Abstract
IFITM3, a protein induced by interferon, restricts reovirus infection in host cells and inhibits the viral replication. It affects the proper penetration of the virus by altering the dynamics of the viral uncoating in the endosomes. This study is the first in characterizing the anti-viral activity of IFITM3 on non enveloped viruses that depend on endosome for cellular entry.
Following contagion with a virus, the body triggers the release of a class of cytokines, interferons that are involved in first line of defense against infection. Type I interferons are specifically induced in the cells following insult with both DNA and RNA viruses. Release of interferon molecules invokes an “antiviral state” in these infected cells. The type I interferon, bind to their specific interferon receptor (type 1) and induce the activity of a number of Interferon stimulated genes (ISG). A large number of ISG are induced following interferon signaling. These genes induce the synthesis of proteins that are involved in various anti-viral pathways, such as protein synthesis inhibition, degradation of viral RNA etc. The identification of antiviral role of these various interferon induced protein products has significance in providing critical knowledge for developing anti viral therapy.
Interferon inducible transmembrane proteins (IFITM) belong to the family of proteins that are induced by Interferon. IFITM belongs to the CD225 family whose membranes share homology in their structure. The anti-viral activity of IFITM -1, -2 and -3 has been demonstrated on Influenza A, West Nile, dengue, SARS and filoviruses. The IFITM proteins prevent and/or restrict the entry of these viruses. The IFITM3 protein demonstrates the highest efficacy in prevention and inhibition of transmission of influenza A virus in-vitro cell culture assays, in vivo mice experiments and in infected human beings. IFITM3 proteins trap the virus particles in the endosomes and prevent proper uncoating resulting in their death by the lysosomes. The authors had developed this model based on their observations on the inhibitory activity of IFITM3 on enveloped viruses such as the influenza and SARS virus.
In this study the authors investigate and characterize the anti-viral activity of the interferon inducible IFITM3 protein on non-enveloped mammalian orthoreovirus. Rotavirus belongs to this family of non-enveloped viruses. These virus particles are made of dsRNA encapsulated by protein shell or capsid. These viruses have segmented genome (10 segments) of variable length. The entry of the reovirus to their host cells is a multistep process. The first step involves binding to carbohydrate (sialic acid) on the host cell followed by interaction with the receptor JAM-A (junction adhesion molecule A). 1 integrin targets the virus to endosomes. It is in these endosomal compartments where uncoating of virus takes place. IFITM3 also localize to the late endosomes and control the replication of reovirus and prevent their infection. The effect of the IFITM3 on the viral machinery occurs at the stage of endosomal penetration only. The dynamics of the endosomal uncoating is modified and resulting in improper penetration through the membrane or degradation by lysosomal acid following IFITM3 signaling.
The experimental procedure involved use of modified cells that were engineered to stably express IFITM-3. Additionally, stable cell lines generated by transduction of control and IFITM3 shRNA lentiviral particles were also used. T1L and T3D were two reovirs strains that were used to infect the cells. Mutants of the viral particles that were resistant to protease cleavage and could generate infectious subvirion particles (ISVP) were also utilized. The virion particles were produced from L-cell lysates, plaque purified and concentrated and titer measured. Antibody tagged with a label with affinity for antigens on the reovirus was used in fluorescent microscopy. Degree of viral replication was measured by a plaque assay. Human cells were incubated with virion particles under different experimental condition and stimuli from interferon and viral yields measured. qPCR was performed to measure the expression of human IFITM-3 following stimulation by interferon in either the control cells or cells transfected with shIFITM-3 particles or cells stably expressing the IFITM-3.
Results
Induction of IFN increased the level of IFITM3 protein and mRNA levels in cells expressing IFITM3 at a much greater level than in control cells. On the other hand, cells expressing shIFITM3 had a much weaker response to IFN induction, both at mRNA and protein expression level. This first experiment verified the efficient working of the modified cells.
Expression of IFITM-3 lowers the degree of infection and viral replication. Hela and U2OS cells that stably expressed the IFITM-3 protein demonstrated a lower percentage of infection as opposed to control cells following incubation with T1L and T3D reovirus strains. The degree of infection was dependent on the viral titer measured by m.o.i. The degree of infection was measured by fluorescent focus assay. Antibody tagged with a label having affinity for antigens on the reovirus was used in the assay. These fluorescently labeled cells were used for measuring viral antigen positive cells as an indicator of degree of infection. Viral particles with very high m.o.i. saturated the degree of infection or in other words the cells expressing IFITM-3 were minimally able to inhibit the infection percentage. Following infection with virion T3D (m.o.i of 1 pfu/cell), viral titers were measured at various time points and mean viral yields calculated. Cells that could stably express IFITM-3 showed significantly lower (5-10 fold) viral yields as compared to control cells. At this m.o.i, the infected cells produced interferon -1 and could prime the uninduced cells for increased IFITM-3 expression. At a very high m.o.i of 100pfu/cell, the priming ability was compromised and lower inhibition of replication was observed. This demonstrates that over expression of IFITM3 restricts but does not completely stop infection.
