Innate immune sensing of HIV-1 by dendritic cells

DC maturation and priming of naïve T cells

Recent research on HIV-1 interaction with dendritic cells (DCs) suggests another hypothesis to explain HIV-1 persistence. DCs are highly heterogeneous, antigen-presenting cells, that initiate acquired immune responses by priming naïve, antigen-specific T cells (Steinman and Idoyaga, 2010). The nature of the T cell response that ensues, whether tolerance or sterilizing antiviral immunity, is determined by the maturation status of the antigen-presenting DC and the array of signals that the DC provides to the naïve, antigen-specific T cell (Figure 1). Among the molecules relevant to T cell priming that DCs elaborate in response to maturation are cell surface MHC Class II, CD80 and CD86, and cytokines such as IL-12 that promote T_H1 responses and the cytotoxic T lymphocytes that clear virus-infected cells (Reis e Sousa, 2006; Altfeld et al., 2011).

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Figure 1

Dendritic cells provide multiple signals to naïve T cells

The innate immune system instructs the acquired immune system via a diverse set of signals that dendritic cells provide to naïve, antigen-specific T cells. A range of outcomes are possible - from tolerance to potent antiviral immunity - depending upon the exact signals provided. Protective antiviral immunity requires that naïve T cells specific for viral antigens receive 3 signals from DCs (Reis e Sousa, 2006). Elaboration of the three signals requires DC maturation, most effectively attained by activation of cell-intrinsic pattern recognition receptors. The first signal involves antigen presentation via MHC. The second signal is provided by cell surface molecules that include CD80 and CD86. The third signal is delivered by cytokines that include IL-12, the structure of which is shown (PDB:1F45) (Yoon et al., 2000); the two IL-12 subunits are distinguished from each other by red (IL12A, p35) and blue (IL12B, p40).

DCs mature when pattern recognition receptors (PRRs) are stimulated by pathogen associated molecular patterns (PAMPS) (Akira et al., 2006). Viruses are obligate intracellular parasites, largely dependent upon host cell machinery for replication. They therefore do not generate completely foreign molecules like lipopolysaccharide that distinguish them from the host. Instead, PRRs alert cells to the presence of viruses by detecting more subtle features such as structured replication intermediates, modified nucleic acids, or viral replication complexes in cellular compartments where nucleic acids are not normally found.

Viral nucleic acids can be detected via cell extrinsic or cell intrinsic mechanisms (Iwasaki and Medzhitov, 2010). The former include TLR activation after phagocytosis of virus-infected, apoptotic cells. Such extrinsic PRRs are not expressed by all cell types but are generally restricted to antigen-presenting or phagocytic cells, such as dendritic cells. Cell intrinsic mechanisms detect viral nucleic acid within the infected cell, and the PRRs that detect these PAMPs are generally expressed on a broad range of cell types, including fibroblasts. RIG-I and MDA-5 are cell intrinsic cytosolic receptors that detect structural features unique to viral RNA. A cell intrinsic mechanism also exists for detecting viral DNA in the host cell cytoplasm. Cytosolic sensors for DNA include IFI16, DDX41, DAI, LSm14A, and AIM2 (Unterholzner et al., 2010; Sharma and Fitzgerald, 2011; Sharma et al., 2011; Zhang et al., 2011; Li et al., 2012; Upton et al., 2012).

Optimal priming of naïve T cells to generate potent CD4⁺T_H1 and CD8⁺ cytotoxic T cell responses requires that PAMP-recognition by PRRs occur within the same DCs that present the antigen, at least under particular experimental conditions (Spörri and Reis e Sousa, 2005; Hou et al., 2008; Kratky et al., 2011). Separation of antigen presentation from PAMP recognition and DC maturation may result in expansion of antigen-specific T cells that lack potent antiviral activity. It is in fact well described that HIV-1-specific T cells express elevated levels of inhibitory molecules such as PD-1, TRAIL, and CTLA-4 (Day et al., 2006; Trautmann et al., 2006; Wherry et al., 2007).

