A specific domain within the 3' untranslated region of Usutu virus confers resistance to the exonuclease ISG20.

Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN-induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3'-5' exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3' untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms.

Hydrophobic Mismatch in the Thylakoid Membrane Regulates Photosynthetic Light Harvesting.

The ability to harvest light effectively in a changing environment is necessary to ensure efficient photosynthesis and crop growth. One mechanism, known as qE, protects photosystem II (PSII) and regulates electron transfer through the harmless dissipation of excess absorbed photons as heat. This process involves reversible clustering of the major light-harvesting complexes of PSII (LHCII) in the thylakoid membrane and relies upon the ΔpH gradient and the allosteric modulator protein PsbS. To date, the exact role of PsbS in the qE mechanism has remained elusive. Here, we show that PsbS induces hydrophobic mismatch in the thylakoid membrane through dynamic rearrangement of lipids around LHCII leading to observed membrane thinning. We found that upon illumination, the thylakoid membrane reversibly shrinks from around 4.3 to 3.2 nm, without PsbS, this response is eliminated. Furthermore, we show that the lipid digalactosyldiacylglycerol (DGDG) is repelled from the LHCII-PsbS complex due to an increase in both the pKa of lumenal residues and in the dipole moment of LHCII, which allows for further conformational change and clustering in the membrane. Our results suggest a mechanistic role for PsbS as a facilitator of a hydrophobic mismatch-mediated phase transition between LHCII-PsbS and its environment. This could act as the driving force to sort LHCII into photoprotective nanodomains in the thylakoid membrane. This work shows an example of the key role of the hydrophobic mismatch process in regulating membrane protein function in plants.

Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.

Resurrection of 2'-5'-oligoadenylate synthetase 1 (OAS1) from the ancestor of modern horseshoe bats blocks SARS-CoV-2 replication.

The prenylated form of the human 2'-5'-oligoadenylate synthetase 1 (OAS1) protein has been shown to potently inhibit the replication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. However, the OAS1 orthologue in the horseshoe bats (superfamily Rhinolophoidea), the reservoir host of SARS-related coronaviruses (SARSr-CoVs), has lost the prenylation signal required for this antiviral activity. Herein, we used an ancestral state reconstruction approach to predict and reconstitute in vitro, the most likely OAS1 protein sequence expressed by the Rhinolophoidea common ancestor prior to its prenylation loss (RhinoCA OAS1). We exogenously expressed the ancient bat protein in vitro to show that, unlike its non-prenylated horseshoe bat descendants, RhinoCA OAS1 successfully blocks SARS-CoV-2 replication. Using protein structure predictions in combination with evolutionary hypothesis testing methods, we highlight sites under unique diversifying selection specific to OAS1's evolution in the Rhinolophoidea. These sites are located near the RNA-binding region and the C-terminal end of the protein where the prenylation signal would have been. Our results confirm that OAS1 prenylation loss at the base of the Rhinolophoidea clade ablated the ability of OAS1 to restrict SARSr-CoV replication and that subsequent evolution of the gene in these bats likely favoured an alternative function. These findings can advance our understanding of the tightly linked association between SARSr-CoVs and horseshoe bats.

Altered lipid acyl chain length controls energy dissipation in light-harvesting complex II proteoliposomes by hydrophobic mismatch.

In plants, the major light-harvesting antenna complex (LHCII) is vital for both light harvesting and photoprotection in photosystem II. Previously, we proposed that the thylakoid membrane itself could switch LHCII into the photoprotective state, qE, via a process known as hydrophobic mismatch. The decrease in the membrane thickness that followed the formation of ΔpH was a key fact that prompted this idea. To test this, we made proteoliposomes from lipids with altered acyl chain length (ACL). Here, we show that ACL regulates the average chlorophyll fluorescence lifetime of LHCII. For liposomes made of lipids with an ACL of 18 carbons, the lifetime was ∼2 ns, like that for the thylakoid membrane. Furthermore, LHCII appears to be quenched in proteoliposomes with an ACL both shorter and longer than 18 carbons. The proteoliposomes made of short ACL lipids display structural heterogeneity revealing two quenched conformations of LHCII, each having characteristic 77 K fluorescence spectra. One conformation spectrally resembles isolated LHCII aggregates, whilst the other resembles LHCII immobilized in polyacrylamide gels. Overall, the decrease in the ACL appears to produce quenched conformations of LHCII, which renders plausible the idea that the trigger of qE is the hydrophobic mismatch.

