NUCLEIC ACID–INDEPENDENT VIRAL RECOGNITION
Recognition of Viral Structural Proteins
In addition to viral nucleic acids, other signatures of viruses or viral infection processes are detected by the host cells to trigger innate defenses. Of these defenses, stimulation of PRRs by viral structural proteins has been the target of intense research (29).
Surface TLRs (Figure 1), including TLR2 and TLR4, are stimulated mainly through surface glycoproteins of a variety of viruses. TLR2 is stimulated by a variety of viruses including HCMV (19), MCMV (95), HSV-1 and HSV-2 (53, 86), HCV (17), LCMV (108), measles virus (13), VZV (101), and VV (110). TLR4 activation is triggered by respiratory syncytial virus (54) and mouse mammary tumor virus (MMTV) (45). Macrophages and DCs that express TLR2 and TLR4 are stimulated by these respective viruses and secrete a variety of cytokines.
However, inflammatory monocytes appear to be the predominant producer of type I IFNs upon recognition of viruses such as VV and MCMV by TLR2 (8). Interestingly, monocyte recognition of viruses by TLR2 requires endocytosis, whereas recognition of bacterial TLR2 ligands does not. In contrast to the usage of TLRs to induce the antiviral state by the host, certain viruses rely upon TLR signaling as a survival mechanism (13, 45).
MMTV persists indefinitely in wildtype mice but is rapidly cleared by the cytotoxic T cell response in mice deficient in TLR4 (45). MMTV stimulates interleukin (IL)-10 production by B cells through DC and macrophage activation mediated by TLR4 signaling. IL-10, which is an immunosuppressive cytokine, protects the MMTV-infected B cells from removal by cytotoxic T cells. Another example in which TLR-virus interaction benefits the virus is the measles virus.
The hemagglutinin (HA) protein of measles virus induces cytokine secretion in a TLR2- dependent manner. Interestingly, this interaction results in upregulated expression of the measles virus receptor, CD150, suggesting that HA-TLR2 interactions in fact benefit the virus at the expense of the host (13).
Innate Recognition of Viral Invasion Activities
In addition to recognizing unique features of viral nucleic acids or viral structural proteins, macrophages and DCs sense damage inflicted by viral invasion. This type of recognition is best characterized for the NLR protein NLRP3 following infection by viruses. NLRP3 forms a complex with ASC and caspase-1 to form inflammasomes (Figure 1).
Two important functions of NLR/ALR-inflammasome activation are (a) to release caspase-1-dependent cytokines such as IL-1β and IL-18, which act on multiple cell types to induce activation of innate leukocytes and lymphocytes, and (b) to induce pyroptosis in order to kill infected cells.
The formation of the NLRP3 inflammasome requires two signals. The first signal is often transduced by TLRs, leading to the expression of NLRP3; the second signal enables the assembly of a multiprotein complex involving NLRP3, ASC, and caspase-1 (62). Although many distinct stimuli act as the second signal for the NLRP3 inflammasome, many of them have in common the ability to perturb membranes (94).
Most viral infections impose by necessity some form of membrane stress or damage during entry, fusion, replication, and budding. Not surprisingly, emerging evidence indicates that host cells sense viral infections through NLRP3 and other innate sensors (Table 1). Table 1 summarizes the current evidence for how each of these viruses activates the inflammasome; in many cases neither the trigger for activation nor the antiviral relevance of inflammasomes is known. One exception is influenza virus, for which we understand the molecular trigger and the immune consequences of inflammasome activation.
Abbreviations: ssRNA, single-stranded RNA; dsDNA, double-stranded DNA; VSV, vesicular stomatitis virus; MCMV, murine cytomegalovirus; KSHV, Kaposi’s sarcoma-associated herpesvirus; EMCV, encephalomyocarditis virus; NLRP, Nod-like receptor protein; ALR, AIM2-like receptor; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; RIG-I, retinoic-acidinducible gene 1; IFI, interferon-inducible protein; AIM2, absent in melanoma 2. Question marks indicate that the exact trigger for a given pathway has not been identified. NT, not tested.