Infection by reovirus induced the expression of IFITM3 measured by its mRNA levels using qPCR. It was observed that a 5 fold increase in mRNA level of IFITM3 occurred 24 hours following infection with the T3D virion particles at m.o.i of 100 pfu/cell, as opposed to cells that were mock infected. The increase in mRNA level was first observed at 12 hours consistent with its requirement for induction by IFN. Similar results were observed at the level of expression of IFITM3 protein by western blot analysis.
Hela cells which stably expressed short hairpin RNA targeting IFITM3 (shIFITM3) had lower amount of IFITM3 expressed at protein and mRNA levels. Subsequently, control and shIFITM3 expressing cells were induced by 100 IU of IFN- 6 hours prior to incubation with the virion particle. The presence of the shIFITM3 made the host Hela and U2OS cells more sensitive to infection. shIFITM3 expressing cells were less effective in controlling infection by the virion particle following induction with IFN as opposed to control cells. Induction with IFN reduced the replication of reovirus on the control cells; however, in cell expressing shIFITM3, induction with IFN did not lower rate and amount of viral replication. This importantly demonstrated that expression of IFITM3 following IFN induction, could restrict viral replication in the host cells. This exhibited the cause and effect phenomenon between IFN induction and inhibition of viral replication and infection by IFITM3.
Cells that stably expressed IFITM3 prevented the viral entry following infection with the T3D or T1L particles. However, when ISVP particles were used to infect the stably expressing cells no inhibition of infection or entry to cells was observed. The ‘ISVP particles’ had the ability to bypass the endosomal proteolysis. This demonstrated that the IFITM3 inhibited the late endosomal processing of reovirus. Following this discovery, the researchers wanted to understand the exact mechanism or step modified or inhibited by IFITM3. The IFITM3 expressing cells were transfected with tagged endosomal markers, Rab 7 and RILP and evaluated for co-localization with reovirus. No difference in control or stably expressing cells was observed in the co-localization indicating that IFITM3 does not alter endosomal trafficking.
An assay utilized the addition of ammonium chloride to various time points. Addition of ammonium chloride inhibited acidification of endosomes. The control cells infected with rsT3D escaped the blockade sooner than cells expressing IFITM3, indicating its role in delaying the “kinetics of endosomal escape”. It was also identified that IFITM3 inhibited the rate of endosomal compartment acidification. A pH sensitive label that fluoresced at acidic pH was used to evaluate the rate of viral entry to acidic compartment of endosomes. The fluorescence of control cells increased much faster than cells expressing IFITM. This occurred to the same degree of difference as colocalization of viral particle and endosome between control and protein expressing cells. The IFITM3 expressing cells also inhibited or slowed the rate of cleavage of viral capsid protein 1 to 1c and as opposed to control cells. This demonstrated that IFITM3 affected the protease activity of endosomes based on band intensity of the 1c and bands in the western blot assay.
This study demonstrates possibly for the first time that the interferon induced proteins IFITM3, inhibit the viral entry and replication of non enveloped reovirus into the host cells. They target an important step utilized in membrane fusion and penetration by the viruses. The reoviruses require entry to late endosomal compartment for proteolytic cleavage or conformational change. The IFITM3 alter the efficiency of delivery of virus to the acidified endosome compartment and capsid cleavage to restrict entry of the virus into the host cell. Expression of the IFITM3 proteins lowers the degree of infection by virus and viral replication in cells.
It is possible that IFITM3 inhibits infection by various types of non enveloped viruses that are dependent on the endosome for proper membrane penetration into host cells. Identifying the various viruses that respond to anti viral activity of IFITM3 would improve understanding of its precise mechanism of action and location of activity. This understanding of the exact mechanism would be of interest to scientists involved in developing broad spectrum antiviral therapeutics.
References
Anafu, A., Bowen, C.H., Chin, C.R., Brass, A.L., Holm, G.H. (2013). Interferon-inducible transmembrane protein 3 (IFITM3) restricts reovirus cell entry. Journal of Biological Chemistry, 14, 288, 7261-71. doi: 10.1074/jbc.M112.438515.
