Coxsackievirus B3 (CVB3) is a positive strand
RNA virus with a non-segmented genome of approx. 7.4 kB in size, encoding a
single polyprotein which is cleaved by cellular and viral proteins to generate
the non-structural and structural proteins. As discussed in a previous post,
following the infection of pancreatic acinar cells with CVB3 autophagosome-like
vesicles can be observed in infected cells. Akin to the role of the induction
of autophagy in the formation of replication centers following the infection of
cells with Corona- or Arterivirus’, the autophagy machinery is required for
forming the replication centers whilst at the same time the degradation of
autophagosomes containing components of the viral replication complex via
fusion with the lysosome and subsequent formation of the autolysosome is
inhibited. Indeed, in pancreatic acinar cells infected with CVB3,
“megaphagosomes” containing components of the viral replication complex can be
observed in the absence of increased autophagic flux. Since the application of
3-Methyladenine (3-MA) or siRNAs targeting components of the autophagic
machinery such as Beclin-1, Vps34, Atg5, or Atg7 not only inhibits the
formation of megaphagosomes but also viral replication and the induction of
autophagy by Rapamycin or starvation increases the number of megaphagosomes as
well as viral titers, the induction of autophagosome formation has been
proposed to be essential for CVB3 replication. Since these structures are
located in close proximity to lysosomes it has been postulated that CVB3
inhibits the fusion of autophagosomes with the lysosome whilst inducing the
formation of autophagosomes either by sequestering of proteins required for the
initiation of phagophore formation or inducing autophagy either as a consequence
of the localisation of viral proteins or by r (potentially) by cleavage of
p62/SQSTM1 to the ER and thus by initiating the ER stress response as discussed before.
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| Model of Coxsackievirus B3 induced induction of autophagy via the induction of the ER stress response |
Bactericidal/permeability-increasing protein
(BPI) fold-containing family B, member 3 (BPIFB3) was recently identified by
RNAi screening whose depletion enhances the replication of CVB3 but not
Poliovirus in human brain microvascular endothelial cells (HBMEC) as determined
by plaque assay of supernatants from infected cells. In contrast to cells
treated with siBPIFB3, U2OS cells transfected with a plasmid allowing the
overexpression of a C-terminal Flag tagged version of BPIFB3, however exhibited
a decrease in viral titres, suggesting that BPIFB3 is required for efficient
viral replication.
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| BPIFB3 domains and anchoring in the ER |
Unlike the rat homologue of BPIFB3, Rya3,
human BPIFB3 localises to the ER in uninfected cells with a boomerang like
structure in which the BIP1-fold 1 and -2 are interacting with the lipids and
the C terminal exposed to the cytosol, although the precise structure is not
known. Being a ER resident protein, BPIFB3 might either inhibit or promote
autophagy by interacting with components of the autophagy machinery in
particular Beclin-1. Indeed, silencing
BPIFB3 enhances both basal and starvation induced autophagy in HBMEC, HeLa as
well as human kidney 786-O cells, suggesting that BPIFB3 binds proteins like
Beclin-1 akin to Bcl-2 although this has not been shown yet. In contrast to
BPIFB3 silencing, the overexpression of BPIFB3 induces the formation of large
LC3B negative structures in the presence of overexpressed LC3 that are negative
for both LAMP1 and BPIFB3, and thus are non-degradative. Given that these
vacuole-like structures are negative for LC3B, their movement is restricted
following the treatment of transfected cells with Rapamycin since they cannot
associate with microtubuli. Interesting, these structures are also negative for
p62/SQSTM1, suggesting that p62/SQSTM1 dependent selective autophagy might be
impaired, although p62/SQSTM1 levels are not reduced in cells transfected with
siBPIFB3. Concomitant to the formation of LC3B negative vacuoles, the
conversion of LC3-I to LC3-II is inhibited probably by inhibition of the Atg4B protease
and thus prevents the cleavage of LC3-I and subsequent conjugation. From a structural point of view, the
expression of BPI-1 fold was sufficient to induce the formation of these
vacuoles and also co-localises with LC3B both in mock and Rapamycin treated
cells.
Since the infection of pancreatic acinar cells
with CVB3 has been associated with the formation of megaphagosomes, it might be
possible that the expression of siBPFB3 increases the formation of
megaphagosomes following CVB3 infection and increases viral replication.
Indeed, BPFB3 silencing promotes the formation of megaphagosomes in HBMC
infected with CVB3 but not in Poliovirus infected cells, as well as increasing
the association of LC3B with viral replication complexes, similar to non-infected
cells treated with siBPFB3.
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| Model of CVB3 induced autophagy in the presence and absence of BPIFB3: smaller vesicles are induced in the presence of BPFB3 which may or may not contain viral RNA |
It remains to be seen if the accumulation of
viral proteins at the ER induces the degradation of BPIFIB3 and thus favours
the formation of megaphagosomes or if viral proteins inhibit the inhibitory
function of BPIFB3 by either binding BPIFB3 directly or by decreasing BPIFB3
expression. Alternatively, the viral proteins might target BPIFB3 for either
proteasome dependent or independent degradation and thus favour the formation
of megaphagosomes.
Further reading
Coyne CB, Bozym R, Morosky SA, Hanna SL, Mukherjee A, Tudor M, Kim KS, & Cherry S (2011). Comparative RNAi screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers. Cell host & microbe, 9 (1), 70-82 PMID: 21238948
Delorme-Axford E, Morosky S, Bomberger J, Stolz DB, Jackson WT, & Coyne CB (2014). BPIFB3 regulates autophagy and coxsackievirus B replication through a noncanonical pathway independent of the core initiation machinery. mBio, 5 (6) PMID: 25491355
Alirezaei M, Flynn CT, Wood MR, & Whitton JL (2012). Pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo. Cell host & microbe, 11 (3), 298-305 PMID: 22423969
Kemball CC, Alirezaei M, Flynn CT, Wood MR, Harkins S, Kiosses WB, & Whitton JL (2010). Coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo. Journal of virology, 84 (23), 12110-24 PMID: 20861268
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