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Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1–5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment—which contributes to virologic failure, resistance generation and viral transmission—as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7–9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV. The small molecule GS-6207, which disrupts the function of the HIV capsid protein, shows potential as a long-acting therapeutic agent for the treatment and prevention of HIV infection.

Five histone deacetylase genes (HDA1, RPD3, HOS1, HOS2,andHOS3) have been cloned fromCandida albicansand characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylasesHDA1andRPD3is down-regulated in the opaque phase of the white-opaque transition in strain WO-1.HOS3is selectively transcribed as a 2.5-kb transcript in the white phase and as a less-abundant 2.3-kb transcript in the opaque phase.HDA1andRPD3were independently deleted in strain WO-1, and both switching between the white and opaque phases and the downstream regulation of phase-specific genes were analyzed. Deletion ofHDA1resulted in an increase in the frequency of switching from the white phase to the opaque phase, but had no effect on the frequency of switching from the opaque phase to the white phase. Deletion ofRPD3resulted in an increase in the frequency of switching in both directions. Deletion ofHDA1resulted in reduced white-phase-specific expression of theEFG13.2-kb transcript,but had no significant effect on white-phase-specific expression ofWH11or opaque-phase-specific expression ofOP4, SAP1,andSAP3.Deletion ofRPD3resulted in reduced opaque-phase-specific expression ofOP4, SAP1,andSAP3and a slight reduction of white-phase-specific expression ofWH11and 3.2-kbEFG1. Deletion of neitherHDA1norRPD3affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein geneMCM1, which has been implicated in the regulation of opaque-phase-specific gene expression. In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genesHDA1andRPD3play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.

SUMMARY: Using degenerate primers of highly conserved regions of two-component response regulators for PCR amplification, a two-component response regulator was cloned from Candida albicans that is homologous to nik-l+ of Neurospora crassa. This two-component hybrid kinase, CaNIKl, also shows features of bacterial two-component response regulators, including a putative unorthodox second histidine kinase motif at the carboxy-terminal end. CaNIKl was expressed at low levels in both the white and opaque switch phenotypes and in the bud and hyphal growth forms of C. albicans strain WO-1, but in both developmental programmes, the level of transcript was modulated (levels were higher in opaque cells and in hyphae). Partial deletion of both CaNIKl alleles, by which the histidine autokinase- and ATP-binding domains were removed, did not inhibit either high-frequency phenotypic switching or the bud-hypha transition in high salt concentrations, but in both cases the efficiency of the developmental process was reduced.

Candida albicans strain WO-1 undergoes two developmental programs, the bud-hypha transition and high-frequency phenotypic switching in the form of the white-opaque transition. The WH11 gene is expressed in the white budding phase but is inactive in the white hyphal phase and in the opaque budding phase. WH11 expression, therefore, is regulated in the two developmental programs. Through fusions between deletion derivatives of the WH11 promoter and the newly developed Renilla reniformis luciferase, the WH11 promoter has been characterized in the two developmental programs. Three transcription activation sequences, two strong and one weak, are necessary for the full expression of WH11 in the white budding phase, but no negative regulatory sequences were revealed as playing a role in either the white hyphal phase or the opaque budding phase. These results suggest that regulation is solely through activation in the white budding phase and the same mechanism, therefore, is involved in regulating the differential expression of WH11 in the alternative white and opaque phases of switching and the budding and hyphal phases of dimorphism.

The Candida albicans gene EFG1 encodes a putative trans -acting factor. In strain WO-1, which undergoes the white-opaque transition, EFG1 is transcribed as a 3.2-kb mRNA in white-phase cells and a less-abundant 2.2-kb mRNA in opaque-phase cells. cDNA sequencing and 5′ rapid amplification of cDNA ends analysis demonstrate that the major difference in molecular mass of the two transcripts is due to different transcription start sites. EFG1 null mutants form opaque-phase colonies and express the opaque-phase cell phenotype at 25°C. When shifted from 25 to 42°C, mutant opaque-phase cells undergo phenotypic commitment to the white phase, which includes deactivation of the opaque-phase-specific gene OP4 and activation of the white-phase-specific gene WH11 , as do wild-type opaque-phase cells. After the commitment event, EFG1 null mutant cells form daughter cells which have the smooth (pimpleless) surface of white-phase cells but the elongate morphology of opaque-phase cells. Taken together, these results demonstrate that EFG1 expression is not essential for the switch event per se, but is essential for a subset of phenotypic characteristics necessary for the full expression of the phenotype of white-phase cells. These results demonstrate that EFG1 is not the site of the switch event, but is, rather, downstream of the switch event.

Although Candida glabrata has emerged in recent years as a major fungal pathogen, there have been no reports demonstrating that it undergoes either the bud-hypha transition or high-frequency phenotypic switching, two developmental programs believed to contribute to the pathogenic success of other Candida species. Here it is demonstrated that C. glabrata undergoes reversible, high-frequency phenotypic switching between a white (Wh), light brown (LB), and dark brown (DB) colony phenotype discriminated on an indicator agar containing 1 mM CuSO 4 . Switching regulates the transcript level of the MT-II metallothionein gene(s) and a newly discovered gene for a hemolysin-like protein, HLP . The relative MT-II transcript levels in Wh, LB, and DB cells grown in the presence of CuSO 4 are 1:27:81, and the relative transcript levels of HLP are 1:20:35. The relative MT-II and HLP transcript levels in cells grown in the absence of CuSO 4 are 1:20:30 and 1:20:25, respectively. In contrast, switching has little or no effect on the transcript levels of the genes MT-I , AMT-I , TRPI , HIS3 , EPAI , and PDHI . Switching of C. glabrata is not associated with microevolutionary changes identified by the DNA fingerprinting probe Cg6 and does not involve tandem amplification of the MT-IIa gene, which has been shown to occur in response to elevated levels of copper. Finally, switching between Wh, LB, and DB occurred in all four clinical isolates examined in this study. As in Candida albicans , switching in C. glabrata may provide colonizing populations with phenotypic plasticity for rapid responses to the changing physiology of the host, antibiotic treatment, and the immune response, through the differential regulation of genes involved in pathogenesis. More importantly, because C. glabrata is haploid, a mutational analysis of switching is now feasible.

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.