Heal The Girls: Porcine Circovirus: Autophagy, Nucleolus, and Apoptosis

Porcine circovirus type 2 (PCV2) is a small non-enveloped single-strand (ss) DNA virus with a genome of 1768 bp in length. Although the infection of pigs with PCV2 by itself only causes a relatively mild diseases, symptoms -including Postweaning Multisystemic Wasting Syndrome (PMWS), congenital tremors, Porcine Dermatitis and Nephropathy Syndrome, reproductive failure, proliferative and necrotizing pneumonia, enteritis, exudative epidermitis, and porcine respiratory disease complex- are alleviated upon co-infection with other porcine viruses such as PRRSV or porcine parvovirus, Mycoplasma hyopneumoniae or following immunostimulation with Interferon-α/-γ (IFN-α/γ). This is reflected by low virus yields in PK-15 cells infected with PCV2 which is increased following the treatment with IFN-γ or agents inhibiting the acidification of endosomes and lysosomes such as Chloroquine, Monesin, or NH₄Cl, suggesting that the release of the genome from internalised virus is inhibited by the acidic pH of the endosome and that the expression of viral genes is stimulated by IFN-α/γ; indeed the PCV2 genome contains a interferon-stimulated response element (ISRE)-like sequence is responsive to both IFN-α and IFN-γ.

Prototype Circovirus particle

The genome itself encodes at least four ORF’s, with ORF1 encoding the replication proteins (Rep and Rep’), ORF2 the Capsid protein (Cap), ORF3 activating the NF-κB pathway by facilitating the ubiquitin-mediated proteasomal degradation of regulator of G protein signalling 16 (RGS16), thus promoting the expression and secretion of IL-6 and -8, and ORF4 being a regulator of both ORF1 and ORF3 expression, with latter being less well characterised.

PCV genome

PCV2: Autophagy, nuclear egress, non-lytic spread and apoptosis

As discussed before, the induction of autophagosome formation by viral proteins can support the formation of viral replication centers that function as the site for the replication of the genome (as discussed in extensio for Coronaviruses) whilst also can be part of the antiviral response by facilitating the degradation of viral components including viral RNA and/or stimulating the immune response by increasing the presentation of viral proteins via MHC Class II on the cell surface. Paradoxically, autophagy can also promote viral replication and in particular egress of viral proteins via the ESCRT pathway, allowing viral egress without cell lysis. Indeed, Hepatitis A virus (HAV) has been shown to exist in extracellular vesicles, whose formation is dependent on two proteins, Alix and VPS4B, that are components of the exosome pathway, but independent of Tsg101 or Beclin-1, suggesting that HAV induces the formation of autophagosome or autophagosome-like particles that contain viral particles that are exported via the ESCRT pathway. Indeed, Alix has been demonstrated to links autophagy to the ESCRT pathway probably by binding a complex consisting of ATG12 and ATG3. In this scenario, autophagosomes containing ATG12 and ATG3 bind to Alix at sites of intraluminal vesicle formation thus allowing membrane curvature and exosome formation. Although in the case of HAV a functional link between Alix and the ATG12-ATG3 complex has not been shown, the formation of virus like particles by a retroviral protein, Murine Leukaemia Virus (MLV) Gag, has recently been shown to be dependent on the formation of ATG12-ATG3-Alix complex. In the case of Poliovirus, the inhibition of autophagy by siLC3 decreases the spread of viral particles in cell culture whereas the stimulation of autophagy by Rapamycin, Loperamide, or Nicarpidine increases cell-to-cell transmission of viral particles without cell lysis. Contrary to these results however, the induction of autophagy can also be detrimental for viral replication, especially if induction of autophagy causes apoptosis early in the replication cycle. A number of viruses therefore encode proteins that prevent the fusion of mature autophagosomes with the lysosomes, such as the M2 protein of Influenza A virus (IAV) or ICP 34.5 of Herpes Simplex Virus (HSV)-1. Stimulating autophagy in cells infected with HSV-1 indeed decreases viral titres (without affecting cell viability), whereas in IAV infected cells (massive) induction of autophagy decreases cell viability.

In the case of PCV2 infected PK-15 cells, treatment of cells with either Chloroquine or NH₄Cl increases viral replication, which is being attributed to facilitating the release of the viral genome. Both reagents however also prevent the degradation of the mature autophagosome suggesting that autophagy -or to be precise autophagic flux- might inhibit viral replication. Indeed, in the studies published both reagents were applied not only during viral entry but also over the whole course of the experiments, making it difficult to separate early from later stages of viral replication.

