Tiny Pests Causing Huge Problems

Why do people put on bug spray when they go outside?  The obvious answer is that having itchy mosquito bites all over your arms and legs is one of the most irritating feelings ever.  However, most people forget that these tiny insects can carry extremely dangerous viruses that can cause severe illness or even death.  West Nile virus (WNV), a well-known mosquito-borne virus, is the major cause of encephalitis (inflammation of the brain), which is a disease characterized by flu-like symptoms, and in severe cases, seizures and sensory/movement issues.  Dengue virus (DENV) is another dangerous mosquito-borne virus that is a major cause of viscerotropic disease, which is a disease that is associated with yellow fever and ultimately leads to organ failure and death.  These viruses cause significant problems all over the world, and currently there are no approved vaccines to prevent or combat WNV and DENV.  In order to develop effective vaccines, we need to understand how our bodies’ immune system recognizes the viruses and provide protection.  “Innate Immune Sensing of Flaviviruses” is a recently published paper in PLoS Pathogens, in which Suthar et al. (2013) discusses recent research findings on the way that the innate immune system interacts with WNV and DENV to trigger an antiviral response.¹ Also, understanding how these viruses can evade our immune response is equally important for developing new treatments.  A group mentioned in “Innate Immune Sensing of Flaviviruses,” Aguirre et al. (2012)², investigate one of the main mechanisms that DENV uses to inhibit inducing an immune response.

           

WNV and DENV are part of the flavivirus family, and they contain a single stranded RNA genome.  In order to recognize these viruses, we encode receptor proteins called pattern recognition receptors (PRRs), which recognize and bind to structures on pathogens that are not present in normal, human cells.  These structures are called pathogen-associated molecular patterns (PAMPs).  Once the virus is recognized, antiviral immune defenses are initiated which leads to inflammation.

  

The two PRRs that recognize the viral, non-self RNA structures are called retinoic-acid inducible gene-1 (RIG-1)-like receptors (RLRs) and myeloma differentiation factor 5 (MDA5).  RIG-1 recognizes and binds to double stranded RNA with a triphosphate group on the 5’ end.  MDA5 interacts with long double stranded RNA.  Double stranded RNA does not exist in normal, host cells, and it is a common viral PAMP.  Once the virus is recognized, RIG-1 and MDA5 induce the production of type 1 interferon (IFN) and proinflammatory cytokines that will migrate to the site of infection and recruit/help produce other factors needed to eradicate the virus.  A study done by Fredericksen et al. (2008) demonstrated that RIG-1 and MDA5 function cooperatively in creating an antiviral response to WNV.³ They found that RIG-1 is activated first by WNV, and then MDA5 is activated to sustain type 1 IFN and ISG expression (initiated by RIG-1).³   It is known that the PRRs RIG-1 and MDA5 are involved in pathogen recognition and initiate the immune response, but it is still unclear what the exact PAMP RNA substrate and nucleic acid sequence that RIG-1 and MDA5 interacts with. 

Interestingly, the genomic RNA in flaviviruses contains methylated caps on the 5’ end, which would hide the 5’ triphosphate structure that RIG-1 recognizes.  The genomic RNA is also single stranded, and long double stranded RNA is the PAMP for MDA5.  This suggests that RIG-1 and MDA5 do not bind the genomic RNA, but instead bind replication intermediates produced during viral RNA synthesis.  In support of this hypothesis, Fredericksen and Gale (2006) found that IFN-3 (produced in response to RIG-1 or MDA5 virus recognition) was only activated when viral synthesis was occurring.³ However, the group found that IFN-3 activation was delayed with respect to the beginning of viral synthesis, suggesting that the virus is able to avoid activating the PRRs early in the infection.³ The virus’s RNA replication complexes are located in vesicle pockets, so newly synthesized RNA and double stranded RNA intermediates are also contained in these compartments.  Suther et al. hypothesize that the compartments could be protecting viral RNA replication intermediates from RIG-1 or MDA5.  Later in the infection, the compartments begin to fall apart exposing the viral RNA replication intermediates to RIG-1 and MDA5, which allows for initiation of the innate immune response.  If our bodies were able to immediately detect the presence of the virus, before it began to replicate, would it lessen the severity of the viruses?

Aguirre et al. (2012)² describe the mechanism that DENV uses to inhibit type 1 IFN production in their paper “DENV Inhibits Type 1 IFN Production in Infected Cells by Cleaving Human STING.”  STING is an adaptor protein that associates with RIG-1 and is necessary to RIG-1 signaling to result in type 1 IFN expression (creating an antiviral state).  It has been found that expression of the DENV protease NS2B3 inhibits type 1 IFN expression, suggesting that NS2B3 is able to block some portion of signaling cascade leading to type 1 IFN expression which facilitates virus proliferation and infection.

