Susceptibility of Toxoplasma gondii to autophagy in human cells relies on multiple interacting parasite loci.

IMPORTANCE: Autophagy is a process used by cells to recycle organelles and macromolecules and to eliminate intracellular pathogens. Previous studies have shown that some stains of Toxoplasma gondii are resistant to autophagy-dependent growth restriction, while others are highly susceptible. Although it is known that autophagy-mediated control requires activation by interferon gamma, the basis for why parasite strains differ in their susceptibility is unknown. Our findings indicate that susceptibility involves at least five unlinked parasite genes on different chromosomes, including several secretory proteins targeted to the parasite-containing vacuole and exposed to the host cell cytosol. Our findings reveal that susceptibility to autophagy-mediated growth restriction relies on differential recognition of parasite proteins exposed at the host-pathogen interface, thus identifying a new mechanism for cell-autonomous control of intracellular pathogens.

Overexpression screen of interferon-stimulated genes identifies RARRES3 as a restrictor of Toxoplasma gondii infection.

Toxoplasma gondii is an important human pathogen infecting an estimated one in three people worldwide. The cytokine interferon gamma (IFNγ) is induced during infection and is critical for restricting T. gondii growth in human cells. Growth restriction is presumed to be due to the induction of interferon-stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFNγ, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFNγ induced ISGs to identify factors that impact T. gondii infection in human cells. In addition to IRF1, which likely acts through the induction of numerous downstream genes, we identified RARRES3 as a single factor that restricts T. gondii infection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFNγ induced factor restricting T. gondii infection, the limited number of ISGs capable of restricting T. gondii infection when individually expressed suggests that IFNγ-mediated immunity to T. gondii infection is a complex, multifactorial process.

Toxoplasma gondii infection and its implications within the central nervous system.

Toxoplasma gondii is a parasite that infects a wide range of animals and causes zoonotic infections in humans. Although it normally only results in mild illness in healthy individuals, toxoplasmosis is a common opportunistic infection with high mortality in individuals who are immunocompromised, most commonly due to reactivation of infection in the central nervous system. In the acute phase of infection, interferon-dependent immune responses control rapid parasite expansion and mitigate acute disease symptoms. However, after dissemination the parasite differentiates into semi-dormant cysts that form within muscle cells and neurons, where they persist for life in the infected host. Control of infection in the central nervous system, a compartment of immune privilege, relies on modified immune responses that aim to balance infection control while limiting potential damage due to inflammation. In response to the activation of interferon-mediated pathways, the parasite deploys an array of effector proteins to escape immune clearance and ensure latent survival. Although these pathways are best studied in the laboratory mouse, emerging evidence points to unique mechanisms of control in human toxoplasmosis. In this Review, we explore some of these recent findings that extend our understanding for proliferation, establishment and control of toxoplasmosis in humans.

Comparative analysis of viral entry for Asian and African lineages of Zika virus.

Zika virus (ZIKV) is an emerging pathogen with global health and economic impacts. ZIKV circulates as two major lineages, Asian or African. The Asian lineage has recently been associated with significant disease in humans. Numerous studies have revealed differences between African and Asian ZIKV strains with respect to cellular infectivity, pathogenesis, and immune activation. Less is known about the mechanism of ZIKV entry and whether viral entry differs between strains. Here, we characterized ZIKV entry with two Asian and two African strains. All viruses exhibited a requirement for clathrin-mediated endocytosis and Rab5a function. Additionally, all ZIKV strains tested were sensitive to pH in the range of 6.5-6.1 and were reliant on endosomal acidification for infection. Finally, we provide direct evidence that ZIKV primarily fuses with late endosomes. These findings contribute new insight into the ZIKV entry process and suggest that divergent ZIKV strains enter cells in a highly conserved manner.

LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step.

Interferons (IFNs) contribute to cell-intrinsic antiviral immunity by inducing hundreds of interferon-stimulated genes (ISGs). In a screen to identify antiviral ISGs, we unexpectedly found that LY6E, a member of the LY6/uPAR family, enhanced viral infection. Here, we show that viral enhancement by ectopically expressed LY6E extends to several cellular backgrounds and affects multiple RNA viruses. LY6E does not impair IFN antiviral activity or signaling, but rather promotes viral entry. Using influenza A virus as a model, we narrow the enhancing effect of LY6E to uncoating after endosomal escape. Diverse mammalian orthologs of LY6E also enhance viral infectivity, indicating evolutionary conservation of function. By structure-function analyses, we identify a single amino acid in a predicted loop region that is essential for viral enhancement. Our study suggests that LY6E belongs to a class of IFN-inducible host factors that enhance viral infectivity without suppressing IFN antiviral activity.

Mucolipin-2 Cation Channel Increases Trafficking Efficiency of Endocytosed Viruses.

Receptor-mediated endocytosis is a cellular process commonly hijacked by viruses to enter cells. The stages of entry are well described for certain viruses, but the host factors that mediate each step are less well characterized. We previously identified endosomal cation channel mucolipin-2 (MCOLN2) as a host factor that promotes viral infection. Here, we assign a role for MCOLN2 in modulating viral entry. We show that MCOLN2 specifically promotes viral vesicular trafficking and subsequent escape from endosomal compartments. This mechanism requires channel activity, occurs independently of antiviral signaling, and broadly applies to enveloped RNA viruses that require transport to late endosomes for infection, including influenza A virus, yellow fever virus, and Zika virus. We further identify a rare allelic variant of human MCOLN2 that has a loss-of-function phenotype with respect to viral enhancement. These findings establish a mechanistic link between an endosomal cation channel and late stages of viral entry.IMPORTANCE Viruses must co-opt cellular processes to complete their life cycle. To enter cells, viruses frequently take advantage of cellular receptor-mediated endocytosis pathways. A growing number of host proteins are implicated in these viral uptake pathways. Here, we describe a new role for the gated cation channel MCOLN2 in viral entry. This endosomal protein modulates viral entry by enhancing the efficiency of viral trafficking through the endosomal system. Thus, MCOLN2-mediated enhancement of infection may represent a key vulnerability in the viral life cycle that could be targeted for therapeutic intervention.