HIV-1 Gag Recruits Oligomeric Vpr via Two Binding Sites in p6, but Both Mature p6 and Vpr Are Rapidly Lost upon Target Cell Entry.

The p12 region of murine leukemia virus (MLV) Gag and the p6 region of HIV-1 Gag contain late domains required for virus budding. Additionally, the accessory protein Vpr is recruited into HIV particles via p6. Mature p12 is essential for early viral replication events, but the role of mature p6 in early replication is unknown. Using a proviral vector in which the gag and pol reading frames are uncoupled, we have performed the first alanine-scanning mutagenesis screens across p6 to probe its importance for early HIV-1 replication and to further understand its interaction with Vpr. The infectivity of our mutants suggests that, unlike p12, p6 is not important for early viral replication. Consistent with this, we observed that p6 is rapidly lost upon target cell entry in time course immunoblot experiments. By analyzing Vpr incorporation into p6 mutant virions, we identified that the 15-FRFG-18 and 41-LXXLF-45 motifs previously identified as putative Vpr-binding sites are important for Vpr recruitment but that the 34-ELY-36 motif also suggested to be a Vpr-binding site is dispensable. Additionally, disrupting Vpr oligomerization together with removing either binding motif in p6 reduced Vpr incorporation ∼25- to 50-fold more than inhibiting Vpr oligomerization alone and ∼10- to 25-fold more than deleting each p6 motif alone, implying that multivalency/avidity is important for the interaction. Interestingly, using immunoblotting and immunofluorescence, we observed that most Vpr is lost concomitantly with p6 during infection but that a small fraction remains associated with the viral capsid for several hours. This has implications for the function of Vpr in early replication. IMPORTANCE The p12 protein of MLV and the p6 protein of HIV-1 are both supplementary Gag cleavage products that carry proline-rich motifs that facilitate virus budding. Importantly, p12 has also been found to be essential for early viral replication events. However, while Vpr, the only accessory protein packaged into HIV-1 virions, is recruited via the p6 region of Gag, the function of both mature p6 and Vpr in early replication is unclear. Here, we have systematically mutated the p6 region of Gag and have studied the effects on HIV infectivity and Vpr packaging. We have also investigated what happens to p6 and Vpr during early infection. We show that, unlike p12, mature p6 is not required for early replication and that most of the mature p6 and the Vpr that it recruits are lost rapidly upon target cell entry. This has implications for the role of Vpr in target cells.

Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis.

The murine leukaemia virus (MLV) Gag cleavage product, p12, is essential for both early and late steps in viral replication. The N-terminal domain of p12 binds directly to capsid (CA) and stabilises the mature viral core, whereas defects in the C-terminal domain (CTD) of p12 can be rescued by addition of heterologous chromatin binding sequences (CBSs). We and others hypothesised that p12 tethers the pre-integration complex (PIC) to host chromatin ready for integration. Using confocal microscopy, we have observed for the first time that CA localises to mitotic chromatin in infected cells in a p12-dependent manner. GST-tagged p12 alone, however, did not localise to chromatin and mass-spectrometry analysis of its interactions identified only proteins known to bind the p12 region of Gag. Surprisingly, the ability to interact with chromatin was conferred by a single amino acid change, M63I, in the p12 CTD. Interestingly, GST-p12_M63I showed increased phosphorylation in mitosis relative to interphase, which correlated with an increased interaction with mitotic chromatin. Mass-spectrometry analysis of GST-p12_M63I revealed nucleosomal histones as primary interactants. Direct binding of MLV p12_M63I peptides to histones was confirmed by biolayer-interferometry (BLI) assays using highly-avid recombinant poly-nucleosomal arrays. Excitingly, using this method, we also observed binding between MLV p12_WT and nucleosomes. Nucleosome binding was additionally detected with p12 orthologs from feline and gibbon ape leukemia viruses using both pull-down and BLI assays, indicating that this a common feature of gammaretroviral p12 proteins. Importantly, p12 peptides were able to block the binding of the prototypic foamy virus CBS to nucleosomes and vice versa, implying that their docking sites overlap and suggesting a conserved mode of chromatin tethering for different retroviral genera. We propose that p12 is acting in a similar capacity to CPSF6 in HIV-1 infection by facilitating initial chromatin targeting of CA-containing PICs prior to integration.

