Alphaviruses, such as Sindbis virus (SINV), are plus-strand RNA viruses with an enveloped virion of about 70 nm diameter. Alphavirus is a broad genus of viruses that include Sindbis virus, chikungunya virus, Eastern, Western, and Venezuelan equine encephalitis virus, Ross River virus, and Semliki Forest virus. Alphaviruses infect millions of humans and animals each year (Strauss and Strauss 1994; de Oliveira Mota et al. 2016). They are mainly transmitted through mosquito vectors, which are rapidly spreading across the globe. Chikungunya virus (CHIKV) has recently gone through a global outbreak resulting in over 2.9 million confirmed and suspected cases and almost 300 fatalities during summer 2016 (Wahid et al. 2017; Luis and De Figueiredo 2014). Alphavirus infections result in a variety of diseases and symptoms including rash, malaise, chronic arthralgia, or even deadly encephalitis (Atkins 2013; Zacks and Paessler 2010; Sun et al. 2014). Nearly 50% of individuals infected with chikungunya virus report having severe and disabling joint pain even 3 years after the initial infection (Simon et al. 2015; Schilte et al. 2013; Marimoutou et al. 2012). Despite the millions of people infected with CHIKV and chronic arthralgia, there are no specific treatments or drugs commercially available for individuals infected with Alphaviruses and it is necessary to evaluate plausible treatment options, one of which, that targets the endocannabinoid system.

The medicinal benefits of targeting the endocannabinoid system have been studied for many decades and have compelling evidence of its clinical efficacy (Park and Wu 2017; Rong et al. 2017; Makriyannis and Rapaka 1987). Cannabinoids have been used to treat a wide variety of diseases and health conditions including physical, emotional, and neurological diseases (Ryan and Sharts-Hopko 2017; Shah et al. 2017). Specifically, cannabinoids have been shown to reduce inflammation and can lower joint pain caused by arthritis or other injury (Mbvundula et al. 2004). These clinical and medical findings are becoming more well-known and accepted, but the molecular and mechanistic reasoning for many of these treatments still remains unclear. Although a dampened inflammatory response caused by cannabinoids may help the individual feel better, the question remains how the cannabinoids directly affects the underlying condition. We are seeking to understand the impact of cannabinoids directly on cells infected with an alphavirus.

Many cells in the human body have cannabinoid (CB) receptors (Pertwee 2008; Pacher et al. 2006). There are two different cannabinoid (CB) receptors that have been found on human cells: CB1 and CB2 (Tuccinardi et al. 2006; Ai and Chang 2012). Neurons, lungs, liver, and other digestive and reproductive tissues have CB1, where CB2 is primarily found on neurons and cells associated with the immune system, including the spleen, bones, and lymphocytes (Liu et al. 2017; Stempel et al. 2016). These CB receptors bind to endogenous, exogenous, or synthetic cannabinoids and trigger specific cellular responses (Tuccinardi et al. 2006). The binding and stimulation of the CB receptors seems to be the key for the clinical and physiological responses induced during cannabinoid use. CB receptor agonists, such as arachidonyl-2-chloroethanolamide (ACEA), bind to CB1 receptors and activate signaling pathways (Ai and Chang 2012; Biernacki and Skrzydlewska 2016; Ma et al. 2015). Antagonists/inverse agonists, such as AM-251, bind and block the stimulation of the receptor and subsequent cellular pathways (Shahidi et al. 2014; Chanda et al. 2011). These synthetic CB1 receptor ligands are powerful tools for studying cannabinoid-induced cellular changes.

Activation of CB1 receptors stimulates fatty acid synthesis and gluconeogenesis in human liver cells (Chanda et al. 2011; Osei-Hyiaman et al. 2005a). This elevated metabolic phenotype has been shown to play roles in obesity and in viral hepatitis (caused by Flaviviruses) (Pacher et al. 2006; Osei-Hyiaman et al. 2008; Toyoda et al. 2011; Shahidi et al. 2014; Osei-Hyiaman et al. 2005b; Agilent 2015). Viruses are completely dependent on the infected host cell for energy and metabolites to synthesize and produce new virions. Many viruses alter the host cell physiology and elevate certain metabolic pathways to enhance the virus replication rate (Sanchez and Lagunoff 2015; Fontaine et al. 2014; Heaton and Randall 2011). Alphaviruses, including Sindbis virus, have been shown to require glycolysis for an optimal replication cycle and without sufficient glucose metabolism, the virus is inhibited (Findlay and Ulaeto 2015; Silva da Costa et al. 2012). These data indicate that both CB1 receptor stimulation and virus infection result in altered cellular physiology. CB1 receptor activation increases metabolism, specifically through a gluconeogenesis pathway which could enhance Sindbis virus infection which relies on glycolytic activity (Chanda et al. 2011). Gluconeogenesis and glycolysis are both glucose metabolic pathways although essentially opposite from each other with gluconeogenesis being an anabolic pathway and glycolysis being catabolic, there could be benefits to viral replication for the synthesis of glucose. We hypothesized that CB1 receptor deactivation and Sindbis virus infection would suppress glycolytic cellular pathways that would result in non-optimal conditions for virus replication and would successfully inhibit viral infection. Previous reports have shown that cannabinoids can affect viral infection, including Kaposi sarcoma-associated herpesvirus, herpes simplex virus, human immunodeficiency virus, and hepatitis (Medveczky et al. 2004; Reiss 2010; Maor et al. 2012). In this study, we have verified that SINV infects liver cells and have investigated the impact of cannabinoid receptor stimulation on Sindbis virus (alphavirus) replication.