Genetic dissection of innate immunity to infection: the mouse cytomegalovirus model

https://doi.org/10.1016/j.coi.2004.11.004Get rights and content

Resistance to infection is largely inherited rather than acquired, and is encoded by a definable set of host genes designated the ‘resistome’. Logically speaking, piecemeal disruption of the resistome gives us the best chance to define it, and the most spectacular advances in understanding innate immunity have grown from spontaneous or induced germline mutations of the resistome. Mutations induced by random germline mutagenesis have now become so numerous that we are nearly in a position to define the size of the resistome, and both random and targeted mutations give us a fairly nice sketch of its components and how they interact. Our own N-ethyl-N-nitrosourea mutagenesis effort, which recently showed that components of Toll-like receptor signaling are essential constituents of the arsenal against MCMV infections, validated the forward genetic approach as a powerful tool to define the resistome.

Introduction

The innate immune system recognizes a restricted collection of molecules that are indispensable for microbial life and are shared by essentially all microbes. The molecular basis of innate immune sensing and response, which not only protects the host but paradoxically also produces many of the symptoms of infectious disease, is now partly understood, and in many instances involves the Toll-like receptors (TLRs) and their signaling pathways. But there are specialized pathways that do not depend upon TLRs 1.•, 2.. Beyond its ability to sense infection, the innate immune system is endowed with effector functions, whereby it creates conditions that are anathema to most microbes. Utilizing a restricted set of genes, far fewer in number than the number of microbes with which it must cope, the innate immune system defends the host very effectively.

A central goal of the science of innate immunity is to identify all of the sensing and effector mechanisms that comprise the innate immune response. It would be desirable to know what resistance entails from the outset. Hence, we may ask, ‘How many genes are involved in innate immunity? How do their products interact with one another? And, which products can be categorized as having sensory versus effector function?’

These questions can best be addressed using a model system in which the host must respond to the execution of a genetic program resident within the pathogen. Viruses, which have lower genomic complexity than bacteria, offer the best opportunity to achieve this. The complement of host genes required for effective containment of viral infection during the early hours following inoculation is currently sought. The tool used to identify these genes and enumerate them is random germline mutagenesis. The mutagen of choice, in the mouse, is N-ethyl-N-nitrosourea (ENU).

Section snippets

Pathogenesis of mouse cytomegalovirus infection: the agent

Mouse cytomegalovirus (MCMV) is a member of the β-herpes virus family, and as such is a relatively large double stranded (ds)DNA virus (230 kb in length). There are approximately 170 genes in the MCMV genome, and simultaneous bidirectional transcription of many of them assures that dsRNA will be produced in abundance during the course of an infection. This, in turn, is certain to alert the host via specialized proteins (including TLR3) that detect dsRNA. The virus is cytosine/guanine rich

Pathogenesis of mouse cytomegalovirus infection: the host

The immune control of MCMV infection requires elements from both innate and adaptive immune systems. Innate defenses (illustrated in Figure 1) provide early protection, whereas adaptive defenses provide delayed protection. Neither alone is sufficient to permit survival of the host.

The genomic footprint of resistance to murine cytomegalovirus

Only a handful of genes have been named in the preceding paragraphs. In all, how many genes are actually required to defend the host against MCMV? Many approaches might conceivably be used to address the question, but few are equal to the task. One might, for example, monitor the changes in gene expression that occur in diverse host tissues in the wake of infection; however, the number of genes that show altered expression might greatly exceed the number that is essential for effective

Progressive destruction of the mouse genome to find the murine cytomegalovirus resistome

Direct sequencing estimates suggest that ENU mutagenesis (performed by administering three injections of ENU at a dose of 100 mg/kg body weight to G0 male mice at weekly intervals) causes the introduction of approximately one mutation per million base pairs of haploid genomic DNA [30]. As the mouse genome is 2600 Mb in length, and about 1.3% of the mouse genome has coding function [31], this rate of mutation corresponds to an alteration of coding sequence in about 45 genes in each G1 mouse born

The nature of the genes that offer protection

Approximately 25 000 genes are annotated in the mouse genome at present. A fraction of them have conditionally lethal alleles, in the sense that, when they are altered, they create susceptibility to infection by MCMV. There are two types of these mutations. On the one hand, there are effector mutations that permit unbounded growth of the pathogen, yet make the host ineffectual in coping with it. On the other hand, some mutations affect the host ability to recognize infection, and also permit

The overlap of resistomes and the degeneracy of the innate immune system: how many genes offer resistance to all infectious agents?

