Human Cytomegalovirus (HCMV) and autophagy: multiple points of interference with the autophagy pathway

Autophagy is a intracellular degradation pathway that targets and delivers cytoplasmic material such as proteins, organelles, bacteria or viral particles to lysosomes where the material is degraded. As such, autophagy is involved in the degradation of damaged organelles such as mitochondria (mitophagy), the removal of misfolded and/or aggregated proteins (aggrephagy) or the removal and processing of viral proteins  (xenophagy) as well as being induced under conditions of cellular stress such as nutrient withdrawal, DNA damage or viral infections.

One of the main characteristics is the formation of isolation membranes and subsequent vesicular structures which are derived from the ER are formed and expanded into double-membrane autophagosomes that engulf the cellular cargo. Mature autophagosomes that are formed by the conversion of LC3-I to LC3-II then fuse either with late endosomes or lysosomes to form the autolysosome where the cargo is degraded by lysosomal enzymes. In general (under conditions of nutrient withdrawal), cellular autophagy is regulated by mammalian target of rapamycin complex-1 (mTORC1) kinase, that regulates a complex consisting of Unc-51-like kinase (ULK-1), focal adhesion kinase family interacting protein of 200 kDa (FIP200), Atg13, and Atg101. ULK-1 activates a downstream complex couch as the class III phosphatidylinositol 3-kinase (PI3-kinase/PI-3K) complex (Atg6/beclin1-Atg14-Vps15-Vps34) and the Atg12 (Atg12-Atg5-Atg16) system as well as the LC-3 ubiquitin-like conjugation system that converts LC3-I to LC3-II in a PI3-kinase dependent manner by recruiting the Atg12-Atg5-Atg16 complex to the phagosome. 

In the absence of starvation, mTORC1 inhibitors such as rapamycin or torin can activate the formation of the autophagosome, whereas PI-3 kinase inhibitors such as Wortmannin can inhibit the recruitment of Beclin1/Atg14/Vps15/Vps34. In contrast to mTORC1 inhibitors, trehalose might act induce autophagy either by inducing autophagy via a different pathway or alternatively by acting on components other than mTORC1 or the ULK1/PI3K complex although an increase in autophagy regulators such as Beclin-1/Atg6, Atg12, Atg7, or Atg5 has been ruled out. It should be noted that trehalose itself does not bind any cellular receptor whose activation might induce autophagy and it has been speculated that in order to induce autophagy, trehalose needs to be internalized via endocytotic processes; it might be therefore possible that autophagy is induced at least partially by the recruitment of UVRAG, VPS15, VPS34 and Rab5GTPase to early endosomes following the uptake of cargo. In this scenario the late endosome might fuse with the mature -late- autophagosome although it can not be ruled out that trehalose does indeed induce autophagy directly as indicated by results that show that in HEK 293T cells synthesizing intracellular trehalose autophagy is induced in the absence of other inducers, most probably by macroautophagy and/or chaperone mediated autophagy (CMA), thus counteracting the effects of proteasome inhibitors, increased levels of ROS, cleaved Caspase-3 or ubiquitinated proteins.

Outline of the autophagy pathway

A number of viral proteins have been shown to interfere with either the formation of autophagosome or with the fusion of the mature autophagosome via different mechanisms, among them the nsp-4 and nsp-6 derived from a number of Coronaviruses', the nsp-5/6/7 protein of Porcine Respiratory Syndrome Virus (PRRSV), or the M2 protein of Influenza A Virus (via a LC3 interacting motif) and autophagy benefits the replication of Newcastle Disease Virus (NDV) in chicken cells and tissues.

Coronaviruses and autophagy: interference at multiple points promotes the formation of replication centres

In the case of members of the Herpesvirus family the ICP34.5 protein of Human Herpesvirus-1 (HSV-1) induces the formation of autophagosomes, as indicated by an increase of GFP-LC3 positive punctae and endogenous lipid action of LC3, whilst preventing the degradation of autophagic substrates as indicated by the failure to reduce levels of p62/SQSTM-1 during the late stages of viral replication via a Beclin-1 interaction domain within ICP34.5, suggesting that HSV-1 inhibits the fusion of the mature autophagosome with the lysosome. Studies using bovine Herpesvirus-1 (BoHV-1) indicate that the bovine ICP-0 protein promotes the clearance of autophagosomes and more importantly prevents the induction of of IRF-3 in infected Mardin Darby Bovine Kidney cells (MDBK), suggesting that viral induced autophagy in Mardin Darby Bovine Kidney cells (MDBK) infected with a bICP-0 null mutant virus might play an important role in evading the immune response. In the case of BoHV-4, autophagy is likewise induced at 48 h p.i as indicated by increased levels of Beclin-1, PI-3-K, and Akt-1/2 whilst p62/SQSTM-1 levels are reduced (compared to mock infected cells), indicating that the induction of autophagy by bovine Herpesvirus' is a common feature. Although there are strong indications that the induction of autophagy prevents the induction of apoptosis in addition to immune evasion, further studies are needed to explore the connection between BoHV induced autophagy and apoptosis.

