Innate Immune Defense Against Pathogens

Eukaryotic cells have numerous mechanisms to protect themselves from pathogens. The innate immune response is the first line of defense against these pathogens. Pattern associated molecular factors (PAMPs) are viral factors including proteins, lipids, and viral nucleic acids that are recognized by pattern recognition receptors (PRRs) within the cell. During viral infection, PAMPs accumulate in different cellular components and once recognized by the PRRs activate the intracellular signaling pathways that are involved in the innate immune response. These pathways are responsible for the expression of genes that function to prevent virus replication and spread. Chemokines, including interferon type 1 and type 3, are secreted during the innate
RIG-I is expressed at low levels in most cells but it becomes more abundant in response to inferferon. During normal physiologic conditions, RIG-I is in the resting state with the repressor domain covering the RNA binding and helicase domains. RIG-I recognizes PAMPs based on length of RNA, presence of 5’ cap, and sequence composition. RNA with a 5’ cap does not activate RIG-I signaling nor does RNA lacking exposed 5’ triphosphates (5’ppp) or 5’ diphosphates (5’pp). The ideal RIG-I PAMP is dsRNA that is less than 300 base pairs with a 5’ppp cap. Once RIG-I recognizes PAMP RNA it hydrolyzes ATP and opens the RNA binding domain for association with PAMP RNA. This also releases the caspase activation and recruitment domain (CARD) of RIG-I to signal the mitochondrial activator of antiviral signaling protein (MAVS). RIG-I oligomerizes with other RIG-I molecules and is ubiquitinated by TRIM25. At this point the entire complex binds to the translocase 14-3-3ε and translocates to the mitochondrial associated membrane (MAM). Once at the MAM, RIG-I CARD domains associate with the CARD domain of MAVS which activates TBK1 and NFkB. TBK1 phosphorylates interferon response factor 3/7 (IRF3/7) and along with NFkB translocates to the nucleus and activates numerous innate immune response genes including direct antiviral genes, pro-inflamatory genes, and interferon (Kell, 2015). Expression of interferon induces the
One way alphaviruses and VEEV in particular can avoid the innate immune system is due to the ability to avoid detection by the RIG-I pathway. One mechanism VEEV employs to avoid detection by RIG-I is through RNA capping. Capping is necessary for translation but it also helps the RNA avoid recognition by RIG-I. The VEEV non-structural protein 1 (nsP1) is responsible for capping the viral RNA. The first step in the reaction is the methylation of GTP via the methyltransferase activity of nsP1. This results in methylated GTP plus S-adenosyl-L-homocysteine (AdoHcy). The newly methylated GTP (m7GTP) forms a link with nsP1 releasing the diphosphate from m7GTP making m7Gp. The triphosphate at the 5’ end of RNA is dephosphorylated and the m7Gp is transferred to the 5’ end of viral RNA creating capped RNA (Li, 2015). This structure is essential for mRNA translation and prevents viral RNA from recognition by RIG-I and degradation by 5’ exonucleases. VEEV RNA also prevents recognition of RIG-I via secondary structure of the RNA. Normally the 2’ O position of the penultimate and antepenultimate nucleotides of eukaryotic RNA (and some