Heal The Girls: Positive strand RNA viruses and the DDR: cell cycle arrest and autophagy

Both DNA and RNA viruses are dependent of cell proliferation for replication. Thus viruses often posses proteins which are able to interact with cellular proteins which are responsible for maintaining the regulation of the cell cycle. In general viruses could either slow the cell cycle or activate the cell cycle.

Figure: Viruses and the DDR: multiple points of action

In the case of HIV-1, the viral R protein (VpR ) was identified to be responsible for accumulation of G2 arrested cells. by preventing the activation of the cyclinB/CDK 1 through the inhibition of a phosphatase, cdc25A, and the activation of a kinase, Wee1. Both enzymes are hypophosphorylated in HIV-1 infected cells and VpR may act through the activation of protein phosphatase 2A (PP2A)

and thus blocks the G2-M transition. In this context it is very interesting to note that VpR -similar to coronaviral N protein is also located in the nucleolus of the cell. Another example is the paramyxovirus Simian parainfluenza virus 5, which is able to slow the progression both G1 into S and G2 into M but not of a G1 or G2 arrest .The V protein of Simian Virus 5 binds to a protein involved in DNA repair, DDB1 (DNA damage binding protein). DDB1 becomes activated upon binding through V. Activation of DDB1 in turn leads to slowing the progression of G2 into M and binds to the Retinoblastoma protein and thus prevents entering of the S phase through non-activation of E2F. Because of the delayed activation of E2F, the transactivation of the genes necessary for successful entry into the S phase is also delayed.

Coronavirus and the cell cycle: Induction of p53 by ATR induces S phase arrest

The expression of the nucleocapsid (N) protein of Infectious Bronchitis Virus (IBV) or Severe Acute Respiratory Syndrome Virus (SARS) in Vero cells decreases not only cell proliferation in the absence of apoptosis but also arrests the cell cycle at S phase as well as inducing in a significant number of cells a mitotic defect as indicated by the presence of a cleavage furrow.

Figure: Localisation of Coronavirus N protein and mitotic arrest

Indeed, bivariate flow cytometry of BrdU incorporation and Propidium iodide content and subsequent dot plot analysis by gating for G0/G1, S and G2/M phase showed that the number of cells in S phase increased by 20% if the cells were transfected with IBV N compared to mock transfected or cells transfected with empty vector as well as an increase in cells with DNA content higher than 4n,indicating not only that the expression of IBV N delays the cell cycle but also might induce mitotic defects. Similar the cell cycle is delayed in Vero cells arrested in G2/M with Nocadazole and released for 24 hrs by replacing the Nocadazole containing medium with fresh DMEM, indicating that the expression of N in Vero cells does delay the progression of Vero cells from S phase into G2 phase of the cell cycle. In Vero cells infected with a Vero adapted IBV Beaudette US strain a similar delay can also be observed (albeit to a lower extent) indicating that viral replication does not antagonise the ability of N to delay the cell cycle. In contrast to Vero cells, the expression of IBV N in Saos-2 cells that are deficient for p53 does not induce a delay or arrest in the cell cycle indicating that p53 is required for inducing the observed delay in cell cycle progression.

Figure: Expression of IBV N in Vero cells but not in Saos-2 cells induces cell cycle arrest
that it can be abrogated by Caffeine

p53 can be activated by ATR as a result of nucleolar stress and since the expression of the expression of the coronaviral N protein has been proposed to induce nucleolar stress, treatment of cells transfected with SARS or IBV N with an inhibitor of ATR should therefore reverse the activation of p53 by ATR. Indeed, Vero cells transfected with either IBV or SARS N protein and treated with 4 mM Caffeine do not exhibit an increase in S phase -indicating that N protein induced S phase arrest is indeed mediated by an activation of ATR- but instead exhibit an increase in G0/G1 phase of the cell cycle indicating that N inhibits the progression of cells from G1 into S phase. Indeed, the expression of N does not only induce nucleolar stress but also inhibits Cyclin D1 as well as Cyclin A and E thus inhibiting entry into S phase in cells treated with Caffeine.

Figure: The expression of SARS N in A549 cells induces a transient
arrest in S phase

In addition, Caffeine treatment of Vero cells transfected with IBV N also increases the percentage of cells in G2/M indicating the activation of the G2/M checkpoint and flow cytometry analysis of Caffeine treated Vero cells transfected with SARS N confirms that the expression of SARS N induces a G2 rather than a mitotic arrest since SARS N does not increase Histone H3(Ser-10) phosphorylation.

In conclusion, the expression of IBV and SARS N induces an arrest primary in S phase of the cell cycle that is at least partially dependent on the induction of p53 by ATR. Abrogation of ATR dependent cell cycle arrest induces an arrest in late G1/early S phase and an arrest in G2 -but not M- phase.