Complicating the assessment of these inhibitory molecules on the HIV-1-specific CD8⁺ T cells is that these same molecules are up-regulated on activated T cells. Co-expression of PD-1 with another negative regulator of T cell activation, CD160, distinguishes a subset of ineffective HIV-1-specific CD8⁺ T cells from activated T cells (Peretz et al., 2012). While these ineffective, HIV-1-specific, CD8⁺ T cells are thought to reflect a state of immune exhaustion that arises during chronic infection with HIV-1 (Khaitan and Unutmaz, 2011), similar phenotypes are observed in short-term experiments ex vivo and in humanized mice (Brainard et al., 2009; Lubong Sabado et al., 2009; Che et al., 2010), suggesting that T cell priming to HIV-1 antigens in the absence of optimal DC maturation might contribute to these ineffective, anti-HIV-1 immune responses.

Innate immune detection of retroviruses

Innate immune detection of viral nucleic acid generally activates type 1 interferon (IFN) and a large number of IFN-stimulated genes with a range of antiviral effector functions. Type 1 IFN also promotes DC maturation and contributes to potent antiviral T cell responses (Longhi et al., 2009). Retroviruses normally do not induce type 1 IFN, perhaps because the level of viral nucleic acid is kept at low level, or because the retrovirus genome is inaccessible to intrinsic PRRs (Fonteneau et al., 2004; Smed-Sörensen et al., 2004; Beignon et al., 2005; Yan et al., 2010; Liberatore and Bieniasz, 2011). This has made it difficult to determine if the innate immune system is capable of detecting retroviruses, and explains why the study of innate immunity against retroviruses has lagged behind that of other viruses. Unlike other viruses that do induce type 1 IFN, retroviruses are generally believed not to encode specific proteins that block innate immune signaling, although some recent reports suggest that HIV-1 disrupts RIG-I and a downstream transcription factor, interferon regulatory factor (IRF3) (Solis et al., 2011; Doehle et al., 2012a; 2012b). Further, under particular experimental conditions in vitro, aborted HIV-1 reverse transcripts in CD4⁺ T cells have been shown to activate apoptosis and inflammation in a process that involves caspase-3 and caspase-1 (Doitsh et al., 2010).

Important information concerning innate immune detection of retroviruses has been obtained by studying rare instances where adaptive immune control of retroviral infection has been observed. Specific strains of inbred mice such as I/LnJ and C57BL/6J, for example, are resistant to infection with the murine retroviruses Murine Leukemia Virus (MuLV) and Mouse Mammary Tumor Virus (MMTV) via both humoral and cellular mechanisms. This experimental system offers an opportunity to identify the innate immune PRRs that contribute to these protective, anti-retroviral acquired immune responses. The protective antibody responses to both viruses in these mice was TLR7-dependent (Kane et al., 2011b), but induction of protective CTL responses was dependent on a separate, yet to be defined, innate immune detection pathway (Browne and Littman, 2009; Kane et al., 2011b).

The importance of innate immune detection of retroviruses for subsequent acquired immunity was also demonstrated in experiments concerning the influence of the route of infection. When transmitted to pups via ingestion of mother’s milk, MMTV acquires a coat of LPS derived from intestinal bacteria (Kane et al., 2011a). Upon engulfment of these virions by the host cells, TLR4 signaling was activated by the virion-associated LPS. The subsequent cascade of signals resulted in production of IL-10, a tolerogenic cytokine that permitted chronic infection. When MMTV was ingested following sterilization of gut bacteria, or when MMTV infection was initiated parenterally, tolerance to the virus was not observed, and chronic infection did not ensue.

HIV-1 sensing by DCs

Several types of DCs have been described, though they can be divided into two major groups, the myeloid CD11c⁺ conventional DC (cDC) and the plasmacytoid DC (pDC) (Steinman and Idoyaga, 2010; Altfeld et al., 2011). The pDC, in contrast to the cDC, constitutively expresses TLR7 and TLR9 (Kadowaki et al., 2001). The pDC endocytoses HIV-1 virions via Env interaction with CD4, and the virion genomic RNA is detected by TLR7 within the endocytic compartment (Fonteneau et al., 2004; Beignon et al., 2005; Smed-Sörensen et al., 2005); this innate immune detection of HIV-1 does not require viral replication and, yet, results in the production of copious amounts of type 1 IFN. A clear role for the pDC in priming of naïve, HIV-1-specific CD4+ T cells has not been established, but the IFN secreted by pDC in response to HIV-1 promotes cDC maturation and NK cell activation (Fonteneau et al., 2004; Romagnani et al., 2005). The complex interplay between NK cells, T cells, and DCs, as well as the direct effects of NK cells on HIV-1-infected cells, has been discussed extensively in a recent review (Altfeld et al., 2011).

