethal influenza infection in mice 
. In addition, treatment of AMPK-activating drugs inhibited infection of HCMV and HIV in cells, and the addition of AMPK-activating drugs such as Metformin to current HCV treatment regimens had promising, albeit modest, effects on reducing patient viral loads 
. Infections with HCMV, HIV, and HCV have also been shown to inhibit AMPK activity 
. AMPK may have multiple effects on these infections since different downstream mechanisms have been implicated 
; however, this suggests the possibility that some viruses have developed mechanisms of immune evasion that target AMPK. Taken together, AMPK plays a broad role in cellular innate immunity through potent inhibition of fatty acid synthesis, which is broadly utilized by viruses, suggesting that AMPK and perhaps other modulators of lipid biosynthesis are potential targets for broad pan-antiviral therapeutics.