Figure: The expression of SARS N in Vero cells induces p53 (A), p21 (B), CHK-1 (C) and but not mitotic arrest (D)

As discussed before, the expression of N might induce DRAM-1 and thus autophagy as a result of nucleolar stress that might promote viral replication by promoting the formation of viral replication centres. In budding yeast, ATR mediated induction of autophagy has been linked to the induction of anaphase and recent evidence suggests that LC3 positive structures -probably mitophagosomes- are present throughout mitosis. Induction of autophagy by N therefore might be responsible for a premature onset of anaphase and thus inducing the formation of the midbody. Since aberrant cytokinesis is only present in a subset of cells expressing N, the majority of cells might not undergo premature mitosis but instead, the induction of autophagy might be a contributing factor to N induced S-G2/M arrest since autophagy can arrest cells by increasing levels of phosphorylated Cdc2(Y15), Cyclin A, E and B1, and Rb (S807/811) and decreasing Cyclin D1. Furthermore, Coronavirus N induced autophagy might also attenuate the DNA damage response by degrading Chk-1 via chaperone-mediated autophagy.

In addition to N, the expression of IBV and SARS nsp-13 has been shown to induce the DNA damage response as well as inducing S phase arrest via ATR. In this case, ATR is activated by stalled replication forks due to the inhibition of p125 subunit of DNA polymerase δ and -similar to IBV and SARS N- the activation of p53 might induce autophagy. Since nsp-13 localises both to the nucleus and the cytoplasm, nsp-13 might trigger distinct events at different times of the viral replication cycle. ER localised nsp-13 might trigger the ER stress response and akin to (and maybe in addition to) nsp-4 and -6, promote the formation of viral RC by initiating the formation of omegasome-like structures. Nuclear nsp-13 in contrast inhibits DNA replication and induces the ATR response which in turn might facilitate the formation of RC indirectly by inducing the formation of autophagosomes and/or by inducing a S-G2/M arrest thus preventing degradation of the viral genome during mitosis as well as extending the cell cycle to allow viral replication. The individual contributions of both N and nsp-13 are however not known and it remains to be seen if the localisation of nsp-13 is altered during viral replication, i.e. if nsp-13 undergoes nuclear-cytoplasmic shuttling. It should also be noted that the expression of SARS N induces the activation of caspase-3 in the absence of depolarised mitochondria and in the absence of apoptosis. N and/or nsp-13 induced autophagy therefore might also promote the degradation of proapoptotic proteins or promote mitophagy and thus prevent Coronavirus induced apoptosis in addition to promoting the formation of viral RC. Interestingly, activated Caspase-3 localises to the nucleolus in A549 cells transfected with SARS N without -as indicated by staining of SARS N transfected cells with HSP-60 and Bcl-2 as well as flow cytometry detection of activated Bax- the presence of damaged mitochondria.

Figure: SARS N induces the localisation of active Caspase-3 to the nucleolus

Figure: Activity of Caspase-3 is not impaired in A549 cells transfected
with SARS N

Figure: SARS N does not impair mitochondrial integrity in A549 cells

In human MRC-7 cells the disintegration of the nucleolus has been shown to precede cell cycle arrest and p53 activation, suggesting that the nucleolar localisation of active Caspase-3 might contribute to nucleolar stress induced by the expression of SARS-CoV N protein, but further studies are needed to determine the pathway of caspase-3 activation and if damaged mitochondria are degraded by mitophagy. Also, it has to be determined of the induction of ATR by N or any other coronaviral protein does prevent the activation of caspase-3 dependent apoptosis. Alternatively, the expression of nsp-13 or N might induce the ER stress response and thus protects cells from caspase-3 dependent apoptosis.

In conclusion, both the expression of N and nsp-13 induces the ATR dependent DNA damage response as well might prevent the execution of the DDR by abrogating downstream signaling pathways thus arresting cells in S and/or G2 phase of the cell cycle. In the case of N, inhibition of G1 and S phase cyclins, namely Cyclin D1, A, and E, contributes to the observed arrest. The induction of p53 via ATR by N or nsp-13 might however contribute to the induction of Interferon type I signaling, which can be counteracted by the expression of the PLP2 catalytic domain of nsp-3 . PLP2 therefore might not only inhibit antiviral singling but also partially prevent N induced cell cycle arrest, namely pathways induced by p53 such as the degradation of CHK-1 and the increase of Cyclin E m A, and B as well as the decrease of Cyclin D1, thus preventing S phase arrest in addition to prevent mitotic defects. So far however this has not been experimentally been proven.

Figure: IBV N, nsp-13, and nsp-14: induction of ATR and ATM dependent pathways

Furthermore, it needs to be determined why the expression of the TGEV N protein in contrast to the N protein derived from IBV, SARS, or MHV does induce p53 dependent apoptosis. Similar to SARS N, TGEV N induces a cell cycle arrest (in S and G2/M) and the activation of Caspase-3; unlike in A549 cells expressing SARS N however, Bax translocates to the mitochondria and induces the depolarisation of the mitochondrial membrane. It might be possible that in PK-15 autophagy is impaired and damaged mitochondria might accumulate. One possibility is that both SARS and IBV N do increase the expression of both Bak and Mcl-1 whereas TGEV N does not.

Finally, it needs to be determined if the nucleolar localisation of other viral proteins such as the core protein of JEV induces the activation of ATR as a result of nucleolar stress.

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Positive strand RNA viruses and the DDR: cell cycle arrest and autophagy

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