Recent Advances in Studies of Host Immune Responses to RV

Initial Virus Recognition of RV Infection

Germline encoded pattern recognition receptors (PRRs) are the first active line of defense against pathogens through the detection of pathogen-associated molecular patterns (PAMPs)94 There are many PRRs that serve to recognize non-eukaryotic and non-self domains, and most notably in the defense against HRV, the recognition of dsRNA is by toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs). With TLRs, RLRs, and NLRs, epithelial cells and immune cells are capable of identifying initial infections and trigger antiviral pathways of the immune system.95

Toll-Like Receptors

Members of the TLR family are the major PRRs in cells. They are type I transmembrane proteins containing leucine-rich repeats that recognize viral and bacterial PAMPs in the extracellular environment (TLR1, TLR2, TLR4, TLR5, TLR6, and TLRll) or endolysosomes (TLR3, TLR7, TLR8, TLR9, and TLR10).96 Multiple TLRs on mononuclear cells are upregulated after RV infection in humans and Gn pigs, including TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9.52-97 The presence of RV dsRNA is primarily detected by the intra-endosomal TLR3.6'-98 TLR7 signaling was found to activate dendritic and В cells after exposure to RV.99 TLR2 has also been found to be activated by RV NSP4 in the extracellular space.63

A study in mice showed that TLR3-mediated production of type III IFN helps limit RV infectivity in adults; there is a strong inverse correlation between TLR3 activity and viral susceptibility in adults but not in neonates.61 This study went on to test healthy human adult and child duodenal biopsies, finding that adults also had an increased TLR3 mRNA expression.61 A higher level of TLR3 in adults relates to the viral epidemiology where adults are significantly less susceptible to HRV infection. However, adult mice remain susceptible to RV infection, indicating that TLR3-mediated signaling is not sufficient to protect against this pathogen. Through the use of RIG-I, MDA5, TLR3, and MAVS knockout mice and siRNA in IECs, it was shown that both RIG-I and MDA-5 were needed for IFN-(3 induction, while the knock-down of TLRs in the infected cells and animals did not affect the outcome of the infection.59100

Downstream from TLR stimulation along the innate immune response signalling pathway is the adaptor myeloid differentiation primary response 88 (MyD88), which mediates signaling for all TLRs, except TLR3, and receptors for inflammasome cytokines IL-1 and IL-18.101102 The role of MyD88 signaling in the initial control of murine RV (EC strain) infection and the induction of RV-specific Ab responses were studied in MyD88 knockout mice.83 MyD88 knockout mice shed significantly higher virus titers in feces at 2-4days post-inoculation and had significantly higher copy numbers of the viral genome in the small intestine, colon, and peripheral blood than wild type mice. The loss of MyD88 also impaired humoral immunity to RV and shifted the IgG subtypes. These results indicate that MyD88-mediated TLR signaling contributes to the control of primary RV infection and the development of a properly polarized adaptive immunity to RV.83

RIG-1 Like Receptors

Two RLRs have been identified with the ability to detect dsRNA and are activated in the presence of replicating RVs. RIG-I is capable of detecting short- to medium-length dsRNA (from 21b to ~ 1 kb), while melanoma differentiation associated protein 5 (MDA5) recognizes dsRNA of lengths >lkb.59103 Both of these proteins are able to activate very similar downstream cascades. Once activated, the RLRs will form a complex with the mitochondrial bound protein MAVS (mitochondrial-associated anti-viral signaling).104 Alternatively, a third RLR can be activated, LGP2 (laboratory of genetics and physiology 2), which has been identified as a negative regulator of RIG-1/MDA5 in response to RV.59

Studies of the interactions between RV and the RLR pathway showed that RV modulates the pathway to decrease the production of IFN-(3.74 IECs infected with various strains of RV showed that the levels of MAVS proteins in the cells decreased as the levels of NSP3 and NSP1 increased throughout the progression of RV infection.74 The addition of NSP1 to IECs drastically reduced cellular MAVS levels in a strain- independent manner. Further work on cell culture indicated that NSP1 is able to inhibit MAVS-mediated production of IFN-(3 in response to RV infection.74

Not only has it been shown that NSP1 can reduce the secretion of type I IFNs through the inhibition of MAVS in the cells, but it has also been shown to degrade RIG-1 in a proteasome-independent manner. This was determined through a multi-step analysis of the interactions of the two proteins. Western blots were used to show that the individual immunoprecipitation of NSP1 or RIG-1 from treated 293FT cells led to the coprecipitation of both proteins. When a truncated form of NSP1 that contained only the N-terminal domain was transfected into cells and translated, it was also sufficient to interact with RIG-1; this elucidated the specific domain of NSP1 that has evolved to interfere with the RLR. Moreover, through the use of a proteasome inhibitor, it was further established that NSP1 can directly degrade the protein even without the assistance of proteasomes.75 These findings illustrate the evolution of RV with the innate immune system. It has been elucidated that RLRs are needed within the infected cells to initiate cytokine production, but for the virus’ survival a non-structural protein, NSP1, has developed the ability to lessen the power of innate signaling throughout the course of infection.