The importance of the cDC for priming of naïve, anti-HIV-1 T cells is better established than it is for the pDC. HIV-1-pulsed, monocyte-derived DCs, the most common experimental source of human cDCs, prime naïve CD4⁺ and CD8⁺ T cells in vitro to generate broad, poly-functional, anti-HIV-1 responses targeting epitopes that mirror those detected after acute infection in vivo (Lubong Sabado et al., 2009). Humanized mice challenged with HIV-1 elaborate similar HIV-1-specific CD4⁺ and CD8⁺ T cell responses (Brainard et al., 2009). But, as has been well described in HIV-1 infected people (Day et al., 2006; Trautmann et al., 2006; Wherry et al., 2007), the HIV-1-specific T cells elicited in these assays express elevated levels of inhibitory molecules such as PD-1, TRAIL, and CTLA-4 that suppress the priming of other naïve T cells in a contact-dependent manner (Brainard et al., 2009; Che et al., 2010).

In contrast to pDCs, cDCs challenged with HIV-1 do not make type 1 IFN, and, additionally, they do not produce IL-12 (Fonteneau et al., 2004; Smed-Sörensen et al., 2004; Beignon et al., 2005). The type 1 IFN produced by pDCs in response to challenge with HIV-1 promotes cDC maturation in trans (Fonteneau et al., 2004), but, as mentioned above, if PAMP recognition and antigen-presentation do not occur within the same cell, the critical signals needed for full maturation of cDCs and antiviral immunity may be lacking (Spörri and Reis e Sousa, 2005; Hou et al., 2008; Kratky et al., 2011). These observations demonstrate that cell intrinsic, innate immune sensing of HIV-1 is insufficient to effect complete cDC maturation. Given the clear importance of the cDC for T cell priming, the discussion that follows below will focus on this DC subtype.

Non-replicative interactions dominate HIV-1 entry into DCs

cDCs fail to mature upon HIV-1 exposure probably because the virus cannot infect these cells (Cameron et al., 1992; Smed-Sörensen et al., 2005). Productive infection by HIV-1 requires virion membrane fusion with the target cell membrane, driven by the interaction between HIV-1 gp120/gp41 and the cell surface entry receptors CD4 and CCR5. This results in delivery of the virion core into the cytoplasm where reverse transcription takes place (McDonald et al., 2002). Levels of cell surface CD4 and CCR5 are relatively low on most DC subsets (Caux et al., 1992; Granelli-Piperno et al., 2006), and when HIV-1 encounters DCs it is more likely to be endocytosed via interaction with DC-SIGN or other C-type lectins (Piguet and Steinman, 2007) (Figure 2). Reverse transcription does not occur within this endocytic compartment, PRRs are not activated, and DCs fail to mature (Granelli-Piperno et al., 2004).

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Figure 2

HIV-1 virion interaction with dendritic cells is dominated by non-productive entry pathways

Productive infection requires that HIV-1 virions gain access to the target cell cytoplasm via HIV-1 glycoprotein (gp120/gp41) interacting with host cell receptors (CD4 and either CCR5 or CXCR4). Cell surface levels of CD4 are low on many types of dendritic cells, and HIV-1 virions are endocytosed via interaction with cell surface lectins such as DC-SIGN. Replication of the HIV-1 genomic RNA (green lines) does not occur within the endocytic compartment and innate immune signaling is not activated within conventional, antigen-presenting DCs. However, the endocytosed virion is degraded and HIV-1 proteins are therefore efficiently presented in the absence of DC maturation. Alternatively, the glycosphingolipid GM3 in the HIV-1 virion membrane is recognized by an unknown receptor on DCs (designated X). This results in virion capture and transfer to susceptible CD4⁺ T cells via infectious synapses.

