We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (γHV68) that is closely-related to human Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. “Reconstituting” the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.
In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine γHV68 is a natural pathogen of murine rodents. γHV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that γHV68 hijacks MAVS and IKKβ, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NFΚB activation and antiviral cytokine production. Specifically, γHV68 infection activates IKKβ and that activated IKKβ phosphorylates RelA to accelerate RelA degradation. As such, γHV68 efficiently uncouples NFΚB activation from its upstream activated IKKβ, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.
Recent studies have outlined the overall signaling cascades in mounting host innate immune responses. Residing within distinct compartments, pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs) of diverse origin to trigger innate immune signaling1. The retinoic acid-induced gene I (RIG-I) and melanoma differentiation antigen 5 (MDA5) proteins are cytosolic sensors that recognize RNA species with specific structural features2. Upon activation, RIG-I interacts with its downstream MAVS (also known as IPS-1, VISA, and CARDIF) adaptor that, in turn, activates the IKK (IKKαβγ) and IKK-related kinase (TBK1 and IKKε, also known as IKKi) complexes3-6. Activated innate immune kinases phosphorylate key regulators of gene expression, including transcription factors and inhibitors thereof, and enable transcriptional activation of host antiviral genes (e.g. IL6, TNFα, CCL5, and IFNβ). These signaling cascades constitute potent intrinsic innate immune responses that establish an anti-microbial state to restrict pathogen propagation during early stages of infection.
Murine γHV68 is closely related to human oncogenic KSHV and EBV. Thus, γHV68 infection of mice provides a tractable small animal model to examine the host immune response to gamma herpes virus infection in vivo7. Using γHV68, our lab has uncovered an intricate strategy whereby γHV68 hijacks host innate immune signaling to enable viral infection. On one hand, γHV68 activates the MAVS-IKKβ pathway to promote viral transcriptional activation via directing activated IKKβ to phosphorylate replication transactivator (RTA), the viral transcription factor key for γHV68 replication8. On the other hand, the IKKβ-mediated phosphorylation primes RelA for degradation and terminates NFΚB activation9. As such, γHV68 infection effectively avoids antiviral cytokine production. Interestingly, a screening utilizing an expression library of γHV68 identified RTA as an E3 ligase to induce RelA degradation and abrogate NFΚB activation10. These findings uncover an intricate immune evasion strategy whereby the upstream immune signaling events are harnessed by γHV68 to negate the ultimate antiviral cytokine production.
Here we describe a protocol to measure the antiviral cytokine production in mice infected with γHV68 both in vivo and ex vivo. In the protocol we further explore the "reconstituted" expression of innate immune components in the knockout embryonic fibroblasts by lentiviral transduction, which pinpoints the function of the specific innate immune molecules in regulating the antiviral cytokine production. This protocol can be easily adapted to other viruses and signaling pathways.
Ethics Statement: All animal work was performed under strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Southern California.
1. Cytokine Gene Expression by Quantitative Real-time PCR and Secretion by ELISA in γHV68-infected Mice
2. MEF Cell Infection and Cytokine Quantification by ELISA and qRT-PCR
3. Lentivirus Production, Infection, and Generation of Stable Cell Lines
Three representative figures are shown here, including cytokine production in the lung of γHV68-infected Mavs+/+ and Mavs-/- mouse, cytokine secretion and gene expression level of γHV68-infected Mavs+/+ and Mavs-/- MEFs, and cytokine mRNA levels of γHV68-infected Mavs-/- MEFs "reconstituted" with MAVS. These representative experiments utilize gene knockout mice to investigate antiviral cytokine production in vivo and explore knockout MEFs to dissect the mechanism of regulated cytokine production ex vivo. Specifically, γHV68 infection of Mavs-/- mice led to significantly higher production of CCL5 in vivo. Mavs-/- MEFs produced more antiviral cytokine than Mavs+/+ MEFs during γHV68 infection, which recapitulates the in vivo phenotype. "Reconstituted" expression of MAVS in Mavs-/- MEFs decreased CCL5 production. Collectively, these results demonstrate that MAVS is necessary for γHV68 to suppress antiviral cytokine production both in vivo and ex vivo.