Aguirre et al. used bioinformatics to locate proteins with cleavage sites involved in the IFN signaling pathway in order to determine all potential targets for NS2B3 mediated cleavage.  STING adaptor protein was identified to be the most likely target of NS2B3.  Interestingly, the hypothesized cleavage site in human STING (determined by bioinformatics) is absent in mouse STING.  To investigate this, the lab members expressed human or mouse STING with functional NS2B3 (wild-type) and a mutated form (nonfunctional) and analyzed them by Western Blot.  The results showed that the cells with wild-type STING expressed full length human STING and also a smaller fragment of STING, suggesting the NS2B3 was able to cleave human STING.  However, the cells containing the mutated version of NS2B3 only expressed full length human STING.  Mouse STING was unable to be cleaved by wild-type and mutated NS2B3, which was consistent with the observation that mouse STING does not contain the same, necessary cleavage sequence as human STING.

In order to confirm that the cleavage of STING reduces type 1 IFN production, they introduced human or mouse STING proteins into cells with 3 different versions of NS2B3 (wild-type, nonfunctional, and the portion of the protein that performs protease activity, also known as proteolytic core), and luciferase genes linked to a type 1 IFN promoter.  Luciferase is a protein that performs reactions that emit light, so by linking it to an IFN promoter, visible expression of luciferase informs the lab members that IFN is being expressed.  They found that wild-type and the proteolytic core of NS2B3 prevented luciferase (and IFN) expression in cells with human STING, but did not effect luciferase expression in cells with mouse STING.  The mutated form of NS2B3 also did not effect luciferase expression.  These results indicate that NS2B3 is inhibiting IFN expression in humans by cleaving STING adaptor protein.

The past two experiments were performed in mammalian 293T cells that are typically used for experiments.  However, in our bodies, STING is naturally expressed in dendritic cells (DCs), which are immune cells that are target to DENV infection. The lab members wanted to ensure that cleavage and decreased IFN expression still occurs in cells that are susceptible to DENV infection.  They infected human DCs with DENV and another virus (SFV) expressing NS2B3, and then used qRT-PCR to determine type 1 IFN levels.  Both viruses decreased IFN expression, consistent with their previous results.  When mouse DCs were infected with both viruses, IFN expression was not affected, and additionally, IFN was produced rapidly post-infection.  This suggests that NS2B3 is also inhibiting the human immune system to rapidly respond to viral infection. 

The lab members were then curious whether mice were able to inhibit viral replication of DENV since mouse STING is not susceptible to NS2B3 cleavage.  They infected embryonic mouse cells with DENV.  They found that the cells that had wild-type STING did not allow for viral replication, but the embryonic mice cells in which STING had been knocked out (no longer present) and viral replication was able to proceed.  This suggests that intact STING also inhibits viral replication, likely by allowing proliferation of type 1 IFN.  They then expressed a mutated form of STING, in which the cleavage sequence is mutated (making it un-cleavable) in human DCs.  Inability of NS2B3 to cleave STING resulted in a decrease in viral replication.  However, when STING was knocked out in human DCs by RNA interference, viral replication increased significantly. 

The lab members also isolated different cell types from the blood, and infected each of them with DENV virus.  After 12 hours, they extracted the RNA from the cells and used qRT-PCR to determine the levels of IFN in the cells.  At this time point, they did not see significant amounts of IFN present.  This suggests that is NS2B3 acting and inhibiting IFN production in many different cell types, not just DCs. 

From these articles, it can be concluded that there are many ways that these resilient viruses can evade our innate immune system.  “DENV Inhibits Type 1 IFN Production in Infected Cells by Cleaving Human STING” describes one method that viruses can use to prevent immune responses.  It is unknown if protein cleavage is a common mechanism that other viruses use, or if it is specific to DENV.  It also raises the question of whether there are other important host proteins that are being cleaved by viral proteases.  In order to develop effective vaccines and eliminate these viruses from the population, we need to have a better understanding of the factors and mechanisms involved in WNV and DENV recognition and elimination. The many research teams mentioned in “Innate Immune Sensing of Flaviviruses” gives me hope that one day we will have a cure for these epidemics, but until then, don’t forget to wear bug spray!

1. Main article à Suthar MS, Aguirre S, Fernandez-Sesma A (2013) Innate Immune Sensing of Flaviviruses. PLoS Pathog 9(9): e1003541. doi:10.1371/journal.ppat.1003541
2. Second main article à Aguirre S, Maestre AM, Pagni S, Patel JR, Savage T, et al. (2012) DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING. PLoS Pathog 8(10): e1002934. doi:10.1371/journal.ppat.1002934
3. Fredericksen BL, Keller BC, Fornek J, Katze MG, Gale M Jr (2008) Establishment and maintenance of the innate antiviral response to West Nile virus involves both RIG-I and MDA5 signaling through IPS-1. J Virol 82: 609–616. doi: 10.1128/jvi.01305-07.
4. Fredericksen BL, Gale M Jr (2006) West Nile virus evades activation of interferon regulatory factor 3 through RIG-I-dependent and -independent pathways without antagonizing host defense signaling. J Virol 80: 2913–2923. doi: 10.1128/jvi.80.6.2913-2923.2006.