The N-terminus of murine leukaemia virus p12 protein is required for mature core stability.

The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

RH5-Basigin interaction plays a major role in the host tropism of Plasmodium falciparum.

Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175-GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175-GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)-Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5-BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host.

Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry.

BACKGROUND: Invasion of host erythrocytes by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion involves recognition events between erythrocyte receptors and ligands on the merozoite, the invasive blood form of the parasite. Identifying and characterizing host-parasite interactions is impeded by the biochemical challenges of working with membrane-embedded glycoprotein receptors. For example, the interaction between P. falciparum erythrocyte binding antigen 175 (PfEBA175) and glycophorin A (GYPA) depends on post-translational modifications that are not easily added in recombinant expression systems, and the use of native GYPA is limited by the hydrophobic transmembrane region, making it difficult to biochemically manipulate. It would, therefore, be desirable to perform quantitative binding assays with receptors embedded within the membranes of intact human erythrocytes. METHODS: The extracellular region of GYPA was over-expressed as a soluble protein in HEK293E cells. This protein was characterized, sialylated and evaluated for binding to the PfEBA175 protein. The label-free and free-solution assay, backscattering interferometry (BSI), was used to perform binding assays of two well-characterized P. falciparum invasion ligands to intact unmodified human erythrocytes. RESULTS: Findings indicate that the post-translational modifications present on native GYPA are required for it to bind recombinant PfEBA175 and that these modifications cannot be recapitulated in vitro using mammalian overexpression methods. Here, BSI was used to obtain quantitative, high fidelity interaction determinations on intact, unmodified erythrocytes. Using BSI and purified recombinant proteins constituting the entire ectodomains of the P. falciparum merozoite ligands PfEBA175 and PfRH5, K Ds of 1.1 µM and 50 nM were measured for the PfRH5-BSG and PfEBA175-GYPA interactions, respectively, in good agreement with previous biophysical measurements of these interactions. CONCLUSIONS: These results demonstrate that BSI can be used to detect and quantify the interactions of two merozoite invasion ligands with their receptors on intact human erythrocytes. BSI assays were performed on unlabelled, free-solution proteins in their native environment, requiring only nanomoles of recombinant protein. This study suggests that BSI can be used to investigate host-parasite protein interactions without the limitations of other assay platforms, and therefore represents a valuable new method to investigate the molecular mechanisms involved in erythrocyte invasion by P. falciparum.

A library of functional recombinant cell-surface and secreted P. falciparum merozoite proteins.

Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.

Biochemical analysis of the Plasmodium falciparum erythrocyte-binding antigen-175 (EBA175)-glycophorin-A interaction: implications for vaccine design.

PfEBA175 has an important role in the invasion of human erythrocytes by Plasmodium falciparum and is therefore considered a high priority blood-stage malaria vaccine candidate. PfEBA175 mediates adhesion to erythrocytes through binding of the Duffy-binding-like (DBL) domains in its extracellular domain to Neu5Acα2-3Gal displayed on the O-linked glycans of glycophorin-A (GYPA). Because of the difficulties in expressing active full-length (FL) P. falciparum proteins in a recombinant form, previous analyses of the PfEBA175-GYPA interaction have largely focused on the DBL domains alone, and therefore they have not been performed in the context of the native protein sequence. Here, we express the entire ectodomain of PfEBA175 (PfEBA175 FL) in soluble form, allowing us to compare the biochemical and immunological properties with a fragment containing only the tandem DBL domains ("region II," PfEBA175 RII). Recombinant PfEBA175 FL bound human erythrocytes in a trypsin and neuraminidase-sensitive manner and recognized Neu5Acα2-3Gal-containing glycans, confirming its biochemical activity. A quantitative binding analysis showed that PfEBA175 FL interacted with native GYPA with a KD ∼0.26 µM and is capable of self-association. By comparison, the RII fragment alone bound GYPA with a lower affinity demonstrating that regions outside of the DBL domains are important for interactions with GYPA; antibodies directed to these other regions also contributed to the inhibition of parasite invasion. These data demonstrate the importance of PfEBA175 regions other than the DBL domains in the interaction with GYPA and merit their inclusion in an EBA175-based vaccine.

Gammaretroviruses tether to mitotic chromatin by directly binding nucleosomal histone proteins.

