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We therefore generated a\n              <jats:italic>C. neoformans<\/jats:italic>\n              strain with a non-functional kinase, referred to as the dead-kinase (dk)\n              <jats:italic>Cn<\/jats:italic>\n              Arg1 strain (dkArg1). We verified that, although dk\n              <jats:italic>ARG1<\/jats:italic>\n              cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP\n              <jats:sub>3-4<\/jats:sub>\n              K activity. Using recombinant\n              <jats:italic>Cn<\/jats:italic>\n              Arg1 and\n              <jats:italic>Ca<\/jats:italic>\n              Ipk2\n              <jats:italic>,<\/jats:italic>\n              we confirmed that, unlike the IP\n              <jats:sub>3-4<\/jats:sub>\n              K homologs in humans and\n              <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n              ,\n              <jats:italic>Cn<\/jats:italic>\n              Arg1 and\n              <jats:italic>Ca<\/jats:italic>\n              Ipk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP\n              <jats:sub>3<\/jats:sub>\n              conversion is blocked in the dkArg1 and\n              <jats:italic>ARG1<\/jats:italic>\n              deletion (\n              <jats:italic>Cnarg1<\/jats:italic>\n              \u0394) strains and that 1-IP\n              <jats:sub>7<\/jats:sub>\n              and a recently discovered isomer (4\/6-IP\n              <jats:sub>7<\/jats:sub>\n              ) are made by wild-type\n              <jats:italic>C. neoformans<\/jats:italic>\n              . Importantly, the dkArg1 and\n              <jats:italic>Cnarg1<\/jats:italic>\n              \u0394 strains had similar virulence defects, including suppressed growth at 37\u00b0C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP\n              <jats:sub>3-4<\/jats:sub>\n              K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to\n              <jats:italic>S. cerevisiae<\/jats:italic>\n              since\n              <jats:italic>Cn<\/jats:italic>\n              Arg1 is dispensable for growth on different nitrogen sources. IP\n              <jats:sub>3-4<\/jats:sub>\n              K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>\n                The World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP\n                <jats:sub>3-4<\/jats:sub>\n                K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens,\n                <jats:italic>Cryptococcus neoformans<\/jats:italic>\n                and\n                <jats:italic>Candida albicans<\/jats:italic>\n                , and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP\n                <jats:sub>3-4<\/jats:sub>\n                K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP\n                <jats:sub>3-4<\/jats:sub>\n                K catalytic activity of\n                <jats:italic>Cn<\/jats:italic>\n                Arg1 promotes all virulence traits in\n                <jats:italic>C. neoformans<\/jats:italic>\n                that are attenuated by\n                <jats:italic>ARG1<\/jats:italic>\n                deletion\n                <jats:italic>,<\/jats:italic>\n                reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP\n                <jats:sub>3-4<\/jats:sub>\n                K catalytic site, as a new antifungal drug class.\n              <\/jats:p>\n            <\/jats:sec>\n          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