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.
|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.
|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.
|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.
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