HIV-1 virions that are endocytosed by DCs are probably transferred to a lysosomal compartment – and a fraction to proteasomes – and rapidly degraded for processing and presentation in the absence of DC maturation (Moris et al., 2004; 2006). Some virions escape degradation by trafficking to immunologic synapses, from which they are efficiently transferred to highly permissive CD4⁺ T cells (Pope et al., 1994; McDonald et al., 2003). Avoidance of the degradation pathway and transfer of infectious virions to immunologic synapses is an Env-independent process that requires incorporation of the host cell-derived, glycosphingolipid GM3 into the HIV-1 virion membrane (Izquierdo-Useros et al., 2012; Puryear et al., 2012). The outcome of interaction with the DC C-type lectin and GM3 pathways would be tolerance to HIV-1 and potentiation of HIV-1 spread to highly permissive, activated CD4+ T cells (Figure 2).

Experimentally, HIV-1 virions can be pseudotyped with heterologous viral glycoproteins (Figure 3). This technique can be exploited to redirect HIV-1 transduction to cells that are normally not targeted by HIV-1. Pseudotyping of HIV-1 with the vesicular stomatitis virus glycoprotein (VSV G) drives fusion of HIV-1 virions from endosomes into the cytoplasm and greatly increases the efficiency with which HIV-1 enters the dendritic cell cytoplasm (Granelli-Piperno et al., 2000). This has become the standard methodology for transducing dendritic cells with HIV-1-based vectors (Berger et al., 2011).

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Figure 3

Bypass of the block to productive entry reveals a block to HIV-1 reverse transcription in DCs due to SAMHD1

The block to productive entry can be overcome experimentally by producing HIV-1 virions bearing the vesicular stomatitis virus glycoprotein (VSV G). Once in the dendritic cell cytoplasm, HIV-1 virions fail to reverse transcribe the viral genomic RNA template (green), into viral cDNA (red), because the dNTP concentration is too low to support cDNA synthesis. This block to reverse transcription is due to the dNTP triphosphohydrolase SAMHD1.

SAMHD1 prevents HIV-1 reverse transcription in DCs

Although VSV G pseudotyping increases the efficiency with which HIV-1 virions enter DCs, a second block to replication occurs at the level of reverse transcription (Figure 3). The first clue to the nature of this block came from the observation that viruses of the human immunodeficiency virus type 2 (HIV-2) and the simian immunodeficiency SIVsm/SIVmac lineage are more efficient than HIV-1 at transducing DCs, and that transduction by SIV_MAC vectors required the vpx accessory gene (Mangeot et al., 2002). When provided in trans, SIV virus-like particles (VLPs) greatly increased the efficiency of HIV-1 reverse transcription and transduction in DCs (Goujon et al., 2006). This increase in HIV-1 infectivity also required the SIV Vpx protein (Goujon et al., 2006) and was mimicked by proteasome inhibitors (Goujon et al., 2007). Vpx was then shown to associate with the CUL4A E3 ubiquitin ligase complex via direct binding to DCAF1 (Sharova et al., 2008; 2008; Srivastava et al., 2008; 2008; Bergamaschi et al., 2009; 2009) and heterokaryon experiments demonstrated the presence of a dominant-acting inhibitor in myeloid cells that was sensitive to Vpx (Sharova et al., 2008). The observation that myeloid cells have 100 times lower dNTP pools than do HIV-1-permissive, activated CD4⁺ T cells (Diamond et al., 2004; Kennedy et al., 2010) was an additional clue to the mechanism of the block to reverse transcription since reverse transcription requires dNTPs for synthesis of viral cDNA.

