Are We Done Monkeying Around With TRIM5α?

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domain would preclude human TRIM5a from inhibiting HIV-1 replication in a meaningful way. If we accept that the mCherry–human TRIM5a fusion is not altering the affinity of the human SPRY for HIV-1 capsid, it seems that this dogma has been challenged. With respect to SPRY domains and specificity, the data presented in the study by Richardson et al. suggest that the field should perhaps begin to look past the primate “SPRY domain envy” that has established the dogma that the human SPRY domain is a nonstarter in approaches designed to inhibit HIV-1 infection. Perhaps the most controversial part of the study is the observation that stabilized human TRIM5a inhibits HIV-1 to an extent comparable to that observed when using rhesus TRIM5a. Many TRIM5a aficionados will find this conclusion difficult to accept, given the depth of the literature in this area. However, debating the validity of this conclusion misses the larger point: human TRIM5a does not need to outperform rhesus TRIM5a to be therapeutically useful. It does need to inhibit HIV-1 in vivo, and the authors have provided the first evidence that this might be possible. If other studies recapitulate and advance this finding, how human TRIM5a stacks up against primate orthologs will be a moot point. Until gene therapy allows for the expression of TRIM5a orthologs of our choosing, we are forced to go to war with the SPRY domain we have, not the SPRY domain we wish we had. These results provide the first evidence that the human SPRY domain might not be as inherently incapable of recognizing HIV-1 as we have come to believe. If human TRIM5a can indeed be leveraged to inhibit HIV-1 replication, what are the types of “performance-enhancing drugs” that might allow TRIM5a to make the jump to the “big leagues”? The mechanistic understanding of the restriction process developed using primate TRIM5a orthologs might be very useful in considering this question. For example, the molecular determinants driving the self-association and assembly of rhesus TRIM5a are established.6,7 If the ability of human TRIM5a to self-associate is increased, this might be predicted to enhance TRIM5a binding to capsid in much the same way that immunoglobulin M utilizes avidity to overcome the

Are We Done Monkeying Around With TRIM5a? Edward Campbell1 doi:10.1038/mt.2014.74

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n 2004, the TRIM5a protein from rhesus macaques was first identified as the restriction factor preventing HIV-1 infection in these monkeys.1 Since then, the antiretroviral activity of a large number of primate TRIM5a orthologs has been examined. Collectively, these studies indicate that human TRIM5a is at the back of the pack when it comes to inhibiting HIV-1 infection. This has established the dogma that retroviruses have evolved to evade the TRIM5a ortholog present in species to which they are endemic. Following this thinking, because HIV-1 has learned to evade human TRIM5a, leveraging TRIM5a as an antiviral therapy will require expression of TRIM5a variants or orthologs not present in the human genome. This dogma is being challenged in the publication by Richardson and co-workers in this issue of Molecular Therapy.2 The authors report that stabilization of human TRIM5a enables it to restrict HIV-1 infection in primary human T cells challenged with HIV-1 in a NOD/SCID mouse model. The findings suggest that a better understanding of the mechanisms of TRIM5a degradation may provide opportunities to reduce its turnover in a more targeted way that could be exploited for therapeutic purposes. TRIM5a is a cellular protein that inhibits retroviral infection by binding to determinants present on the incoming capsid core. This ability to inhibit infection varies remarkably between members of the primate lineage and is thought to

1 Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University, Chicago, IL, USA Correspondence: Edward Campbell, 2160 South First Avenue, Maywood, IL 60153, USA. E-mail: [email protected]

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be responsible for the species-specific tropism of many retroviruses, including HIV1 and other primate lentiviruses. The AIDS pandemic is strong evidence that our endogenous restriction factors, including TRIM5a, are not effective at inhibiting HIV-1 infection. Unlike other restriction factors, which are often targeted for degradation by viral proteins, the failure of human TRIM5a to inhibit HIV-1 seems to stem from inefficient recognition of the viral capsid. Numerous studies from many groups have revealed that genetic variability in the C-terminal SPRY domain defines the species-specific restriction spectrum observed among primate TRIM5a orthologs.3 In the case of humans, a previous encounter with a retrovirus appears to have forced intense selective pressure on our SPRY domain. Unfortunately, these changes have left us poorly equipped to inhibit HIV-1 infection, especially when compared to our primate cousins. Indeed, changing a single amino acid within the human TRIM5a SPRY to the corresponding rhesus residue (R332P) confers potent anti-HIV activity upon human TRIM5a.4 However, Richardson and colleagues’ study reported in this issue shows that expressing human TRIM5a as a fusion protein to mCherry stabilizes the protein, which is otherwise turned over quite rapidly.5 This stabilization unexpectedly allows human TRIM5a to protect primary human T cells from infection. Although this method of stabilization is unlikely to be part of any therapeutic strategy, it establishes a critical proof of principle: human TRIM5a is capable of inhibiting HIV-1 infection in vivo. Clearly, this outcome is achieved with the benefit of performance enhancement. As noted above, however, prevailing dogma would suggest that inherent inadequacies of the human SPRY

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low-affinity interactions inherent in many immunoglobulin M antibodies. Similarly, the authors have demonstrated that reducing turnover of human TRIM5a generates a pool of protein sufficiently large to allow restriction. To this point, most studies of TRIM5a degradation have focused on degradation occurring during restriction, although the mechanism of TRIM5a degradation appears to be different in the absence of restriction-sensitive virus.5,7 These are all questions that are now worth asking. Hopefully, the answers will demonstrate that human TRIM5a can indeed play in the big leagues. Unlike professional athletes, few people will care if success requires the use of performance-enhancing drugs. ACKNOWLEDGMENTS The author thanks Sherry Campbell for fruitful discussions.

