HPV, DNA damage response, and autophagy: complex

Human Papillomavirus (HPV) are non-enveloped viruses with a small circular genome of about 8 kB in size that infect the epithelium of the skin or mucosal surfaces. Although most infections with HPV are benign, malignant tumours of the cervix or the oral cavity have been associated with the high-risk HPV types 16, 18, 31, and 58; indeed, HPV 16 and 18 alone cause two thirds of cervical cancers. In general, HPV infects undifferentiated cells in the basal layer of stratified epithelium and viral replication occurs in differentiated suprabasal epithelial cells that are prevented from exiting the cell cycle by the viral E6 and E7 proteins dependent on their ability to target the cellular Retinoblastoma (RB) and p53 proteins.

Initially, damaged DNA is recognised by ATM which facilitates the recruitment of the MRN (Mre11-Rad50-Nbs1) complex which in turn increases the affinity of ATM for DNA, leading not only to the formation monomers via autophosphorylation at Ser-1981 and fully activated by Tip60 mediated acetylation. Following the formation of the MRN complex and full activation of ATM, Histone H2AX is phosphorylated at Ser-189 leading to the formation of γH2AX. γH2AX in turn recruits the adaptor protein MDC1, the latter being phosphorylated, thus allowing the recruitment of the ubiquitin ligase proteins RNF8 and RNF168. Both RNF8 and RNF168 deubiquitinate γH2AX without degradation, promoting binding of BRCA1 (promoting HR) or 53BP1 (promoting A-NHEJ), promoting the activation of p53 and subsequently p53 dependent signalling. In a similar way, ATR binds to ssDNA breaks at ssDNA/dsDNA junctions, via RPA and ATR interacting protein (ATRIP) and activates the phosphorylation of downstream effectors including Claspin, BRCA1, and p53 (via Chk1).

In both undifferentiated human keratinocyte cells and differentiated suprabasal epithelial cells infected with HPV31 the DNA damage response -particularly the ATM response- is constitutively activated, suggesting that the activation of the DDR plays an important role in maintenance of the genome as well as viral replication with components (including the MRN complex and γH2AX) of the DDR co-localise in foci resembling DNA repair foci which differ from foci induced by HTLV-1 Tax that do not contain γH2AX due to the attenuation of ATM mediated signalling.

HPV and autophagy: ATM dependent?

Similar to HTLV-1 infected cells, the formation of autophagosomes is inhibited in human cervical squamous cell carcinoma cells as evidenced by reduced expression of LC3-B and Beclin-1 and increased expression of ATPase family AAA domain containing 3A (ATAD3A), an anti-autophagy factor, the latter also conferring resistance to apoptosis. The formation of autophagosomes however increases following viral entry probably as part of the antiviral response since blocking autophagy increases viral replication in primary human foreskin keratinocytes but not in 293-FT, HaCat, or NIKS cells indicating that at least in primary keratinocytes autophagy inhibits viral replication. Indeed the treatment of HeLa, CaSki, and SiHa cells with Resveratrol induces apoptosis via the intrinsic -mitochondrial- apoptosis as well as an increase in autophagy induced apoptosis in C33A, Hela and CaLo (HPV18 positive) cell lines following an arrest in G1 phase of the cell cycle, independent of NF-κB activation.

Akin to endothelial cells expressing KSHV vCyclin, the deletion of both HPV E6 and E7 in HPV16 positive W12 cells increases the formation of autophagosomes and the degradation of p62/SQSTM-1 concomitant with an increase in senescence as well as upregulating the expression of anti-apoptotic genes. Whilst E7 has been implicated in mediating the acetylation of the RB via recruitment of p300/CBP -thus deregulating the cell cycle- the viral E6 protein inactivates p53 by increased degradation via the proteasomal pathway. Depletion of E6 in W12 cells therefore might increase DRAM-1 dependent formation of the phagophore as a result of p53 activation via the activation of ATM by the viral E7 protein. Consequently, it would be interesting to investigate if the deletion of both E6 and E7, or the inhibition of ATM dependent signalling in ΔE6 W12 cells, increases the formation of autophagosomes compared to wt/mock treated W12 cells. Deletion of E7 on the other hand might induce autophagic flux independent of the induction of DRAM-1 by inducing autophagy via binding of RB to E2F1, upregulating the expression of autophagy related genes. These results need to be compared to β-HPV 5 and 8 derived E6 since in cells infected with HPV5 p53 is not degraded but the activation of p53 is inhibited by inhibiting the activation of ATM via binding of p300/CBP, preventing ATM dependent phosphorylation of p300/CBP and subsequent stabilisation of NBS1in addition to preventing the acetylation of p53 although it remains possible that viral NBS1 protein might stabilise NBS1 by forming a complex similar to HPV31 E7.

