ORF3c localizes to mitochondria during SARS-CoV-2 infection by inhibiting innate immunity by limiting IFN-β production

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A new international led by researchers from University of Cambridge – UK has found that the SARS-CoV-2 ORF3c protein localizes to mitochondria, inhibiting innate immunity by restricting IFN-Β production.

The study team previously identified ORF3c using comparative genomics to be conserved among Sarbecoviruses.
https://pubmed.ncbi.nlm.nih.gov/32667280/

Coronaviruses encode a variety of accessory proteins in their genomes, which are non-essential for RNA replication but confer advantageous properties to the virus allowing efficient viral propagation in the host. Many of these accessory proteins are known antagonists of the innate immune response and are useful targets for antiviral treatment strategies.

However their variability across the Coronaviridae often means that studies cannot be extrapolated to other members; for example, SARS-CoV-1 ORF3b, which overlaps the 3′ region of ORF3a, is truncated in SARS-CoV-25; and ORF10 in SARS-CoV-2 is entirely lacking in SARS-CoV-1.

Previously, using comparative genomics, we identified ORF3c, an accessory protein conserved across the Sarbecovirus subgenus10. Here we have presented a functional analysis of ORF3c, revealing it to be a tail-anchored transmembrane protein that appears to be inserted into the mitochondrial outer membrane, where it interacts with MAVS and PGAM5, and reduces IFN-β signalling.

To the best of our knowledge, this is the first report of PGAM5 being involved in coronaviral-host protein:protein interactions, although it had been identified as a potential target for viral-induced proteasomal degradation66. PGAM5 localises to the MOM60, although there are also reports of PGAM5 within the inner mitochondrial membrane with the C-terminal catalytic domain facing the intermembrane space78,79.

It has been suggested that its location may depend on cellular stress levels: PGAM5 is known to activate the MAP kinase pathway by dephosphorylating ASK1 (associated with cellular stress)80 and is involved in numerous cell death-related processes. Primarily, it is thought to regulate mitochondrial dynamics (fusion versus fission).

Its ability to promote or suppress various cell death pathways is a contentious issue (recently reviewed in Cheng, 202181): whilst reported initially as a pro-necrotic factor 59,82,83, this has been disputed84. Equally controversial are its role(s) in apoptosis: it has been reported to suppress apoptosis in certain models 85, yet be essential for apoptosis induction in others 86-88.

What does appear consistent – and dependent on the phosphatase function of PGAM5 – is a role in the induction of mitophagy, an organelle-specific form of autophagy that protects the cell against necroptosis by selectively degrading damaged mitochondria87,89-92.

It is unlikely that ORF3c affects the phosphatase function of PGAM5, given this is thought to be independent of the role of PGAM5 in immune signalling. PGAM5 multimerisation has been shown to be required for both IFN-β upregulation and induction of many cell death-related events.

One possibility is that the ORF3c-induced decrease in IFN-β may be at least partially caused by a reduction in PGAM5 multimerisation due to its interaction with ORF3c, and a subsequent redirection of PGAM5 to the proteasome.

Equally, it is possible that PGAM5 multimerisation continues in the presence of ORF3c, but the PGAM5:MAVS interaction is ablated.

A third hypothesis involves the formation of a potential trimeric complex (ORF3c:MAVS:PGAM5), resulting in the functional abrogation of both host proteins. As the self-multimerisation of MAVS and PGAM5 are independent events 57, this would be an efficient mechanism of sequestering both potential innate response activators with a single viral protein.

In addition to the observed ORF3c:MAVS and ORF3c:PGAM5 interactions that may inhibit PGAM5:MAVS stimulation of IFN-β production, we also observed cleavage of MAVS when ORF3c was overexpressed and this cleavage appeared to be driven by caspase-3 suggesting a link to apoptosis.

Whether ORF3c-driven apoptosis is an artefact of overexpression outside of the context of virus infection (where other viral proteins might inhibit apoptosis) is currently unknown. While we found a tantalising suggestion of differences in MAVS cleavage between delta and non-delta infections, the overall strong downregulation of full-length MAVS in either infection compared to mock and the presence of cleaved MAVS even in mock-infected cells in this system, besides potential effects of other differences between delta and non-delta viruses, make it difficult to draw robust conclusions at this stage.