Regarding the murine γHV68 (MHV68), the viral M11 gene encodes for a protein that functions as a homolog for the cellular Bcl-2 protein which binds to the BH3 domain of Beclin-1 and thus inhibits Beclin-1 mediated autophagy. Interestingly, recently reported results from mice deficient for Fip200, Beclin-1, ATG14, ATG16L1, ATG7, ATG3, and ATG5  and infected with MHV68 suggest that MHV68 reactivation is inhibited in macrophages derived from these mice. Furthermore, chronic infection with MHV68 in these mice triggered systemic inflammation, suggesting that autophagy plays an important role in dampening viral induced inflammation similar to BoHV.

As discussed in an earlier post, Kaposi Sarcoma Herpesvirus (KSHV) proteins vGPCR, vIL-6 K1, and vBcl-2 inhibit the formation of autophagosomes via activation of mTORC1 and by inhibiting Beclin-1, whereas the viral (v0 Cumin D activates autophagy potentially via DRAM-1. The viral vIAP and K7 proteins however inhibit the fusion of the mature autophagosome, thus inhibiting autophagic flux.

KSHV and autophagy

                              HCMV and Autophagy

Human Cytomegalovirus (HCMV), the prototype member of the herpesvirus subfamily Betaherpesvirinae, is the major cause of birth defects caused by viral infections, pose a serious problem for immunocompromised patients and has more recently also been associated with the onset of artherosclerosis. Upon birth, between 0.5 and 2.5% of all newborns are infected with HCMV with up to 5% being symptomatic, i.e. presenting themselves with symptoms ranging from microcephaly, motor disabilities to chorioretinitis and hearing loss. About 15% of asymptomatic patients later developing disabilities as well, as hearing loss. In addition to neonatal disease, HCMV has also been implicated in the development of vascular diseases and has been associated with glioblastoma. The dsDNA genome of HCMV has a size of about 235 kb and consists of three unique regions, the unique-long UL) and unique-short (US) regions, which are flanked by inverted repeats, terminal/internal repeat long (TRL/IRL or RL) and internal/terminal repeat short (IRS/TRS, or RS) respectively, encoding for a total of approx. 165 proteins.

HCMV virion and outline of genome 

The interference of HCMV with host cell pathways has been well established for the blockage of apoptosis by the viral IE1, IE2, vICA, vMIA and UL38 proteins and the viral UL97 and pp71 proteins have been demonstrated to stimulate the cell cycle progression by interfering with the Rb-E2F pathway. A first indication that HCMV might also interfere with autophagy stems from observations published in 1978 showing that in WI-38 fibroblasts, cytoplasmic capsids resembling HCMV particles colocalises with lysosomal enzymes in the absence of autophagosomes. At the time it was postulated that these cytoplasmic bodies are involved in the release of mature virions into the medium, an interesting hypothesis especially in the light of recent research that demonstrated that the release of infectious HCV particles via the exosome pathway requires the autophagy machinery.

More recently it was demonstrated that in MRC-5 cells infected with the laboratory adapted AD169 strain of HCMV, the degradation of p62/SQSTM-1 is markedly decreased as early as 6 hrs p.i.; if however the formation of omegasomes is affected or not is not clear since only GFP-LC3 was used to detect the formation of LC3-positive punctae and not GFP-DFCP1 which would have allowed the detection of early autophagosomes and omegasomes. Also the levels of LC3-I and LC3-II respectively were not measured in this study, but more recently (2016) reported results indicate that in human foreskin fibroblasts (HFF) as well as in infected primary human aortic endothelial cells (HAEC) LC3B-II are increased at 24 hrs p.i., indicating that HCMV might not interfere with the formation of omegasomes but rather with the maturation and budding of the omegasome. Further studies involving high resolution microscopy might provide further insight and indeed the accumulation of omegasomes might explain the observation published in 1978 that in HCMV infected WI-38 cells autophagosomes are absent. Interestingly in infected human H9 neural stem cells (H9 NSC) an increase in LC3B-II cannot be observed. Further evidence that HCMV inhibits the formation of mature autophagosomes and autolysosomes rather accelerating autophagic flux is supported by findings that neither E64D nor Bafilomycin increases the levels of p62/SQSTM-1 or LC3B-II respectively as well as using a GFP-RFP tandem LC3 reporter plasmid.

Most importantly, treatment of HCMV AD169 infected MRC5 cells with known inducers of autophagy such as LiCl or Rapamycin does not induce the formation of autophagosomes as measured by the presence of GFP-LC3 positive punctae nor does the starvation of infected cells decrease levels of p62/SQSTM-1, indicating that HCMV does not only decrease autophagic flux but also renders infected (fibroblast) cells insensitive to mTORC1 inhibition. Closer examination revealed that upon infection of MRC5 cells with HCMV AD169, both 4EBP1 and p70 S6 Kinase (p70 S6K) are phosphorylated, implying that not only the expression of autophagy related genes might be increased (via eIF4) but the formation of autophagosomes might increase through the phosphorylation of proteins that form the ULK complex, particular PI-3K. This would suggest that HCMV induces the formation of phagosomes and/or omegasomes and additionally induces a block at later stages in the absence of apoptosis. If the application of Necrostatin induces apoptosis remains to be seen (as it is the case in MDCK cells infected with Influenza A/WSN/33). Also, the viral protein involved in regulating the phosphorylation of both p70 S6K and 4EBP1 has not been identified - one candidate might be the viral UL97 kinase since it is a Ser/Thr kinase.

