Current systems to study essential genes in the human malaria parasite Plasmodium falciparum are often inefficient and time intensive, and they depend on the genetic modification of the target locus, a process hindered by the low frequency of integration of episomal DNA into the genome. Here, we introduce a method, termed selection-linked integration (SLI), to rapidly select for genomic integration. SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, a strategy known as knock sideways, floxing to induce diCre-based excision of genes and knocking in altered gene copies. We demonstrated the power and robustness of this approach by validating it for more than 12 targets, including eight essential ones. We also localized and inducibly inactivated Kelch13, the protein associated with artemisinin resistance. We expect this system to be widely applicable for P. falciparum and other organisms with limited genetic tractability.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic 1 – 8 . Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3) 8 – 10 . It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers) 5 , 11 – 18 . Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1 JR-FL strain in complex with the antibody PGT151 19 . Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pretriggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies—and thus of interest for the design of immunogens—remains unknown.

Highlights d Inactivation of VPS45 abolishes the growth of malaria blood stage parasites d VPS45 is located near the parasite's food vacuole and Golgi d VPS45 is needed for the transport of host cell cytosol to the parasite's food vacuole d Host cell cytosol-filled transport vesicles display the endosomal marker PI(3)P

The HIV-1 envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4+ cells. Single-molecule Fluorescence Resonance Energy Transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pre-triggered conformation (State 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (State 2) into the three-CD4-bound open conformation (State 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream States 2 and 3. The structure of State 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pre-triggered State 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S), or by introducing a disulfide bridge between gp120 and gp41 designated “SOS” (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream towards States 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in State 1 by specific ligands such as the BMS entry inhibitors. The here-described identification of State 1-stabilizing conditions may enable structural characterization of the State 1 conformation of HIV-1 Env. IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pre-triggered (State 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pre-triggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in State 1, potentially facilitating structural characterization of this unknown conformational state.

Homologous recombination-based integration of plasmids into the genome of Plasmodium falciparum parasites is inefficient. The traditionally used drug cycling to obtain parasites with such integrations ('integrants') is time consuming and not always successful. Here we provide a protocol for the rapid selection of integrants and describe how to use it for endogenous gene tagging or to select parasites with specific gene disruptions. Using the appropriate tags, the gene product can then be functionally analysed using knock sideways. A protocol for a flow cytometry (FC) assay to assess the impact of inactivating the gene product on parasite development is also provided. These protocols accompany

The following paragraph should be added to the Acknowledgments. Bristol Myers Squibb (BMS) entry inhibitors BMS-626529/temsavir and prodrug BMS-663068/GSK3684934/fostemsavir were licensed to ViiV Healthcare in 2015. Fostemsavir is now an approved drug in the United States for highly treatmentexperienced patients with multidrug-resistant HIV-1 infection who are failing their current antiretroviral therapy regimen due to resistance, intolerance, or safety considerations.

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