Abstract
Reoviruses are double-stranded RNA viruses that infect the mammalian respiratory and gastrointestinal tract. Reovirus infection elicits production of type I interferons (IFNs), which trigger antiviral pathways through the induction of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified, the functions of many of these genes are unknown. The interferon-inducible transmembrane (IFITM) proteins are one class of ISGs that restrict the cell entry of some enveloped viruses, including influenza A virus. One family member, IFITM3, localizes to late endosomes, where reoviruses undergo proteolytic disassembly; therefore, we sought to determine whether IFITM3 also restricts reovirus entry. IFITM3-expressing cell lines were less susceptible to infection by reovirus, as they exhibited significantly lower percentages of infected cells in comparison to control cells. Reovirus replication was also significantly reduced in IFITM3-expressing cells. Additionally, cells expressing an shRNA targeting IFITM3 exhibited a smaller decrease in infection after IFN treatment than the control cells, indicating that endogenous IFITM3 restricts reovirus infection. However, IFITM3 did not restrict entry of reovirus infectious subvirion particles (ISVPs), which do not require endosomal proteolysis, indicating that restriction occurs in the endocytic pathway. Proteolysis of outer capsid protein μ1 was delayed in IFITM3-expressing cells in comparison to control cells, suggesting that IFITM3 modulates the function of late endosomal compartments either by reducing the activity of endosomal proteases or delaying the proteolytic processing of virions. These data provide the first evidence that IFITM3 restricts infection by a nonenveloped virus and suggest that IFITM3 targets an increasing number of viruses through a shared requirement for endosomes during cell entry.
Highlights
The interferon-stimulated gene (ISG) IFITM3 restricts endosomal entry of enveloped viruses
As the mechanism for this restriction is not defined, we tested whether IFITM3 restricts entry of a non-enveloped virus with similar membrane penetration requirements, mammalian orthoreovirus
Expression levels of IFITM3 in mock- and IFN-␣-treated cells were determined by Quantitative Reverse Transcriptase Polymerase Chain Reaction (qPCR) and by immunoblotting (Fig. 1 and data not shown)
Summary
The interferon-stimulated gene (ISG) IFITM3 restricts endosomal entry of enveloped viruses. Proteolysis of outer capsid protein 1 was delayed in IFITM3-expressing cells in comparison to control cells, suggesting that IFITM3 modulates the function of late endosomal compartments either by reducing the activity of endosomal proteases or delaying the proteolytic processing of virions These data provide the first evidence that IFITM3 restricts infection by a nonenveloped virus and suggest that IFITM3 targets an increasing number of viruses through a shared requirement for endosomes during cell entry. IFITM3 likely alters the dynamics of endosomal uncoating, either leading to inefficient membrane penetration or lysosomal degradation of viral particles This represents the first evidence that IFITM3 can restrict the infection of non-enveloped viruses that utilize endosome-dependent cell entry mechanisms and provides further mechanistic evidence for how IFITM3 restricts an increasing number of viruses by targeting a shared requirement for endosomes for cell entry
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