In addition to the phosphorylation of p70 S6K and 4EBP1, the viral UL38 protein has been shown to inhibit TSC1/2, increasing the levels of Rheb-GTP and increasing mTORC1 activity as early as 8h p.i.

Interference of HCMV with the autophagy pathway early in the replication cycle:
Induction of autophagy by UL38 and UL97, inhibition by an unknown protein

The formation of both autophagosomes and autolysosomes particularly at late stages of infection is inhibited by the viral TRS1 protein that -similar to the Influenza Virus M2 protein- binds Beclin-1 via a N-terminal Beclin-1 binding domain and thus blocks the formation of phagosomes but not the fusion of mature autophagosomes with lysosomes in infected MRC5 cells, since Beclin-1 forms part of both autophagy promoting complexes containing Atg14L and part of a complex consisting of Rubicon/Vps15/UVRAG/Beclin-1 (and thus an autophagy inhibitory complex). Accordingly, HeLa cells expressing TRS1 and a tandem GFP-RFP LC3 plasmid exhibit a lower number of total LC3 positive punctae as well as a lower number of GFP+/RFP+ and GFP-/RFP+ positive punctae.

Autophagy promoting and inhibitory complexes

Taken together, HCMV both induces the formation of at least omegasomes -if not LC3-II positive autophagosomes- early in the replication cycle and inhibits the formation of autophagosomes late in the replication cycle whilst inhibiting the clearance of autophagosomes early in the replication cycle and the possibly also the formation of autolysosomes at later stages of the replication cycle (maybe by stabilising the Rubicon containing complex via IRS1/TRS1 at the ER or at the Golgi whilst inhibiting the formation of an autophagy promoting complex?).   

        

Inducing autophagy by trehalose in HFF, HAEC and H9 derived midbrain dopaminergic neurons (H9 mDA) infected with HCMV strain TB40E (a clinical strain as opposed to the laboratory adapted AD169 strain) results not only in acidification of autophagosomes and a decrease of LC3B-II (and unchanged levels of LC3B-I) in both infected and mock-infected cells but also in reduced viral titres, reduced expression of viral genes and reduced viral titres prticulary at 96 and 120 hrs p.i., indicating that autophagy might indeed bean antiviral mechanism and the inhibition of autophagy by HCMV promotes viral replication by allowing the assembly of viral replication centres, a notion supported by previous findings that indicate the formation of gB/pp28/mTOR positive vacuoles at 72 hrs p.i. in HCMV infected U273 cells.

If the fusion of autophagosomes with the lysosome in HCMV infected treated with trehalose however can be prevented by the expression of either mutant Rab7GTPase or Rab5GTPase as well as in Rab5-/- or Rab7 -/- cells has not been investigated.

Interference of HCMV with the autophagy pathway late in the replication cycle:
Inhibition of autophagy by the viral IRS1/TRS1 proteins

Treatment of infected cells with trehalose therefore might promote the fusion of replication centres with lysosomes and the subsequent degradation of viral proteins as well as the induction of an antiviral response. Also, fusion of the autophagosome with the lysosome is probably not influenced by the expression nor of the viral IRS1 nor of the viral TRS1 protein due to the inability of HCMV infected cells to form a Rubicon or Atg14L containing complex (see above), but since (to my knowledge) the mechanism of the promotion of autophagy by trehalose has not been elucidated this remains a speculation at this point.

Additionally, inducing autophagic flux might protect infected cells from apoptosis due to viral replication as indicated by studies conducted in MCMV infected RPE cells, and it is not clear if the treatment of HCMV infected cells with trehalose induces or sensitizes cells to apoptosis or not.

In contrast to HCMV, retinal pigment epithelial (RPE) infected with Murine Cytomegalovirus to (MCMV) remain sensitive to rapamycin and rapamycin induced autophagy protects MCMV infected RPE cells from viral induced apoptosis. Conversely, treatment of MCMV infected RPE cells with Chloroquine not only inhibits autophagic flux but also increases apoptosis, suggesting that low level autophagic flux is essential for the survival of MCMV infected cells. These results would also explain earlier observations that intracellular HCMV virions partially co-localise with lysosomes in infected cells.

In the case of KSHV -as discussed before-, viral induced autophagy promotes apoptosis and senescence of infected cells. Despite belonging to a different subfamily, the infection of fibroblast, endothelial or neuronal cells with HCMV followed by the induction of autophagy via exogenous agents might induce apoptosis and/or senescence. Treatment with autophagy inducing agents such as drugs or oncolytic viruses that promote autophagy therefore might inhibit viral replication. Since UL38 is not expressed in HCMV positive glioblastoma, treatment of these tumours using oncolytic viruses that induce autophagy might be beneficial.

Gene expression profile of HCMV infected glioblastoma cells

                 

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