Jean-Baptiste Marq

Functional Dissection of the Apicomplexan Glideosome Molecular Architecture

The glideosome of apicomplexan parasites is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45. We identify GAP40, a polytopic protein of the IMC, as an additional glideosome component and show that GAP45 is anchored to the PM and IMC via its N- and C-terminal extremities, respectively. While the C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. The orthologous Plasmodium falciparum GAP45 can fulfill this dual function, as shown by transgenera complementation, whereas the coccidian GAP45 homolog (designated here as) GAP70 specifically recruits the glideosome to the apical cap of the parasite.

Activation of the Beta Interferon Promoter by Unnatural Sendai Virus Infection Requires RIG-I and Is Inhibited by Viral C Proteins

ABSTRACT As infection with wild-type (wt) Sendai virus (SeV) normally activates beta interferon (IFN-β) very poorly, two unnatural SeV infections were used to study virus-induced IFN-β activation in mouse embryonic fibroblasts: (i) SeV-DI-H4, which is composed mostly of small, copyback defective interfering (DI) genomes and whose infection overproduces short 5′-triphosphorylated trailer RNAs (pppRNAs) and underproduces viral V and C proteins, and (ii) SeV-GFP(+/−), a coinfection that produces wt amounts of viral gene products but that also produces both green fluorescent protein (GFP) mRNA and its complement, which can form double-stranded RNA (dsRNA) with capped 5′ ends. We found that (i) virus-induced signaling to IFN-β depended predominantly on RIG-I (as opposed to mda-5) for both SeV infections, i.e., that RIG-I senses both pppRNAs and dsRNA without 5′-triphosphorylated ends, and (ii) it is the viral C protein (as opposed to V) that is primarily responsible for countering RIG-I-dependent signaling to IFN-β. Nondefective SeV that cannot specifically express C proteins not only cannot prevent the effects of transfected poly(I-C) or pppRNAs on IFN-β activation but also synergistically enhances these effects. SeV-Vminus infection, in contrast, behaves mostly like wt SeV and counteracts the effects of transfected poly(I-C) or pppRNAs.

Plasticity and redundancy among AMA–RON pairs ensure host cell entry of Toxoplasma parasites

Malaria and toxoplasmosis are infectious diseases caused by the apicomplexan parasites Plasmodium and Toxoplasma gondii, respectively. These parasites have developed an invasion mechanism involving the formation of a moving junction (MJ) that anchors the parasite to the host cell and forms a ring through which the parasite penetrates. The composition and the assembly of the MJ, and in particular the presence of protein AMA1 and its interaction with protein RON2 at the MJ, have been the subject of intense controversy. Here, using reverse genetics, we show that AMA1, a vaccine candidate, interacts with RON2 to maintain the MJ structural integrity in T. gondii and is subsequently required for parasite internalization. Moreover, we show that disruption of the AMA1 gene results in upregulation of AMA1 and RON2 homologues that cooperate to support residual invasion. Our study highlights a considerable complexity and molecular plasticity in the architecture of the MJ.

Short Double-stranded RNAs with an Overhanging 5′ ppp-Nucleotide, as Found in Arenavirus Genomes, Act as RIG-I Decoys

Arenavirus RNA genomes are initiated by a “prime and realign” mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended 5′ pppdsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that 5′ pppdsRNAs with a single unpaired 5′ ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended 5′ pppdsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5′ ppp. In contrast, 5′ pppdsRNAs with a single unpaired 5′ ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5′ ppp group.

The Amino-Terminal Extensions of the Longer Sendai Virus C Proteins Modulate pY701-Stat1 and Bulk Stat1 Levels Independently of Interferon Signaling

ABSTRACT The Sendai virus (SeV) C proteins are known to interact with Stat1 to prevent interferon (IFN)-induced pY701-Stat1 formation and IFN signaling. Nevertheless, pY701-Stat1 levels paradoxically increase during SeV infection. The C proteins also induce bulk Stat1 instability in some cells, similar to rubulavirus V proteins. We have found that SeV infection increases pY701-Stat1 levels even in cells in which bulk Stat1 levels strongly decrease. Remarkably, both the decrease in bulk Stat1 levels and the increase in pY701-Stat1 levels were found to be independent of the IFN signaling system, i.e., these events occur in mutant cells in which various components of the IFN signaling system have been disabled. Consistent with this, the C-induced decrease in Stat1 levels does not require Y701 of Stat1. We present evidence that C interacts with Stat1 in two different ways, one that prevents IFN-induced pY701-Stat1 formation and IFN signaling that has already been documented, and another that induces pY701-Stat1 formation (while decreasing bulk Stat1 levels) in a manner that does not require IFN signaling. These two types of Stat1 interaction are also distinguishable by C gene mutations. In particular, the IFN signaling-independent Stat1 interactions specifically require the amino-terminal extensions of the longer C proteins. The actions of the SeV C proteins in counteracting the cellular antiviral response are clearly more extensive than previously appreciated.