Influenza virus infection triggers NLRP3 inflammasome activation through the action of the M2 ion channel (43). The M2 ion channel transports H+ ions across lysosomal and Golgi membranes so that the virus can enter the host cells and maintain HA protein in a nonfusogenic conformation while transiting through the acidic trans-Golgi network, respectively (74). The latter activity is recognized by the influenza-infected macrophages and DCs via NLRP3 (43). Influenza virus is capable of inducing both signal 1 (via TLR7) and signal 2 (via M2) within the infected phagocytes.
Adenovirus, on the other hand, activates the NLRP3 inflammasomes in phorbol myristate acetate–differentiated Pam3CysK (TLR2 agonist)-primed THP-1 cells through disruption of the lysosomal membrane (9). Activation of NLRP3 by adenovirus (9) and myxomavirus lacking an ortholog of PYRIN-domain-containing protein, M013 (77), depends on reactive oxygen species and cathepsin B activity. Similarly, modified vaccinia Ankara (MVA) activates NLRP3 in a replication-independent, endocytosis-dependent manner (21).
Although the precise mechanisms are unclear, other viruses including encephalomyocarditis virus (EMCV) (75, 78) and VSV (78) trigger the NLRP3 inflammasome in Pam3CysKprimed THP-1 cells. Unlike the influenza viruses that activate both signal 1 and signal 2, priming of THP-1 cells through TLR activation is required to activate the NLRP3 inflammasomes upon adenovirus, MVA, EMCV, and VSV infection. HIV-1 infection has been reported to induce expression of NLRP3 and stimulates IL-1β release from healthy human peripheral blood mononuclear cells (76).
Recognition of virus-inflicted damage through NLRP3 leads to the activation of caspase-1 and release of its substrates including IL-1β and IL-18, which activate adaptive immune responses to influenza virus (42). IL-1R is expressed by DCs and can signal to activate their migration, antigen presentation, and costimulation {(human} 58). Thus, these cytokines may be particularly important in stimulating noninfected DCs to prime immune responses against viruses that incapacitate directly infected antigen-presenting cells.
UNEXPLORED AREAS OF INNATE VIRUS RECOGNITION
Although progress in the past decade has brought us enormous insights into the molecular and cellular mechanisms of innate viral sensing and the ensuing effector functions, many areas of host viral recognition remain unaddressed. In particular, PRR-independent sensing of viral infections, likely through engaging cellular stress responses, remains largely undefined.
Lytic virus infection almost always results in the shutdown of host transcription and translation, converting the infected cells into a virus factory. This conversion is accompanied by a sudden drop in cellular mRNA in the cytosol and a blockade of synthesis of cellular proteins in general. Instead, cellular translation machinery is taken over by the virus to produce large amounts of viral proteins. For some viruses, translation is mediated exclusively by the internal ribosomal entry site (Figure 3).
I hypothesize that host cells must be equipped to sense drastic changes in cellular mRNA and protein levels through unknown mechanisms and to induce an antiviral program, likely through the induction of apoptosis. Most viruses encode factors to block apoptosis at multiple levels in order to maximize viral production. Similarly, the unfolded protein response induced by viral protein synthesis may induce an antiviral program (34), although studies indicate that the unfolded protein response may also block antiviral responses (56).
In addition to virus recognition systems through these general features of viral infections, there may also be location-specific recognition systems. For instance, most DNA viruses replicate in the nucleus. Nuclear domain 10 (ND10, or nuclear bodies), small nuclear substructures defined by the presence of promyelocytic leukemia protein (PML), becomes the early replication site of many DNA viruses (25).
A number of proteins involved in DNA repair reside in or are recruited to ND10, and they may be utilized by DNA viruses for efficient replication. Structures such as the PML body may contain viral sensors and/or effectors of antiviral defense. In fact, PML and several other ND10 proteins are upregulated by type I IFNs. Consistent with this idea, ND10 structures are disrupted by viral proteins. Viruses deficient in genes capable of ND10 disruption are repressed for viral gene expression or DNA replication. Another possible location-based recognition system might exist to recognize viral replication that occurs on membranous structures.
Many Group IV viruses (positive-strand ssRNA) generate unique 70- to 100-nm membrane vesicles that wrap around the active replicating viral RNA, providing a microenvironment optimal for viral replication (60). Such membrane structures are usually generated by a viral nonstructural protein, using full complement of cellular machinery. It is tempting to speculate whether host cells might be capable of sensing viral infection by detecting unusual membrane formation or pre-existing antiviral defense mechanisms in sites such as the PML bodies.