Figure 1. γHV68 acute infection induced higher cytokine production in the lungs of Mavs-/- mice than that in Mavs+/+ mice. Age- and gender-matched Mavs+/+ and Mavs–/- mice were intranasally infected with 40 PFU of γHV68 and CCL5 in the infected lungs at indicated days post-infection was measured by ELISA. γHV68 infection of Mavs-/- mice led to significantly higher production of antiviral cytokine CCL5, which suggested that MAVS is necessary for γHV68 to suppress antiviral cytokine production. Data are presented as the mean ± the standard error of the mean (SEM). The statistical significance: *,p<0.05; **,p<0.02. Click here to view larger image.
Figure 2. Upon γHV68 infection, Mavs-/- MEFs produced more antiviral cytokine than Mavs+/+ MEFs. Mavs+/+ and Mavs-/- MEFs were infected with γHV68 and harvested at indicated hours post-infection (h.p.i.), CCL5 in the supernatant was assessed by ELISA (A) and gene expression in MEF cells by quantitative real-time PCR (B). The results of Mavs-/- MEFs infected with γHV68 recapitulate those of γHV68-infected Mavs-/- mice. Data are presented as the mean ± SEM of three independent experiments. The statistical significance: **,p<0.02, ***,p<0.005. Click here to view larger image.
Figure 3. "Reconstituted" expression of MAVS in Mavs-/- MEFs decreased CCL5 production. (A) Mavs-/- MEFs were infected with control or MAVS-expressing lentivirus and the expression of MAVS was analyzed by immunoblotting. (B) Mavs-/- MEFs "reconstituted" with MAVS were infected with γHV68. MEFs were harvested at the indicated hours post infection (h.p.i.) and RNA was analyzed by quantitative real-time PCR with primers specific for CCL5. Data are presented as the mean ± SEM of three independent experiments. The statistical significance: **,p<0.02. Click here to view larger image.
Viral immune evasion is one of the most dynamic and fascinating interactions interfacing viral offense and host defense9. The host innate immune components are structured such that signal transduction is effectively initiated and faithfully transmitted. Delineating the hierarchy and regulation of signaling cascades is a preeminent topic of innate immunity. Here, we introduce a protocol to identify the regulatory roles of an innate immune component, MAVS, in viral evasion of cytokine production. The protocol comprises a method determining cytokine production in virus-infected mouse and assessing cytokine gene expression and production in MEFs. The gene knockout MEFs and mouse provide tools that enable the genetic and biochemical dissection of protein function in vivo and ex vivo. Built on the genetically modified mouse strains, many of which are commercially available and number of which are increasing at an accelerating pace, this protocol further explores the "reconstituted" expression of wild-type or mutated protein of interest by lentivirus. One potential limitation of the "reconstituted" expression of innate immune components is the undesired immune signaling triggered by exogenous expression, which may impact viral infection and signal transduction thereof. Optimization to achieve low level of expression comparable to endogenous levels may reduce the side effect. In the event a mutant protein carrying targeted "lesions" is used, it will allow us to pinpoint the function of a particular domain, post-translational modification, or protein-protein interaction in immune regulation during viral infection. If "reconstituted" cells are transplanted into knockout mouse, in vivo function of selected immune components or targeted mutant can be examined.
Compared to the "knock-in" or transgenic expression of mutant proteins, "reconstituted" expression of wild-type or mutated protein of interest by lentivirus is more cost- and time-effective, permitting the investigation with diverse gain- or loss-of-function mutants. The critical step of the whole protocol is that the reconstituted protein should be comparable to endogenous protein level, especially when the protein is an enzyme or transcription factor. Over-expression of some proteins may lead to aberrant activation of the signaling pathway and ambiguous results.
Although our protocol focuses primarily on mouse fibroblasts, which is largely due to the intrinsic regulation of innate immune component on γHV68 replication, similar applications can be extended to other immune constituents, including macrophage and dendritic cells. Additionally, these approaches can be adapted to mechanistic studies entailing diverse pathogens. The application is emerging12 and will likely take more center-staged roles in the future.
The authors have nothing to disclose.
Wild-type (Mavs+/+) and knockout (Mavs−/−) mice, Wild-type (Mavs+/+) and knockout (Mavs−/−) MEFs were kindly provided by Dr. Zhijian J. Chen (University of Texas Southwestern Medical Center)13. This publication is based on work funded by grants from NIH (R01 CA134241 and R01 DE021445) and American Cancer Society (RSG-11-162-01-MPC).