Intriguingly, ORF3a of both SARS-CoV-1 and SARS-CoV-2, which localises to the plasma membrane, has been implicated in apoptosis induction; however in the case of SARS-CoV-1 this was mapped to the cytosolic C-terminal domain and therefore could not have been an incorrectly attributed function of ORF3c93,94.

PGAM5 has been reported to be cleaved (within the N-terminal transmembrane domain) in response to mitochondrial dysfunction and mitophagy, specifically during outer membrane rupture, resulting in its release into the cytosol 78,79,95. Although we did not observe cleavage of PGAM5 in the presence of ORF3c (indicating mitophagy is not occurring), it would be of interest to confirm this with other methods.

Equally, it would be of interest to analyse the phosphorylation patterns of PGAM5 when bound to ORF3c given that the phosphorylation status of this protein has numerous effects upon the downstream signalling pathways that it activates. During preparation of this manuscript, data became publicly available indicating ORF3c overexpression does not affect mitophagy, despite its mitochondrial localisation, but rather blocked autophagy by causing autophagosome accumulation 96. This finding supports our preliminary evidence that ORF3c may direct cells towards apoptosis in preference to other cell death pathways, possibly by sequestration of PGAM5 and prevention of mitophagy activation.

Among SARS-CoV-2 proteins, ORF3c is not alone in having an inhibitory effect on IFN-β expression; however the mode of action differs significantly between the proteins involved.

Some proteins directly reduce IFN-β mRNA or protein levels: ORF6, ORF8 and N all have similar effects to ORF3c and reduce IFN-β mRNA (and hence protein) levels although, unlike ORF3c, ORF6 and ORF8 simultaneously reduce expression from ISRE-containing promoters67,68,97.

ORF6 has also been shown independently to reduce IRF3 and STAT1 nuclear translocation 17,36,69,98; in comparison, N is thought to inhibit the TRIM25:RIG-I interaction99,100. NSP136,69 and NSP669 bind TBK1 directly, preventing IRF3 phosphorylation. Other sarbecoviral proteins inhibit type I IFN activation as an indirect result of their enzymatic function: SARS-CoV-1 NSP16 reduces MDA5 and IFIT activation by capping the viral RNA101; NSP14 reduces IFN levels by shutting down host translation 102.

Still others (NSP1 and NSP6) suppress the signalling induced by type I IFN, whilst leaving protein levels unaffected69,103. The convergent effects of these viral antagonistic proteins, which collectively target multiple layers of the immune signalling cascade, no doubt combine to reduce the host antiviral response and increase virus fitness in the natural host.

Despite the redundancy in IFN antagonists, those that operate directly from a mitochondrial location are uncommon amongst characterised coronaviral proteins. ORF9b and ORF10 are the exceptions. Similar to our observations for ORF3c, these proteins localise to the mitochondria upon overexpression and are able to dampen the immune response in the absence of other coronaviral proteins28,40,104,105, indicating they may each act in an unassisted fashion and not from within a virally encoded protein complex. ORF9b does, however, interact with host Tom70 6,26,104,105 which in turn is known to interact with MAVS10 6,107.

It has been suggested that the ORF9b:Tom70 interaction may lead to either apoptosis or mitophagy, as the levels of functional Tom70 will affect both of these processes; but these hypotheses have not been validated experimentally105. This provides an interesting parallel to ORF3c, which appears to operate at the same cellular location as Tom70 (specifically, the MOM108), yet we did not observe ORF3c and Tom70 to co-immunoprecipitate (data not shown).

It remains possible that an indirect, transient interaction may occur between ORF9b, ORF3c, Tom70 and MAVS. Additionally, unlike ORF3c, ORF9b has been observed to inhibit the IKK-γ (NEMO) cascade27, suggesting that ORF9b has additional functions downstream of MAVS, specifically inhibiting the NF-κB pathway. Thus it appears that sarbecoviruses have evolved complementary approaches, mediated by ORF9b and ORF3c respectively, to subvert IFN-β signal transduction and reduce mitochondrial innate immune pathway activation from within the mitochondrion itself.