Vpx was then shown to promote the degradation of SAMHD1 (Goldstone et al., 2011; Powell et al., 2011; Lahouassa et al., 2012), a triphosphohydrolase that converts deoxynucleoside triphosphates (dNTPs) to the constituent deoxynucleosides and inorganic triphosphate (Goldstone et al., 2011; Powell et al., 2011; Lahouassa et al., 2012). It was reasonable to hypothesize that, by depleting dNTPs, SAMHD1 would block reverse transcription. Indeed, the kinetics of SAMHD1 degradation that followed Vpx delivery correlated with rising dNTP levels, and subsequent increase in HIV-1 reverse transcription (Kim et al., 2012). SAMHD1 knockdown permitted HIV-1 reverse transcription to proceed in myeloid cells in the absence of Vpx (Hrecka et al., 2011; Laguette et al., 2011). Cells from Aicardi-Goutières Syndrome (AGS) patients that bear mutations in SAMHD1 were similarly permissive for HIV-1 in the absence of Vpx. This inflammatory disease will be discussed further below. The antiviral effect of SAMHD1 has also been reported more recently in non-dividing, resting CD4⁺ T cells (Baldauf et al., 2012).

Consequences of DC transduction

Efficient HIV-1 transduction of DCs can be achieved by pseudotyping particles with VSV G in the presence of Vpx-bearing VLPs or exogenous nucleosides. One group has reported that DC maturation can occur in response to such high-efficiency transduction by HIV-1, and that DC maturation was associated with increased responsiveness of HIV-1-specific T cell clones (Manel et al., 2010). DC maturation required interaction between the cellular protein cyclophilin A and HIV-1 capsid protein synthesized de novo from the provirus (Manel et al., 2010). Cyclophilin A is a well characterized HIV-1 capsid-binding protein that regulates HIV-1 infectivity (Sokolskaja and Luban, 2006).

Other groups have failed to detect maturation of DCs after HIV-1 transduction (Fonteneau et al., 2004; Granelli-Piperno et al., 2004; Beignon et al., 2005; Smed-Sörensen et al., 2005; Granelli-Piperno et al., 2006; Pertel et al., 2011a; 2011b), even when transduction was pushed to high levels with Vpx. Perhaps this discrepancy is due to variation in additional host factors that regulate HIV-1 replication in DCs, such as TREX1 (discussed below), or other myeloid-specific, innate immune factors that are yet to be discovered. For example, Vpx rescues HIV-1 transduction of DCs from the antiviral state established by prior treatment with exogenous type 1 IFN via a mechanism that is independent of SAMHD1 and DCAF1-CUL4A. (Pertel et al., 2011b).

Infection with HIV-2, a virus that expresses Vpx, is less likely to cause AIDS than HIV-1 (de Silva et al., 2008). Perhaps by permitting HIV-2 to complete reverse transcription within DCs, Vpx enables cell-intrinsic PRRs within these cells to sense HIV-2. Signaling from the PRRs would direct the antigen-presenting DCs to mature and thus render acquired immune responses against HIV-2 more effective. Consistent with this idea, polyfunctional, virus-specific, T cell responses are more commonly observed with HIV-2 than with HIV-1 (Duvall et al., 2008). Further evidence in support of this model comes from experiments in which an HIV-1 provirus was modified to express and package SIV Vpx; the resulting virus infected myeloid cells more efficiently than did the parental HIV-1 virus (Sunseri et al., 2011). Additionally, type 1 IFN was detected in these spreading infections (Sunseri et al., 2011), something that was not seen with wild-type HIV-1.

The correlation between Vpx and immune control is not perfect, in that SIV_MAC239, another virus that encodes Vpx, is highly pathogenic in a non-native host monkey species. However, SIV_SM the naturally occurring virus from which SIV_MAC239 was derived, is not pathogenic in its native host (Chahroudi et al., 2012).