So far, we have considered resistance to MCMV. But the innate immune system has inherent degeneracy. Some of the receptors utilized by the innate immune system (for example, receptors of the TLR class) are known to initiate responses to such diverse pathogens such as bacteria and viruses. In mice there are twelve TLRs. Three of these have functions that are still unknown. But there are only five adaptors serving TLR responses, and the destruction of two of these adaptors is sufficient to

Conclusions

We provide here a first approximation of the MCMV resistome size, on the basis of the number of mutations recovered after a germline mutagenesis screen encompassing more than 9000 G3 mice. The discrepancy between our estimate of resistome size and the number of genes that have been experimentally proven to be required for antiviral resistance emphasizes the need for unbiased, phenotype-driven methods to uncover those molecules that serve non-redundant functions in antiviral immunity. ENU

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by a grant from the National Institutes of Health (5U54 AI054523-02).

References (39)

  • K.A. Daniels et al.

    Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H

    J Exp Med

    (2001)
  • S.H. Lee et al.

    Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily

    Nat Genet

    (2001)
  • H. Arase et al.

    Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors

    Science

    (2002)
  • H.R. Smith et al.

    Recognition of a virus-encoded ligand by a natural killer cell activation receptor

    Proc Natl Acad Sci USA

    (2002)
  • S.H. Lee et al.

    Transgenic expression of the activating natural killer receptor Ly49H confers resistance to cytomegalovirus in genetically susceptible mice

    J Exp Med

    (2003)
  • H. Sjolin et al.

    Pivotal role of KARAP/DAP12 adaptor molecule in the natural killer cell-mediated resistance to murine cytomegalovirus infection

    J Exp Med

    (2002)
  • K. Tabeta et al.

    Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection

    Proc Natl Acad Sci USA

    (2004)
  • A. Krug et al.

    TLR9-Dependent Recognition of MCMV by IPC and DC Generates Coordinated Cytokine Responses that Activate Antiviral NK Cell Function

    Immunity

    (2004)
  • K. Hoebe et al.

    Identification of Lps2 as a key transducer of MyD88-independent TIR signalling

    Nature

    (2003)
  • Cited by (43)

    • Emerging Insights into Noncanonical Inflammasome Recognition of Microbes

      2018, Journal of Molecular Biology
      Citation Excerpt :

      In addition to the above-described canonical inflammasomes, a noncanonical inflammasome was recently discovered, which is activated in response to intracellular lipopolysaccharide (LPS) [13,14]. It was previously thought that LPS was solely recognized by the innate immune system through toll-like receptor 4 (TLR-4) in conjunction with MD2 and CD14 at the cell surface [15]. However, these proteins are not responsible for the cytosolic recognition of LPS [13,14].

    • Murine Cytomegalovirus and Other Herpesviruses

      2007, The Mouse in Biomedical Research
    • Murine Cytomegalovirus and Other Herpesviruses

      2007, The Mouse in Biomedical Research: History, Wild Mice, and Genetics: Volume 1-4, Second Edition
    • Pentraxin 3 protects from MCMV infection and reactivation through TLR sensing pathways leading to IRF3 activation

      2006, Blood
      Citation Excerpt :

      Both IL-12 and IFN-γ were produced at high levels in IFN-αβR–/– mice treated with PTX3 (Figure 5), a finding confirming the preminent role of the IL-12p70/IFN-γ axis in the protective effect of PTX3. Effective anti-MCMV immune surveillance required functional TLR2, TLR3, and TLR9 signaling through MyD88-dependent and -independent pathways.7,9-11 To assess the role of TLR and MyD88 in the efficacy of PTX3 in the acute infection, TLR- or MyD88-deficient mice were challenged with MCMV and followed for viral replication in the lung.

    View all citing articles on Scopus
    View full text