The mechanism employed by ORF10 is different yet again; however this protein is not conserved across the subgenus. ORF10 localises to the mitochondrion where it interacts with the mitophagy receptor NIX, to activate mitophagy and thereby eliminate aggregated MAVS28. This may in part explain why downregulation and degradation of MAVS is still visible during infection with a delta variant lacking ORF3c (Figure 6D). Downregulation of MAVS has also been reported from proteome-wide studies of SARS-CoV-2 infected cells (although the level of reduction appears to depend on the model system)109,110.

Prior to the identification of ORF3c, a screen of viral and host protein:protein interactions did not identify either MAVS or PGAM5 as probable interaction partners for any SARS-CoV-2 protein6. This was reflected in a thorough literature review111. Equally, there are remarkably few confirmed direct interactions of MAVS with SARS-CoV-2 proteins.

Although there are some reports of the M protein interacting with MAVS25,70, this is not reconciled with the lack of a mitochondrial localisation for the M protein which is found consistently at the ER and Golgi70,105. As such, the relevance of this potential interaction during an actual viral infection is open to question. In short, ORF3c is the only conserved sarbecoviral protein that has been shown to bind directly to MAVS within the MOM, where the majority of activated MAVS would be located during a viral infection.

There are several obstacles currently impeding further progress. For example, analysis of ORF3c-HA transfected cell lysates following digestion with trypsin or chymotrypsin and LC-MS/MS analysis failed to identify ORF3c-derived peptides even when inclusion lists of predicted ORF3c peptides were employed, likely due to high hydrophobicity of the peptides. This explains why multiple published analyses using trypsin digestion have failed to identify the ORF3c protein during infection (data not shown)9,112.

Equally, our own work has been limited by the poor immunogenicity of ORF3c: a peptide-raised rabbit antibody was not reactive against transfected cell lysates, nor was a sheep polyclonal antibody raised against the entire ORF3c protein (data not shown). However, we are confident that these hurdles will be overcome with time.

The discovery of ORF3c necessitates a reassessment of previous sarbecoviral ORF3a-targeted studies, which may have also included ORF3c during protein overexpression. DNA-based constructs (although useful and often necessary) create an artificial system, because these vectors generally exclude viral untranslated regions and are designed to optimise expression from the desired AUG codon. Thus the degree of ORF3c expression, alongside the desired ORF3a, remains an unknown factor for most previous studies.

It is also probable that ORF3c-mediated effects were overlooked in historical SARS-CoV-1 studies due to the extensive use of Vero cells, which allow efficient replication of many coronaviruses but are deficient in type I IFN production113. For example, inadvertent deletion of ORF3c via ORF3a mutation results in only minor attenuation in these cells, as measured by viral infectious titre114,115 (although, notably, deletion of the entire ORF3a region reduced cytopathic effect and cell death116).

Sarbecoviruses appear to be primarily bat viruses (Figure S1) and ORF3c appears to be conserved throughout this clade (with the exception of two different sarbecovirus sequences from Rhinolophus hipposideros where ORF3c is truncated; Figure S1B). In the human host, ORF3c is clearly not essential (as demonstrated by the success of the delta variant where ORF3c is disrupted). However, whether or not ORF3c provides a selective advantage in the human host is unclear.

It is possible that the observed loss, restoration and variations of ORF3c in the human host may be random events whose effects on virus fitness are outweighed by increases in virus fitness conferred by other mutations (e.g. in the spike protein) with ORF3c variations being “carried along”. The importance of ORF3c in the human host presumably will become clearer as the virus adapts to long-term persistence in the human population.

Similarly, other hosts such as the palm civet Paguma larvata and the Malayan pangolin Manis javanica may be intermediate hosts to which these viruses have not fully adapted. The observed Q5Y pseudorevertant of the delta PTC truncation is curious and may reflect a selective advantage of restoring ORF3c protein expression, but it might also have been a random event. Notably Q5 is perfectly conserved across the sarbecovirus ORF3c sequences (Figure S1A) suggesting that a Q at this position is functionally important, at least in bats.

Sarbecoviruses have many different ways to antagonise host innate immunity and it may be that ORF3c is redundant in the human host (or its relative importance may also depend on cell type, host genetic background or disease state). Although not essential, ORF3c may still lead to an increase in virus fitness in the human host. Future work will be needed to compare WT and ORF3c knockout viruses in both human and bat cell lines, besides animal models.

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