TREX1 conceals HIV-1 cDNA from detection by the innate immune system

The presence of DNA in the cytoplasm of DCs and other cell types, whether from a transfected plasmid, synthetic dsDNA, intracellular bacteria, or infection with a DNA virus, activates type 1 IFN (Sharma and Fitzgerald, 2011). The nuclease TREX1 was identified in a biochemical screen intended to determine the identity of the PRR that activates type 1 IFN in response to DNA within the cytoplasm of macrophages (Stetson et al., 2008). However, in the follow-up functional test, TREX1 disruption did not block type 1 IFN induction by DNA, demonstrating that TREX1 was not the sought-after PRR. Paradoxically, TREX1-deficient animals had elevated type 1 IFN and presented with inflammatory myocarditis (Stetson et al., 2008). Additionally, cDNA from endogenous retroelements was increased in TREX1-deficient cells, consistent with a role for TREX1 in suppressing retroviral cDNA accumulation and blocking cell intrinsic detection of DNA (Stetson et al., 2008). Amazingly, reverse transcriptase inhibitors that prevent synthesis of retroviral cDNA limited the inflammatory disease in TREX1-null mice (Beck-Engeser et al., 2011).

Subsequently it was reported that when TREX1 is disrupted, challenge with HIV-1 resulted in elevated levels of HIV-1 cDNA and type 1 IFN induction (Yan et al., 2010). This study demonstrated that HIV-1 cDNA can be sensed by the innate immune system via an intrinsic mechanism. However, the cDNA resulting from HIV-1 reverse transcription does not normally accumulate to sufficient levels to be detected unless TREX1 is disrupted (Figure 4). The PRR responsible for detecting HIV-1 cDNA under these conditions has not been determined, although the cytosolic viral DNA sensor IFI16 is a possible candidate (Unterholzner et al., 2010; Sharma et al., 2011).

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Figure 4

The human exonuclease TREX1 limits the level of HIV-1 cDNA

HIV-1 reverse transcriptase generates a cDNA (red lines) from the HIV-1 genomic RNA (green) in the target cell cytoplasm. Normally, the HIV-1 cDNA is not detected by pattern recognition receptors (PRRs) that recognize DNA in the cytoplasm. When the host cell TREX1 nuclease is disrupted, either by mutation or by siRNA, HIV-1 cDNA accumulates to supra-normal levels and activates type 1 IFN. Though several candidate receptors exist (e.g., IFI16), the cell intrinsic PRR that recognizes HIV-1 cDNA under these conditions has not been identified.

Mutations in the gene encoding TREX1, as well as in SAMHD1, the HIV-1 restriction factor specific to dendritic cells, macrophages, and nondividing T cells, are associated with Aicardi-Goutières Syndrome (AGS). This rare, lethal, inflammatory syndrome is characterized by elevated type 1 IFN (Crow and Rehwinkel, 2009; Rice et al., 2009). AGS-associated mutations are also found in genes encoding each of the three proteins that constitute the enzyme RNASEH2 (Crow et al., 2006). This enzyme cleaves ribonucleotides within RNA-DNA duplexes like those found in the intermediate products of reverse transcription. Whether RNASEH2 influences innate immune recognition of HIV-1 in DCs remains to be determined. The current model for AGS pathophysiology, whether due to mutations in TREX1, SAMHD1, or RNASEH2, is that inflammation results from activation of cytosolic PRRs in response to the increased levels of endogenous reverse transcripts or reaction intermediates that accumulate when either of these factors is mutated. By demonstrating that the human innate immune system is indeed capable of detecting HIV-1, the AGS factors offer a new paradigm in AIDS vaccine research.

TRIM5, CA-specific restriction, and innate immune signaling in DCs

TRIM5 was identified in expression screens seeking the factor responsible for an anti-HIV-1 activity in diverse cell types from macaques and owl monkeys (Sayah et al., 2004; Stremlau et al., 2004). The TRIM5 orthologues from these two non-human primates were then shown to potently block HIV-1 transduction (10–100-fold). The viral determinant for sensitivity to TRIM5 restriction is the capsid protein (CA), but TRIM5 binds to the capsid lattice, not free capsid protein (Grütter and Luban, 2012) (Figure 5) and binding induces TRIM5 to form a complementary lattice (Ganser-Pornillos et al., 2011). Because the TRIM5-CA interaction is so complicated, a robust binding assay has not yet been developed, and conclusions about binding strength are extrapolated from restriction activity assays or non-quantitative, particulate association assays (Stremlau et al., 2006).