NOD-Like Receptors

Although it was originally suspected in previous decades that NLRs played little to no role in the control of RV infection, recent studies have been able to uncover correlations and molecular interactions between specific NLRs and the RV genomic material. For other viral infections, it has been established in previous reviews that the pyrin and caspase domain containing NLR (NLRP and NLRC, respectively) subfamilies are able to respond to viral infection and activate programmed cell death pathways.95105 Using CRISPR-Cas9

technology, knockout mice were generated with diminished different sections of the NLR pathways, deducing the importance of the specific activation of Nlrp9b on IECs in the host defense against RV.106 Nlrp9b deficiency results in increased susceptibility to RV infection. Increased RV susceptibility was also observed by eliminating Asc and Caspl, two proteins that are required for the formation of infiammasomes. Furthermore, upon establishing the NLR pathway activated by RV, its role in pathogenesis was analyzed. Using knockout mice and a mouse intestinal organoid model, Zhu et al. found that by the activation of Gsdmd by the Nlrp9b pathway pyroptosis, an inflammatory programmed cell death is activated to induce premature death of infected IECs, maintaining intestinal homeostasis, as shown through TUNEL staining of organoids that had been infected with RV in vitro.106 The studies highlight an important anti-viral innate immune signaling pathway, the novel Nlrp9b NLR specifically expressed by IECs, in the host defense against RV infection.

Antiviral Cytokines and Their Associated Transcription Factors

In early innate responses of host cells to a viral infection, after the initiation of PRR cascades, antiviral genes are upregulated leading to the production and release of type I and III IFNs and pro-inflammatory cytokines through transcription factors such as NF-кВ and IRF3 and 7. RVs have evolved multiple strategies to modulate IFN responses in infected cells107, including the strategy to limit the expression of type I IFNs. NSP1 has also been known to bind to IRF3, 5, and 7 and mediate their degradation by protea- somes.108109 Figure 8.1 (adapted from the review by Arnold et al., PLoS Pathog 2013, with permission110) summarizes the interactions of RV with innate signaling pathways.

Many RV strains are able to inhibit the nuclear accumulation of NF-кВ and the transcription factors STAT1 and STAT2.76 RVs utilize a novel method for preventing the translocation of the STAT proteins.1" Studies have found that the inhibition occurred after the STAT proteins were activated and bound to nuclear import proteins, without causing dysregulation to the import proteins or nuclear pores given that other transcription factors were unaffected. Although the viral mediator of this effect has not been identified, it would seem likely to be NSP1.1"

To better understand the role of cytokines in RV infection, many animal studies have been conducted to evaluate the role of IFNs in controlling RV infections in vivo. While one study showed that the administration of INF-a was able to decrease the severity of RV infection in newborn calves, other studies found that knockout infant and adult mice without IFN-ot/p receptors did not see a significant change in illness or infection.79-81

Type III IFNs have been found in recent years to play a substantial role in controlling enteric virus infection in intestinal epithelial cells. Engaging type III IFN receptors activates redundant pathways to type I; however it was also shown that mice without INF-7. receptors suffered from a much more severe infection than mice without type I (IFN-a/p) receptors.79-80 One explanation for this differential effect is found in the fact that type I IFN receptors are nearly ubiquitous in nucleated cells, but type III IFN receptors are largely confined to epithelial cells.112 Beyond these findings, it was also shown that there was an important synergistic effect generated by the combined production of INF-7, and IL-22. Both cytokines have receptors that are preferentially expressed by intestinal epithelial cells allowing for this effect to be critical for RV control."3 Further studies are required to determine the exact nature of IFN-7. action against RV and whether IFN-7. is important for limiting RV infection in humans and other animals.

Though there are studies that suggest that type I and type III IFN play a critical role in RV infection clearance, it was later found that replication of the virus in intestinal epithelial cells was independent of either group IFN’s function and an adaptive response was required to clear the infection."4 What was found though is that these IFNs are able to control the rate of replication and severity of illness until an adaptive response can be generated, while type I IFNs were able to prevent the extra-intestinal spread of RV."4 Altogether, these studies on IFNs offer an explanation to the early control of RV.

Rotavirus interactions with innate signaling pathways

FIGURE 8.1 Rotavirus interactions with innate signaling pathways. Viral nucleic acids may be recognized in a host cell by membrane-bound TLR3 or cytoplasmic RLRs. When activated by nucleic-acid binding, RLRs recruit and activate the signaling adaptor molecule IPS-1 (IFN-p promoter stimulator 1, also known as MAVS), which recruits a signaling complex that activates latent cytoplasmic transcription factors such as IRF3 and NF-kB. TLR3 activation stimulates the recruitment of the adaptor TRIF, which acts as a platform for a variety of signaling molecules that also phosphorylate IRF3 or NF-kB. When signaled, the C-terminus of IRF3 is phosphorylated, causing a conformational change that leads to dimerization and nuclear translocation. NF-kB is held inactive by the inhibitor of NF-kB (IkB). NF-kB subsequently translocates to the nucleus. IRF3 and NF-kB bind to the IFN-p promoter in a cooperative manner with c-Jun/ATF-2 forming an enhanceosome complex initiating the transcription of IFN-p mRNA. Additional transcription factors, including IRF7, are induced by IFN-p and can also bind to the IFN-p promoter to enhance the transcription of IFN-p and IFN-a genes. RV can antagonize innate signaling pathways through several avenues, the primary one is the NSP1-induced degradation of IRF3 and IRF7. Some NSP1 proteins are also known to induce the degradation of p-TrCP. RV NSP3 can also impede antiviral responses by suppressing the translation of host mRNAs generated from IFN-stimulated genes. By sequestering viral RNAs within viroplasms, the virus can prevent their recognition by protein kinase R (PKR), RIG-I, MDA-5, and other sensors that upregulate antiviral responses. (Adapted from Arnold MM. et al. PLoS Pathog 2013; 9:e1003064, with permission from authors and publisher [110].)

 
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