Analyzing the formation of autophagosome by determining the levels of LC3-II however indicates that PCV2 induces the formation of (LC3-II positive) mature autophagosomes and increasing autophagic flux as measurement of p62/SQSTM-1 levels by 24 hrs p.i. , suggesting that PCV2 indeed does induce autophagy. Inhibiting either the formation of autophagosomes with siATG5 or 3-Methyladenine (3-MA), or the fusion of mature autophagosomes with Chloroquine decreases levels of viral DNA and viral titres, suggesting that autophagy is indeed required for efficient viral replication. In contrast to IAV, the increase in autophagy however is not associated with decreased cell viability. Closer examination of the viral proteins revealed that only the Capsid protein but not the proteins derived from ORF1 or ORF3 (with ORF4 not being examined) induces autophagy. Paradoxically, the Capsid protein also induces apoptosis in PK-15 cells which have been pre-treated with IFN-γ, suggesting

the expression of the Capsid protein might sensitize cells to IFN-γ induced apoptosis.

Since so far the precise mechanism of autophagy induction by the Capsid protein has not been determined. The PCV2 Cap localises both to the nucleoplasm and the nucleolus by interacting with NPM-1/B23 in transfected HEK 293T cells. Similar to the coronaviral N protein, the localisation of Cap might induce the redistribution of nucleolar protein(s) and thus nucleolar stress, which might induce autophagy and apoptosis in a p53 dependent manner, thus sensitizing cells expressing Cap to IFN-γ induced apoptosis. The observed subnucleolar localisation of the Capsid protein in infected cells might be necessary for the formation of viral particles which egress from the nucleus in a process involving components of the autophagy machinery akin to HSV-1, thus explaining the dependent of PCV2 replication on an intact autophagy pathway. Expressing viral proteins from other viruses, such as PRRSV or Coronavirus’, that induce the formation of autophagosomes in cells infected with PCV2 might therefore increase nuclear egress by recruitment components of the autophagy machinery in close proximity to the nuclear membrane, especially in a situation in which autosis occurs as a result of increased ER stress induced autophagy. Alternatively, the expression of viral proteins inducing the formation of autophagosomes might facilitate the release of PCV virions from the infected cell using the autophagy and ESCRT pathway in a Alix dependent manner similar to MLV Gag. So far however, none of these hypotheses has been tested.

In the context of viral infection, autophagy is induced via the AMPK/ERK/TSC2, mTOR pathway, namely by inhibiting mTOR by activating the ERK-1/-2 pathway in TSC-2 dependent manner. It might be possible however that either the replication of viral DNA or the presence of other pathogens induces the activation of ATM and thus inducing autophagy in a ATM dependent manner. Again, further studies are needed. Activation of ATM might also exacerbate nucleolar stress by re-localising E2F1, thus linking viral induced activation of ATM to nucleolar stress induced autophagy and sensitizing infected cells to IFN-α/γ treatment. Alternatively, it might be possible that the nucleolar accumulation of the Capsid protein is required for the export of the viral RNA, similar in function to the HTLV-1 Rex protein whose nucleolar localisation is required for the export of tax and rex mRNA, and in infected cells both functions may not be mutually exclusive.

Model of PCV2 ORF3 and Capsid protein mediated
induction of autophagy, cell cycle delay, and apoptosis

Additionally, the ORF3 protein of both PCV1 and 2 have been demonstrated to induce caspase dependent apoptosis as a result of induction of porcine p53, whereas the expression of ORF4 counteracts ORF3 induced apoptosis by regulating the expression of ORF3. The ability of ORF4 to inhibit ORF3 mediated apoptosis and thus promote viral replication is particular evident early in the infection by restricting ORF3 mediated via inhibition of ORF3 expression. In addition, ORF4 also regulates the expression of ORF1 -and thus Rep and Rep’- as evidenced by the increase in ORF1 levels and increased viral replication in cells infected with a ΔORF4 virus. The expression of ORF4 therefore has both a beneficial effect by preventing apoptosis as well as a inhibitory effect on viral replication by regulating the expression of ORF1.

PCV2 ORF3 itself is a protein of 37 kDa in size, localised both in the cytoplasm and the nucleolus. Both nucleolar and cytoplasmic PCV2 ORF3 co-localises and interacts with the p53 binding domain of porcine ubiquitin E3 ligase Pirh2, thus degrading Pirh2 and activating p53, subsequently inducing p53 dependent apoptosis. Since ORF3 also activates the NF-κB pathway, it might also be possible that ORF3 inhibits ASK1 induced apoptosis, thus contributing to the antiapoptotic signalling induced by PCV2 via activation of Akt and subsequent phosphorylation of ASK1. It might therefore be possible that the nucleolar -but not cytoplasmic- localisation of ORF3 is necessary for promoting apoptosis (or vice versa). Interestingly, PCV1 ORF3 localises exclusively to the cytoplasm in a pattern resembling the ER, indicating that nuclear localisation of PCV1 ORF3 is not necessary to induce apoptosis. Further studies comparing ORF3 derived from PCV1 and 2 are therefore warranted. In addition, the recent identification of novel Circoviruses in mosquitoes and bats might offer further insights into the biology of Circoviruses’.

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Porcine Circovirus: Autophagy, Nucleolus, and Apoptosis

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