The gammaretroviral gag cleavage product, p12, is essential for replication at both early and late stages of the virus life cycle. During the early stage of infection, the viral core is released into the cytoplasm, the viral RNA genome is reversed transcribed to cDNA and this viral DNA is then integrated into the host cell chromatin to form a provirus. The p12 protein has N- and C-terminal domains (NTD and CTD) that are required for steps leading up to integration, but the molecular details of their functions remain poorly characterised. Using the prototypic gammaretrovirus, murine leukemia virus (MLV) as a model, we recently showed that the NTD of p12 directly binds to and stabilises the capsid (CA) lattice of the viral core. Alterations to the CTD of MLV p12 prevented the viral pre-integration complex (PIC) tethering to host chromatin in mitosis, and this could be partially rescued by addition of a heterologous chromatin binding motif. In this study we demonstrated that the CTD of p12 directly binds to nucleosomal histone proteins, targeting not only p12 but also CA to mitotic chromatin. Additionally, cell-cycle-dependent phosphorylation of p12 appeared to increase the affinity of p12 for chromatin in mitosis relative to interphase. Thus, we have revealed how p12 can link the CA-containing PIC to mitotic chromatin, ready for integration. Importantly, we observed that direct binding to nucleosomes is a conserved feature of p12 orthologs across the gammaretrovirus genus and that the nucleosomal docking site is potentially shared with that of spumaretroviral Gag proteins.

New antigens for a multicomponent blood-stage malaria vaccine.

An effective blood-stage vaccine against Plasmodium falciparum remains a research priority, but the number of antigens that have been translated into multicomponent vaccines for testing in clinical trials remains limited. Investigating the large number of potential targets found in the parasite proteome has been constrained by an inability to produce natively folded recombinant antigens for immunological studies. We overcame these constraints by generating a large library of biochemically active merozoite surface and secreted full-length ectodomain proteins. We then systematically examined the antibody reactivity against these proteins in a cohort of Kenyan children (n = 286) who were sampled at the start of a malaria transmission season and prospectively monitored for clinical episodes of malaria over the ensuing 6 months. We found that antibodies to previously untested or little-studied proteins had superior or equivalent potential protective efficacy to the handful of current leading malaria vaccine candidates. Moreover, cumulative responses to combinations comprising 5 of the 10 top-ranked antigens, including PF3D7_1136200, MSP2, RhopH3, P41, MSP11, MSP3, PF3D7_0606800, AMA1, Pf113, and MSRP1, were associated with 100% protection against clinical episodes of malaria. These data suggest not only that there are many more potential antigen candidates for the malaria vaccine development pipeline but also that effective vaccination may be achieved by combining a selection of these antigens.

A full-length recombinant Plasmodium falciparum PfRH5 protein induces inhibitory antibodies that are effective across common PfRH5 genetic variants.

The lack of an effective licensed vaccine remains one of the most significant gaps in the portfolio of tools being developed to eliminate Plasmodium falciparum malaria. Vaccines targeting erythrocyte invasion - an essential step for both parasite development and malaria pathogenesis - have faced the particular challenge of genetic diversity. Immunity-driven balancing selection pressure on parasite invasion proteins often results in the presence of multiple, antigenically distinct, variants within a population, leading to variant-specific immune responses. Such variation makes it difficult to design a vaccine that covers the full range of diversity, and could potentially facilitate the evolution of vaccine-resistant parasite strains. In this study, we investigate the effect of genetic diversity on invasion inhibition by antibodies to a high priority P. falciparum invasion candidate antigen, P. falciparum Reticulocyte Binding Protein Homologue 5 (PfRH5). Previous work has shown that virally delivered PfRH5 can induce antibodies that protect against a wide range of genetic variants. Here, we show that a full-length recombinant PfRH5 protein expressed in mammalian cells is biochemically active, as judged by saturable binding to its receptor, basigin, and is able to induce antibodies that strongly inhibit P. falciparum growth and invasion. Whole genome sequencing of 290 clinical P. falciparum isolates from across the world identifies only five non-synonymous PfRH5 SNPs that are present at frequencies of 10% or more in at least one geographical region. Antibodies raised against the 3D7 variant of PfRH5 were able to inhibit nine different P. falciparum strains, which between them included all of the five most common PfRH5 SNPs in this dataset, with no evidence for strain-specific immunity. We conclude that protein-based PfRH5 vaccines are an urgent priority for human efficacy trials.