Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved July 23, 2014 (received for review March 18, 2014)

Author contributions: M.L., E.O.F., and S.-L.L. designed research; M.L., S.D.A., C.M., Y.-M.Z., and M.S.F. performed research; P.D.R. and W.M. contributed new reagents/analytic tools; M.L., S.D.A., Y.-M.Z., W.M., M.C.J., E.O.F., and S.-L.L. analyzed data; and M.L., E.O.F., and S.-L.L. wrote the paper.

Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved July 23, 2014 (received for review March 18, 2014)

Significance

TIM-family proteins have been recently shown to promote viral entry into host cells. Unexpectedly, we discovered that human TIM-1, along with TIM-3 and TIM-4, potently inhibits HIV-1 release. We showed that TIM-1 is incorporated into HIV-1 virions and retains HIV-1 particles on the plasma membrane via phosphatidylserine (PS), a phospholipid that is exposed on the cellular plasma membrane and the viral envelope. Expression of TIM-1 inhibits HIV-1 replication in CD4^{T cells, and knockdown of TIM-3 in monocyte-derived macrophages enhances HIV-1 production. We extended this function of TIMs to other PS receptors, and demonstrated that they also inhibited release of additional viruses, including murine leukemia virus and Ebola virus. The novel role of TIMs in blocking viral release provides new insights into viral replication and AIDS pathogenesis.}

Significance

Abstract

Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors.

Abstract

The T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play essential roles in cellular immunity (1, 2). Certain human pathologies, in particular allergic diseases, are associated with TIM protein dysfunctions and polymorphisms (3 –5). Viral infection has recently been linked to TIM proteins, with some TIMs acting as key factors for viral entry. Human TIM-1 was initially discovered as the receptor for hepatitis A virus (HAV), and has been recently shown to function as a receptor or entry cofactor for Ebola virus (EBOV) and Dengue virus (DV) (5 –8). TIM-1 polymorphisms have been reported to be associated with severe HAV infection in humans (9). More recent studies revealed that TIM-family proteins promote entry of a wide range of viruses, possibly by interacting with virion-associated phosphatidylserine (PS), highlighting a more general role of TIMs in viral infections (10, 11).

TIM-family proteins are classical type I transmembrane proteins, with the N terminus containing the variable Ig-like (IgV) domain extending from the plasma membrane and the C-terminal tail largely mediating intracellular signaling oriented toward the cytosol (2, 12). Human genes encode three TIM proteins, i.e., TIM-1, TIM-3, and TIM-4, whereas the mouse genome encodes eight TIM members, but only TIM-1, TIM-2, TIM-3 and TIM-4 are expressed. Despite significant sequence variations, the IgV regions of all TIM proteins contain a PS binding site that is absolutely conserved (2). Notably, the functions of TIM-family proteins differ greatly, depending on cell type-specific expression as well as the interactions of these TIMs with other molecules, including TIM-family members (2). Human TIM-1 is predominantly expressed in epithelial and T helper 2 (T_H2) cells, and is involved in cell proliferation and apoptotic body uptake, whereas human TIM-3 is expressed in activated T helper cells (T_H1), and functions as a negative costimulatory signal, often resulting in immune tolerance and apoptosis (13, 14). Human TIM-4 has been found to be mainly expressed in macrophages and dendritic cells (DCs), and possibly acts as a ligand for TIM-1, thereby facilitating T-cell activation (15, 16).

TIM-1 has been reported to be expressed in activated CD4^{T cells (}13, 17), which are the major targets of HIV-1 infection. However, it is currently unknown if TIM-1 plays a role in HIV-1 replication and infection, although reduced TIM-3 expression on NK cells has been reported to be associated with chronic HIV-1 infection (18). Here we report that TIM-1 inhibits HIV-1 release, resulting in decreased virus production. Notably, TIM-1 mutants deficient for PS binding are incapable of blocking HIV-1 release. Similar to human TIM-1, we show that human TIM-3 and TIM-4 also potently inhibit HIV-1 production. The inhibitory effect of TIM-family proteins as well as some PS receptors can be extended to murine leukemia virus (MLV) and EBOV. Our study has revealed a novel and general function of TIMs, and likely other PS receptors, in the release of HIV-1 and other viruses.

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Acknowledgments

We thank Kunio Nagashima and Ferri Soheilian at the Leidos Electron Microscopy Laboratory for their expert assistance with the TEM analysis and Kartik Chandran for providing the VP40-GFP construct. This work was supported by National Institutes of Health (NIH) Grants R01 AI112381, R21 AI109464, R21 AI105584, R56 AI107095, and U54 {"type":"entrez-nucleotide","attrs":{"text":"AI057160","term_id":"3331026","term_text":"AI057160"}}AI057160 (to S.-L.L.), R01 GM110776 (to M.C.J.), and R01 AI1077519 (to W.M.). Research in the E.O.F. laboratory is supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, NIH, and by the Intramural AIDS Targeted Antiviral Program.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1404851111/-/DCSupplemental.

Footnotes

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