2 0 3 0 -cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral stimulator of interferon genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen, we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2 0 3 0 -bisphosphosphothioate-cyclic-di-AMP (2 0 3 0 -CDA S ). These discoveries will provide insight into cGAMP's role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

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Administration of exogenous CDNs to activate the cGAMP-STING pathway is a promising therapeutic strategy to unleash the full potential of cancer immunotherapy. This strategy mirrors the role of endogenous extracellular cGAMP, an immunotransmitter that is transferred from cancer cells to cGAMP-sensing cells in the host, promoting immunity. However, the CDN import mechanisms used by host cells within tumors remain unknown. Here we identified the protein SLC46A2 as the dominant cGAMP importer in primary human monocytes. Furthermore, we discovered that monocytes and M1-polarized macrophages directly sense tumor-derived extracellular cGAMP in murine tumors. Finally, we demonstrated that SLC46A2 is the dominant cGAMP importer in monocyte-derived macrophages. Together, we provide the first cellular and molecular mechanisms of cGAMP as an immunotransmitter, paving the way for effective STING pathway therapeutics.

We have examined the interactions of wild-type (WT) and matrix protein-deleted (⌬MA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ⌬MA PrGag proteins were tagged with the Escherichia coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins in trans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ⌬MA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference.

The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation. T he matrix (MA) domain of the human immunodeficiency type 1 (HIV-1) precursor Gag (Pr55Gag) protein serves several assembly-related functions. One role is to direct Pr55Gag proteins to assembly sites at cell plasma membranes (PMs) that are enriched for phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2) and cholesterol (1-8). This function is accomplished in part by virtue of an amino-terminal myristate moiety and, in part, through direct binding to PI(4,5)P2 (1). Evidence indicates that the PI(4,5)P2-binding site on MA overlaps a binding site for RNA, which suggests a chaperone model in which MA-RNA binding protects the MA domain of Pr55Gag from associating with inappropriate intracellular membranes prior to delivery to the PI(4,5)P2-rich PM assembly sites (9-16). In some experimental systems, it has been shown that Pr55Gag proteins with MA deletions (⌬MA) or swaps of MA with other membrane binding domains are capable of directing the assembly of conditionally infectious viruses, but in these cases, assembly and replication efficiencies have tended to be compromised (17)(18)(19)(20)(21). IMPORTANCE The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of EnvA second role of HIV-1 MA is to facilitate the incorporation of envelope (Env) proteins into virions (22-25). How MA accomplishes this is complicated. One confounding issue is that some cell surface proteins can be assembled into HIV-1 particles in what appears to be a passive fashion (26). Indeed, this is how glycoproteins from other viruses can pseudotype with the cores of HIV-1 (17, 27-31). Presumably, PM proteins that localize to HIV-1 assembly sites can be incorporated into viruses as...