 

Proposed effect of HPV E6 on DRAM-1 induced autophagy:
inhibition of OIS?

As discussed before, HTLV-1 Tax also prevents the acetylation of p53 by binding p300/CBP; if however binding of p300/CBP by HTLV-1 Tax also prevents the stabilisation of NBS1 remains to be seen. 

Interestingly, the deletion of both E6 and E7 in SiHa cells not only induces autophagy but also apoptosis (autosis?), suggesting that the expression of E6 and E7 in cervical cancer cells not only deregulates the DDR by activating ATM signalling via E7 and inactivating p53 by E6 but also prevents autophagy and thus autophagy induced senescence and apoptosis by decreasing the expression of autophagy related genes and preventing p53/DRAM-1 dependent autophagy and/or preventing the activation of NF-κB (see below).

It should be noted however that the inhibition of autophagy and autophagy-induced apoptosis by HPV16/18 E6 and E7 proteins might dependent on the cell type since the combined expression of both HPV16 E6 and E7 as well as the expression of E7 alone in immortalized human tonsillar epithelial (HTE) cells increases autophagy concomitant with radio resistance in E7 expressing cells.

In contrast to HTLV-1 infected cells, components of the DDR response, in particular of the MRN complex, are located to sites of HPV replication and the induction of ATM has been proposed to be required for the replication of the viral genome. Similar to the viral replication centers of positive strand RNA viruses, degradation of these by autophagy or autophagy-related processes would decrease viral replication. Although in the case of HPV replication sites they are localised in the nucleus and thus are not encompassed by a membrane it might be possible that ubiqutinated components are recognised by nuclear p62/SQSTM-1 and degraded by selective (p62/SQSTM-1 dependent) autophagy. Alternatively, the inhibition of autophagy itself by HPV E6 and E7 might be sufficient to induce the DDR and thus contribute indirectly to viral replication as well as promoting tumourgenesis. In Atg5 -/- EGFP-p62-expressing iBMK tumour cells genes related to the host defense pathways including antigen presentation, Toll-like receptor and Natural Killer (NK) cell mediated cytotoxicity pathways are downregulated, suggesting that autophagy inhibition prevents the activation of the immune response.

 

HPV E6 and E7 mediated inhibition of autophagy: inhibition of selective autophagy via downregulation of Atg related genes and DRAM-1 ?

In conclusion, the expression of both HPV and HTLV-1 derived proteins deregulates the DNA damage response by either activating (HPV16/18/31) or attenuating the ATM dependent pathway (HPV5/8) whilst preventing the induction of p53 dependent signalling pathways. It remains to be seen to which extent targeting these pathways influences the induction of the formation of the autophagosome. In the case of HTLV-1 Tax, the DDR is attenuated downstream of ATM by increasing the expression of the WIP-1 phosphatase thus decreasing the levels of phosphorylated H2AX Ser-139 (γH2AX) and sequestering components of the DDR such as γH2AX, MDC1 and BRCA1 into “pseudo-foci” or Tax Speckled Structures (TSS) without interfering with ATM activation per se.  Activated ATM however also might increase the levels of NF-κB, promoting autophagy (or at least the formation of autophagosomes) via Bcl-3. HPV in contrast, might inhibit the formation of autophagosomes by inhibiting not only p53 dependent induction of DRAM-1 (in the case of HPV16 E6) but also by decreasing the stability of NBS1, subsequent reduction of ATM levels as well as preventing the acetylation of p53 by binding p300/CBP (in the case of HPV5/8 E6) or by preventing the formation o the autophagosomes via inactivating p53 and inhibiting the expression via binding to RB (HPV16/18 E6 and E7 proteins).

 

Tax and the DDR: Inhibiting the DDR and promoting autophagosome formation

HPV and the DDR: activating the DDR and inhibiting autophagosome formation in cells
infected with high-risk HPV

Since the activation of the DDR is however necessary at certain stages during the replication of oncogenic viruses -in particular   to prevent the accumulation of stalled replication forks during increased DNA replication- a model might be proposed in which during infection the DDR is activated allowing not only the integration of the viral genome into the host genome but also potentially inducing an antiviral response characterised by the activation of autophagy whilst at the same time preventing the activation of p53. Inhibiting autophagy at this stage in the viral replication cycle however is necessary to prevent oncogene-induced senescence (OIS), which in the case of KSHV is prevented by the expression of vFLIP (but not LANA).