commentary REFERENCES

1. Stremlau, M, Owens, CM, Perron, MJ, Kiessling, M, Autissier, P and Sodroski, J (2004). The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427: 848–853. 2. Richardson, MW, Guo, L, Xin, F, Yang, X and Riley, JL (2014). Stabilized human TRIM5a protects human T cells from HIV-1 infection. Mol Ther 22: 1084–1095. 3. Johnson, WE and Sawyer, SL (2009). Molecular evolution of the antiretroviral TRIM5 gene. Immunogenetics 61: 163–176. 4. Yap, MW, Nisole, S and Stoye, JP (2005). A single amino acid change in the SPRY domain of human Trim5alpha leads to HIV-1 restriction. Curr Biol 15: 73–78. 5. Wu, X, Anderson, JL, Campbell, EM, Joseph, AM and Hope, TJ (2006). Proteasome inhibitors uncouple rhesus TRIM5alpha restriction of HIV-1 reverse transcription and infection. Proc Natl Acad Sci USA 103: 7465–7470. 6. Diaz-Griffero, F, Qin, X, Hayashi, F, Kigawa, T, Finzi, A, Sarnak, Z et al. (2009). A B-box 2 surface patch important for TRIM5alpha self-association, capsid binding avidity, and retrovirus restriction. J Virol 83: 10737–10751. 6. Sastri, J, O’Connor, C, Danielson, CM, McRaven, M, Perez, P, Diaz-Griffero, F et al. (2010). Identification of residues within the L2 region of rhesus TRIM5alpha that are required for retroviral restriction and cytoplasmic body localization. Virology 405: 259–266. 7. Rold, CJ and Aiken, C (2008). Proteasomal degradation of TRIM5alpha during retrovirus restriction. PLoS Pathog 4: e1000074.

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Boolean Immunotherapy: Reversal of Fortune Richard A Morgan1 doi:10.1038/mt.2014.75

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n this issue of Molecular Therapy, Leen et al. describe an approach to exploit the inhibitory tumor microenvironment to enhance antitumor immune responses.1 Tumors produce a range of inhibitory cytokines and signals that reduce the ability of infiltrating T cells to recognize and destroy the tumor (reviewed in refs. 2–4). One such tumor-derived signal is the cytokine interleukin 4 (IL-4), and in this report Leen and colleagues express on T cells a chimeric cytokine receptor comprising

1 Immunotherapy, bluebird bio, Cambridge, Massachusetts, USA Correspondence: Richard A Morgan, Immunotherapy, bluebird bio, 150 Second Street, Cambridge, Massachusetts 02141, USA. E-mail: [email protected]

Molecular Therapy vol. 22 no. 6 june 2014

a fusion between the extracellular binding domain of the IL-4 cytokine receptor and the intracellular signaling portion of the receptor for IL-7, a cytokine that potentiates the growth and activity of effector T cells. The approach has potential application to the field of adoptive immunotherapy using genetically engineered T cells, in that it presents a strategy to reverse the immunoinhibitory components of the tumor microenvironment and instead use them to activate T cells and augment cellmediated antitumor efficacy. The new study describes an approach to cancer therapy that involves the transfer of ex vivo–expanded T cells with antitumor activity into a tumor-bearing host in a process called adoptive cell therapy (ACT). ACT had been shown to be clinically effective in autologous lymphocyte

infusions for viral-associated malignancies5 and in melanoma using autologous tumor-infiltrating lymphocytes,6 but did not generate significant interest until it was shown that genetic modification of normal T cells with anti­tumor antigen receptors is effective in a variety of tumor types. The accelerated pace of development of genemodified T-cell ACT has been largely driven by excellent clinical responses, first reported by the National Cancer Institute7 and later confirmed by others,8 that chimeric antigen receptors (CARs) targeting the B-cell antigen CD19 could yield durable complete responses in a high proportion of patients. In general, these results have not been repeated in solid tumors, whose inhibitory micro­ environment has been suggested as a major element in this lack of success. Beyond the inhibitory environment, the lack of truly tumor-specific antigens on solid tumors has led to safety issues with regard to “on-target antigen but off-target tumor” toxicities. The data presented in the report by Leen et al. begin to address both negative issues and turn them into positive attributes. The investigators fused the extra­ cellular binding domains of the IL-4 receptor a-chain (IL-4Ra) with the intracellular signaling domains of the IL-7 receptor a-chain (IL-7Ra) (Figure 1) to create a chimeric cytokine receptor.9 IL-4 is one of several tumor-associated cytokines that can have inhibitory effects on antigen-reactive T cells, for example, by initiating a gene expression cascade that can be associated with immune suppression. Thus, when IL-4 binds to its natural receptor it activates transcription factors, which in turn can lead to the suppression of the important effector cytokine interferon g. Conversely, signaling through the normal IL-7 receptor transmits signals that promote T-cell growth and survival. When the investigators introduced the chimeric IL-4Ra/IL-7Ra gene into T cells and then exposed them to IL-4, microarray analysis demonstrated that the gene expression profile resembled that obtained from T cells that had been exposed to IL-7. Thus, a potentially negative cytokine signal was reversed and turned into a positive signal! To test the activity of their approach in a tumor system, the authors introduced the IL-4Ra/IL-7Ra gene into naturally occurring T cells specific against Epstein-Barr virus (EBV). They then 1073