RIG-I and dsRNA-Induced IFNβ Activation

Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses), most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral pppRNAs remain unblocked during the infection. Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5. We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5′ tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5. We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.

Unpaired 5′ ppp-Nucleotides, as Found in Arenavirus Double-stranded RNA Panhandles, Are Not Recognized by RIG-I

Arenavirus and bunyavirus RNA genomes are unusual in that they are found in circular nucleocapsids, presumably due to the annealing of their complementary terminal sequences. Moreover, arenavirus genome synthesis initiates with GTP at position +2 of the template rather than at the precise 3′ end (position +1). After formation of a dinucleotide, 5′ pppGpCOH is then realigned on the template before this primer is extended. The net result of this “prime and realign” mechanism of genome initiation is that 5′ pppG is found as an unpaired 5′ nucleotide when the complementary genome ends anneal to form a double-stranded (dsRNA) panhandle. Using 5′ pppRNA made in vitro and purified so that all dsRNA side products are absent, we have determined that both this 5′ nucleotide overhang, as well as mismatches within the dsRNA (as found in some arenavirus genomes), clearly reduce the ability of these model dsRNAs to induce interferon upon transfection into cells. The presence of this unpaired 5′ ppp-nucleotide is thus another way that some viruses appear to use to avoid detection by cytoplasmic pattern recognition receptors.

Plasticity between MyoC- and MyoA-Glideosomes: An Example of Functional Compensation in Toxoplasma gondii Invasion

The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.

A short peptide at the amino terminus of the Sendai virus C protein acts as an independent element that induces STAT1 instability.

ABSTRACT The Sendai virus C protein acts to dismantle the interferon-induced cellular antiviral state in an MG132-sensitive manner, in part by inducing STAT1 instability. This activity of C maps to the first 23 amino acids (C1-23) of the 204-amino-acid (aa)-long protein (C1-204). C1-23 was found to act as an independent viral element that induces STAT1 instability, since this peptide fused to green fluorescent protein (C1-23/GFP) is at least as active as C1-204 in this respect. This peptide also induces the degradation of C1-23/GFP and other proteins to which it is fused. Most of C1-204, and particularly its amino-terminal half, is predicted to be structurally disordered. C1-23 as a peptide was found to be disordered by circular dichroism, and the first 11 aa have a strong potential to form an amphipathic α-helix in low concentrations of trifluoroethanol, which is thought to mimic protein-protein interaction. The critical degradation-determining sequence of C1-23 was mapped by mutation to eight residues near its N terminus: 4FLKKILKL11. All the large hydrophobic residues of 4FLKKILKL11, plus its ability to form an amphipathic α-helix, were found to be critical for STAT1 degradation. In contrast, C1-23/GFP self-degradation did not require 8ILKL11, nor the ability to form an α-helix throughout this region. Remarkably, C1-23/GFP also stimulated C1-204 degradation, and this degradation in trans required the same peptide determinants as for STAT1. Our results suggest that C1-204 coordinates its dual activities of regulating viral RNA synthesis and counteracting the host innate antiviral response by sensing both its own intracellular concentration and that of STAT1.

Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii.

Toxoplasma gondii possesses 11 rather atypical myosin heavy chains. The only myosin light chain described to date is MLC1, associated with myosin A, and contributing to gliding motility. In this study, we examined the repertoire of calmodulin‐like proteins in Apicomplexans, identified six putative myosin light chains and determined their subcellular localization in T. gondii and Plasmodium falciparum. MLC2, only found in coccidians, is associated with myosin D via its calmodulin (CaM)‐like domain and anchored to the plasma membrane of T. gondii via its N‐terminal extension. Molecular modeling suggests that the MyoD–MLC2 complex is more compact than the reported structure of Plasmodium MyoA–myosin A tail‐interacting protein (MTIP) complex. Anchorage of this MLC2 to the plasma membrane is likely governed by palmitoylation.