Deregulation of the DDR however is not limited to the ATM dependent HR pathway. HTLV-1 Tax has been proposed to inhibit the (conservative) Non-Homologues End Joining (NHEJ) pathway via Ku80 inhibition and sequestering 53BP1 in TSS and -via attenuating ATM - probably also the alternative NHEJ pathway. In the case of HPV, so far I am not aware of any results indicating that the conservative NHEJ pathway is inhibited, activated, or not affected (whilst activating ATM by HPV 16/18 or 31 might effect A-NHEJ). In addition to inhibition of ATM mediated signalling, the expression of HPV5/8 E6 also decreases levels of ATR similar to ATM, whereas HTLV-1 Tax does affect the ATR-Chk1 pathway only by preventing the activation of p53. Therefore HPV 5/8 but not HPV 16/18 or 31 might inhibit ATR as well as ATM dependent signalling pathways, including the alternative NHEJ pathway.

 

Tax and NHEJ: Blocking A- and C-NHEJ pathways

HPV and NHEJ: activating A-NHEJ and neutral on C-NHEJ?

HPV and NF-κB: inhibition of autophagy via NF-κB inhibition?

Comparing the NF-κB activity in primary epithelial cells isolated from three different cervical regions -ectocervix, endocervix and the transformation zone- to HPV16 positive cells from the same regions indicate that NF-κB activity is higher non-infected cells, suggesting that HPV16/18 not only prevent but downregulate NF-κB.  Further experiments comparing primary epithelial cells transduced with lentivirus’ allowing the expression of HPV16 E6, E7, or E6/E7 to cells transduced with a vector control revealed that the observed decrease is due to the expression of E7 either alone or in combination with E6, suggesting that E7 activates ATM but not NF-κB, thus providing a link between the observation that the deletion of E7 in HPV16 positive W12 cells promotes the formation of autophagosomes.

Although a direct link has not been demonstrated, it might be possible that the activation of downstream factors of the DDR prevents the translocation of ATM into the cytoplasm and thus cytoplasmic activation of NF-κB.

Further reading

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Luftig, M. (2014). Viruses and the DNA Damage Response: Activation and Antagonism Annual Review of Virology, 1 (1), 605-625 DOI: 10.1146/annurev-virology-031413-085548 

Turnell, A., & Grand, R. (2012). DNA viruses and the cellular DNA-damage response Journal of General Virology, 93 (Pt_10), 2076-2097 DOI: 10.1099/vir.0.044412-0 

Jansma AL, Martinez-Yamout MA, Liao R, Sun P, Dyson HJ, & Wright PE (2014). The high-risk HPV16 E7 oncoprotein mediates interaction between the transcriptional coactivator CBP and the retinoblastoma protein pRb. Journal of molecular biology, 426 (24), 4030-48 PMID: 25451029

Moody CA, & Laimins LA (2009). Human papillomaviruses activate the ATM DNA damage pathway for viral genome amplification upon differentiation. PLoS pathogens, 5 (10) PMID: 19798429 

  

Gillespie KA, Mehta KP, Laimins LA, & Moody CA (2012). Human papillomaviruses recruit cellular DNA repair and homologous recombination factors to viral replication centers. Journal of virology, 86 (17), 9520-6 PMID: 22740399 

Anacker DC, Gautam D, Gillespie KA, Chappell WH, & Moody CA (2014). Productive replication of human papillomavirus 31 requires DNA repair factor Nbs1. Journal of virology, 88 (15), 8528-44 PMID: 24850735 

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Griffin LM, Cicchini L, & Pyeon D (2013). Human papillomavirus infection is inhibited by host autophagy in primary human keratinocytes. Virology, 437 (1), 12-9 PMID: 23290079 

  

Chen TC, Hung YC, Lin TY, Chang HW, Chiang IP, Chen YY, & Chow KC (2011). Human papillomavirus infection and expression of ATPase family AAA domain containing 3A, a novel anti-autophagy factor, in uterine cervical cancer. International journal of molecular medicine, 28 (5), 689-96 PMID: 21743956 

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