Zika virus prM protein contains cholesterol binding motifs required for virus entry and assembly.

For successful infection of host cells and virion production, enveloped viruses, including Zika virus (ZIKV), extensively rely on cellular lipids. However, how virus protein-lipid interactions contribute to the viral life cycle remains unclear. Here, we employ a chemo-proteomics approach with a bifunctional cholesterol probe and show that cholesterol is closely associated with the ZIKV structural protein prM. Bioinformatic analyses, reverse genetics alongside with photoaffinity labeling assays, and atomistic molecular dynamics simulations identified two functional cholesterol binding motifs within the prM transmembrane domain. Loss of prM-cholesterol association has a bipartite effect reducing ZIKV entry and leading to assembly defects. We propose a model in which membrane-resident M facilitates cholesterol-supported lipid exchange during endosomal entry and, together with cholesterol, creates a platform promoting virion assembly. In summary, we identify a bifunctional role of prM in the ZIKV life cycle by mediating viral entry and virus assembly in a cholesterol-dependent manner.

Identification of host dependency factors involved in SARS-CoV-2 replication organelle formation through proteomics and ultrastructural analysis.

IMPORTANCE: Remodeling of the cellular endomembrane system by viruses allows for efficient and coordinated replication of the viral genome in distinct subcellular compartments termed replication organelles. As a critical step in the viral life cycle, replication organelle formation is an attractive target for therapeutic intervention, but factors central to this process are only partially understood. In this study, we corroborate that two viral proteins, nsp3 and nsp4, are the major drivers of membrane remodeling in SARS-CoV-2 infection. We further report a number of host cell factors interacting with these viral proteins and supporting the viral replication cycle, some of them by contributing to the formation of the SARS-CoV-2 replication organelle.

The Compound SBI-0090799 Inhibits Zika Virus Infection by Blocking De Novo Formation of the Membranous Replication Compartment.

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Toward this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single-amino-acid substitutions in the N-terminal region of nonstructural protein NS4A, arguing this is the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as a selective and potent inhibitor of Zika virus (ZIKV) replication using a high-throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel nonenzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this nonstructural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.

Replication-Independent Generation and Morphological Analysis of Flavivirus Replication Organelles.

Positive-strand RNA viruses replicate in distinct membranous structures called replication organelles (ROs). Mechanistic studies of RO formation have been difficult because perturbations affecting viral replication have an impact on viral protein amounts, thus affecting RO biogenesis. Here, we present a detailed guide on how to use a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation), inducing bona fide flavivirus ROs in transfected cells. This will be useful for mechanistic studies of viral and cellular factors driving flavivirus RO biogenesis. For complete details on the use and execution of this protocol, please refer to Cerikan et al. (2020).

Evolutionarily distant I domains can functionally replace the essential ligand-binding domain of Plasmodium TRAP.

Inserted (I) domains function as ligand-binding domains in adhesins that support cell adhesion and migration in many eukaryotic phyla. These adhesins include integrin αß heterodimers in metazoans and single subunit transmembrane proteins in apicomplexans such as TRAP in Plasmodium and MIC2 in Toxoplasma. Here we show that the I domain of TRAP is essential for sporozoite gliding motility, mosquito salivary gland invasion and mouse infection. Its replacement with the I domain from Toxoplasma MIC2 fully restores tissue invasion and parasite transmission, while replacement with the aX I domain from human integrins still partially restores liver infection. Mutations around the ligand binding site allowed salivary gland invasion but led to inefficient transmission to the rodent host. These results suggest that apicomplexan parasites appropriated polyspecific I domains in part for their ability to engage with multiple ligands and to provide traction for emigration into diverse organs in distant phyla.

A Non-Replicative Role of the 3' Terminal Sequence of the Dengue Virus Genome in Membranous Replication Organelle Formation.

Dengue virus (DENV) and Zika virus (ZIKV), members of the Flavivirus genus, rearrange endoplasmic reticulum membranes to induce invaginations known as vesicle packets (VPs), which are the assumed sites for viral RNA replication. Mechanistic information on VP biogenesis has so far been difficult to attain due to the necessity of studying their formation under conditions of viral replication, where perturbations reducing replication will inevitably impact VP formation. Here, we report a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation) that induces bona fide DENV and ZIKV VPs that are morphologically indistinguishable from those in infected cells. Using this system, we demonstrate that sequences in the 3' terminal RNA region of the DENV, but not the ZIKV genome, contribute to VP formation in a non-replicative manner. These results validate the pIRO system that opens avenues for mechanistically dissecting virus replication from membrane reorganization.

Parasites shape community structure and dynamics in freshwater crustaceans.

Parasites directly and indirectly influence the important interactions among hosts such as competition and predation through modifications of behaviour, reproduction and survival. Such impacts can affect local biodiversity, relative abundance of host species and structuring of communities and ecosystems. Despite having a firm theoretical basis for the potential effects of parasites on ecosystems, there is a scarcity of experimental data to validate these hypotheses, making our inferences about this topic more circumstantial. To quantitatively test parasites' role in structuring host communities, we set up a controlled, multigenerational mesocosm experiment involving four sympatric freshwater crustacean species that share up to four parasite species. Mesocosms were assigned to either of two different treatments, low or high parasite exposure. We found that the trematode Maritrema poulini differentially influenced the population dynamics of these hosts. For example, survival and recruitment of the amphipod Paracalliope fluviatilis were dramatically reduced compared to other host species, suggesting that parasites may affect their long-term persistence in the community. Relative abundances of crustacean species were influenced by parasites, demonstrating their role in host community structure. As parasites are ubiquitous across all communities and ecosystems, we suggest that the asymmetrical effects we observed are likely widespread structuring forces.

Trypanosoma brucei infection protects mice against malaria.

Sleeping sickness and malaria are parasitic diseases with overlapping geographical distributions in sub-Saharan Africa. We hypothesized that the immune response elicited by an infection with Trypanosoma brucei, the etiological agent of sleeping sickness, would inhibit a subsequent infection by Plasmodium, the malaria parasite, decreasing the severity of its associated pathology. To investigate this, we established a new co-infection model in which mice were initially infected with T. brucei, followed by administration of P. berghei sporozoites. We observed that a primary infection by T. brucei significantly attenuates a subsequent infection by the malaria parasite, protecting mice from experimental cerebral malaria and prolonging host survival. We further observed that an ongoing T. brucei infection leads to an accumulation of lymphocyte-derived IFN-γ in the liver, limiting the establishment of a subsequent hepatic infection by P. berghei sporozoites. Thus, we identified a novel host-mediated interaction between two parasitic infections, which may be epidemiologically relevant in regions of Trypanosoma/Plasmodium co-endemicity.

A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy.

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies.

Ultrastructural Characterization of Zika Virus Replication Factories.

A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.