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Figure 5

TRIM5 is a pattern recognition receptor specific for the retrovirus capsid lattice

The cytoplasmic E3 ubiquitin ligase TRIM5 exists as a dimer in the cytoplasm where it signals weakly or not at all. Upon infection with a retrovirus, if avidity for the capsid lattice is great enough, TRIM5 multimerizes to form a complementary lattice (Ganser-Pornillos et al., 2011) and E3 ubiquitin ligase activity is increased (Pertel et al., 2011a). In complex with the heterodimeric E2 (UBC13/UEV1A), TRIM5 synthesizes unattached K63-linked ubiquitin chains that activate the TAK1 kinase complex and innate immune signaling (Pertel et al., 2011a). Most HIV-1 strains (blue capsid) are only weakly detected by the human TRIM5 orthologue. HIV-1 capsid variants that are better recognized by human TRIM5 (orange capsid) would increase innate immune signaling, and might make better immunogens.

Phylogenetic comparisons have shown that the sequences encoding the capsid binding domain of TRIM5 are evolving at rates faster than any coding sequence in the primate genome (Sawyer et al., 2005). It has been suggested that this positive selection is driven by challenges in the remote past from pathogenic retroviruses. The human TRIM5 orthologue weakly inhibits lab strains of HIV-1, on the order of two-fold, in quantitative, single-cycle infectivity assays (Sokolskaja et al., 2006; Battivelli et al., 2010; 2011). The weak inhibition of HIV-1 by human TRIM5 is remedied by replacing a few capsid binding residues in the human orthologue with those from the macaque (Stremlau et al., 2005). It is possible that human TRIM5 is an active restriction factor in vivo and that it just does not recognize lab strains of HIV-1 very well. One group has recently cloned HIV-1 sequences directly from infected people and found impressive variation in TRIM5 sensitivity. Some HIV-1 isolates were 10 times more sensitive than the lab strain standard (Battivelli et al., 2010; 2011). Interestingly, the variant amino acids in these TRIM5-sensitive strains were located in dominant CTL epitopes within capsid, suggesting that escape from CTL forced the acquisition of increased TRIM5 sensitivity.

TRIM5 promotes innate immune signaling (Tareen and Emerman, 2011; Pertel et al., 2011a) via a mechanism that involves synthesis of unattached K63-linked ubiquitin chains and activation of the TAK1 kinase complex (Pertel et al., 2011a) (Figure 5). This biochemical activity, and correspondingly the innate immune signaling within DCs, was amplified greatly by infection with retroviruses that bear restriction-sensitive capsids (Pertel et al., 2011a). The results demonstrating that TRIM5 acts as a PRR specific for the retrovirus capsid lattice were obtained using select combinations of TRIM5 orthologues with specific retrovirus capsids (Pertel et al., 2011a). For example, human TRIM5 in DCs or macrophages signaled in response to the restricted N-tropic MLV, but not to the isogenic, non-restricted, B-MLV. Owl monkey TRIM5 signaled in response to restricted HIV-1, but not to the unrestricted SIV.

Induction of cytokines was not detected in human DCs challenged with HIV-1, most probably because the avidity of human TRIM5 for capsid from standard strains of HIV-1 is too weak to activate signaling. For signaling to occur in human dendritic cells in response to HIV-1 capsid, the avidity of the interaction will need to be increased. This might be achieved by using HIV-1 capsid variants with CTL escape mutations that confer greatly increased HIV-1 sensitivity (Battivelli et al., 2010; 2011) (Figure 5). Alternatively, if robust binding assays are developed for measuring the interaction of TRIM5 with soluble capsid lattice components (Zhao et al., 2011), screens might be undertaken to identify small molecules that increase the avidity of the interaction.

This review was written in fond memory of a very inspiring teacher, Ralph Steinman. Thanks to Tatyana Golovkina for critical reading of the manuscript and to Rahm Gummuluru, Rick Koup, Harmit Malik, Douglas Nixon, and Celia Schiffer for informative discussions. Apologies to the many investigators whose discoveries and influence is not adequately acknowledged due to space limitations or my ignorance. The author is funded by grants from the Swiss National Science Foundation, NIAID (USA), and a NIDA Avant-Garde Award.