A Journal Club on the Mechanism and

Implications of IFITM3 Shuttling of

Virus Particles to Lysosomes
What is IFITM3?
● part of the intrinsic immune response
against viral pathogens
● response against type I interferons →
transmembrane proteins mainly
localized in endosomal and
endolysosomal compartments
● IFITM3 = prevents a range of viruses,
particularly those that exploit the
endocytic pathways, from penetrating
host cell’s lipid bilayer
○ first line of antiviral defense

Spence et al., 2019

IFITM3 + Influenza
● viruses like influenza depend on
low-pH environments to
complete fusion
● believed that S-palmitoylated
IFITM3 is able to inhibit
influenza by two mechanisms
○ physical restriction
○ accelerated degradation in the
lysosome
● rate + mechanism of IFITM3
shuttling viral particles to
lysosome → implicate its ability
to inhibit influenza fusion

Spence et al., 2019

 Hypothesis
IFITM3 is on endocytic vesicles that
fuse with incoming virus particles
and enhances the trafficking of this
pathogenic cargo to lysosomes.
Key Findings
1. IFITM1, IFITM2, and IFITM3 act
cooperatively to enhance trafficking of late
endocytic cargo to lysosomes in
IFN-stimulated cells.
2. IFITM2 and IFITM3 increase the rate of
virus trafficking to lysosomes.
3. IFITM3 vesicles directly fuse with restricted
virus particles, and functional IFITM3 is
required for virus particle fusion.
 1. IFITM1, IFITM2, and IFITM3 act
cooperatively to enhance trafficking of late
endocytic cargo to lysosomes in
IFN-stimulated cells
 Expression and Antiviral Activity of IFITM1/2/3 KO Cell Lines

● IFITM1, IFITM2, and IFITM3

expression in IFN-treated HeLa and
A549 cell lines (figures b, c)
● following infection with IAV, cell lines
lacking IFITM2/3 tended to have a
greater fold infection compared to
WT cells (figure d)
● this could be rescued when IFITM2/3
were reconstituted in those same cell
lines (figures e, f)
Spence et al., 2019
IFITM3 in IFN-a stimulated A549 WT and IFITM2/3-KO Cells
● top row =
endogenous IFITM3
+ CD63 in WT cells;
PCC = 0.58
● middle =
endogenous IFITM3
+ CD63 in KO cells
● bottom = IFITM2/3
KO cells w/ stable
EGFR levels in IFN-a
re-expression of stimulated WT,
IFITM3; PCC = 0.46 IFITM2/3-KO cells, and
KO cells with stable
re-expression
Spence et al., 2019
IAV-DiD Imaging in HeLa WT and IFITM2/3-KO Cells
● DiD → tracks viral
membrane fusion
● LAMP1 → localization to
the lysosomes

● key takeaway = IFITM2/3

increase the rate of virus
trafficking to lysosomes

Spence et al., 2019

2. IFITM2 and IFITM3 increase the rate of virus
trafficking to lysosomes.
Trafficking of DiD-IAV and IFITM3 in IFITM2/3-KO Cells
● DiD-labeled IAV and epifluorescence
imaging → BODIPY-IFITM3
colocalization w/ majority of
internalized virus particles before lipid
mixing

● key takeaway: although virus particles

may fuse with IFITM3-positive vesicles,
IFITM3 does not significantly inhibit
IAV lipid mixing or hemifusion w/
endocytic membranes
Spence et al., 2019
3. IFITM3 vesicles directly fuse with restricted
virus particles and functional IFITM3 is
required for virus particle fusion.
IFITM3-N21Δ LoF Mutant in HeLa IFITM2/3-KO Cells, part 1
● features of IFITM3 that are
required for trafficking to
incoming IAV particles →
loss-of-function mutants
● rs12252-C allele → N21Δ
mutant
● IFITM3-N21Δ = decreased
antiviral activity towards IAV, in
contrast to similar full-length
IIFTM2 or IFITM3. Spence et al., 2019
IFITM3-N21Δ LoF Mutant in HeLa IFITM2/3-KO Cells, part 2

Spence et al., 2019

● 55% reduction in the number of IAV particles colocalized with IFITM3-N21Δ at

the time of lipid mixing relative to WT IFITM3
● truncated protein = less likely to be incorporated into, but not completely absent
from, endosomal compartments supporting viral fusion
Site-specific S-palmitoylation regulates IFITM3 trafficking to IAV particles

● C71A, C72A, and C105A mutations

→ most prominent site(s) of
S-palmitoylation

● trafficking + colocalization of C72A

mutant with dequenced DiD-IAV
particles → decreased to ~20%
compared to WT, C71A, or C105A

Spence et al., 2019

Site-specific S-palmitoylation regulates IFITM3 trafficking to IAV particles

● key takeaway: site-specific

S-palmitoylation of IFITM3 at
Cys72 = important for rate of
trafficking to IAV particles during
infection!

Spence et al., 2019

Summary of Main Findings
● IFITM1, IFITM2, and IFITM3 were
shown to act cooperatively to enhance
trafficking of late endocytic cargo to
lysosomes
● specifically, IFITM3 was shown to
increase the rate of virus trafficking to
lysosomes
● IFITM3 vesicles directly fused with
restricted virus particles
● functional IFITM3 was required for
(Influenza) virus particle fusion
Spence et al., 2019
Study Limitations / Implications for my Project
● key questions still remain re: the cellular pathway and biochemical mechanism by
which these IFN-effectors prevent pathogen entry and accelerate trafficking of
incoming viral particles to lysosomes for degradation

● specific protein-protein interaction partners and reconstitution of lipidated

isoforms for biochemical and structural studies in the future

● only IAV was tested for all conditions → different viruses may initiate fusion in
different subsets of late endo/lysosomal compartments + exhibit differential
sensitivity to IFN effector expression levels and trafficking properties
○ interactions not readily apparent from bulk viral entry studies
 Thank You :)

Please feel free to ask questions / give feedback!

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