How Does Physician (Non)accommodation Affect Patient Behavioral Intention? Using a Web-Based Experiment to Examine Indirect Effects of Language Type on Behavioral Intention Through Goal Inferences and Source Appraisals.

Drawing on communication accommodation theory (CAT), we investigated how physician (non)accommodation indirectly affects participants' intention to engage in advocated health behaviors through participant goal inferences and source appraisals. We conducted a 3 (language type: medical jargon, analogies, literal language) × 2 (health topic: coronary artery disease, influenza vaccine) web-based experiment. Participants recruited from an online research panel (N = 545) were randomly assigned to a condition and watched a video featuring a physician explaining medical information and providing health recommendations. In a serial mediation analysis, results suggested two parallel indirect effects (relational vs. informational). Relative to underaccommodation (i.e. medical jargon), physician accommodation (i.e. literal language, analogies) had positive, indirect effects on participant health behavioral intention through goal inferences and assessment of physicians (i.e. warmth, expertise). Compared to the use of literal language, physician use of analogies had a positive, indirect effect on participant behavioral intention solely through the relational path, not the informational path. These findings extend CAT by explicating a mechanism underlying physician (non)accommodation and patient outcomes, offering practical implications for physicians to foster relationships with patients and facilitate patient comprehension.

Ferredoxin C2 is required for chlorophyll biosynthesis and accumulation of photosynthetic antennae in Arabidopsis.

Ferredoxins (Fd) are small iron-sulphur proteins, with sub-types that have evolved for specific redox functions. Ferredoxin C2 (FdC2) proteins are essential Fd homologues conserved in all photosynthetic organisms and a number of different FdC2 functions have been proposed in angiosperms. Here we use RNAi silencing in Arabidopsis thaliana to generate a viable fdC2 mutant line with near-depleted FdC2 protein levels. Mutant leaves have ~50% less chlorophyll a and b, and chloroplasts have poorly developed thylakoid membrane structure. Transcriptomics indicates upregulation of genes involved in stress responses. Although fdC2 antisense plants show increased damage at photosystem II (PSII) when exposed to high light, PSII recovers at the same rate as wild type in the dark. This contradicts literature proposing that FdC2 regulates translation of the D1 subunit of PSII, by binding to psbA transcript. Measurement of chlorophyll biosynthesis intermediates revealed a build-up of Mg-protoporphyrin IX, the substrate of the aerobic cyclase. We localise FdC2 to the inner chloroplast envelope and show that the FdC2 RNAi line has a disproportionately lower protein abundance of antennae proteins, which are nuclear-encoded and must be refolded at the envelope after import.

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Overexpression of LHCSR and PsbS enhance light tolerance in Chlamydomonas reinhardtii.

Nonphotochemical quenching (NPQ) is a crucial mechanism for fine-tuning light harvesting and protecting the photosystem II (PSII) reaction centres from excess light energy in plants and algae. This process is regulated by photoprotective proteins LHCSR1, LHCSR3, and PsbS in green algae, such as Chlamydomonas reinhardtii. The det1-2 phot mutant, which overexpresses these photoprotective proteins, resulting in a significantly higher NPQ response, has been recently discovered in C. reinhardtii. Here, we analysed the physiological impact of this response on algal cells and found that det1-2 phot was capable of efficient growth under high light intensities, where wild-type (WT) cells were unable to survive. The mutant exhibited a smaller PSII cross-section in the dark and showed a detachment of the peripheral light-harvesting complex II (LHCII) antenna in the NPQ state, as suggested by a rise in the chlorophyll fluorescence parameter of photochemical quenching in the dark (qPd > 1). Furthermore, fluorescence decay-associated spectra demonstrated a decreased excitation pressure on PSII, with excess energy being directed toward PSI. The amount of LHCSR1, LHCSR3, and PsbS in the mutant correlated with the magnitude of the protective NPQ response. Overall, the study suggests the mechanism by which the overexpression of photoprotective proteins in det1-2 phot brings about an efficient and effective photoprotective response, enabling the mutant to grow and survive under high light intensities that would otherwise be lethal for WT cells.

Emerging Therapies and Advances in Sickle Cell Disease with a Focus on Renal Manifestations.

The underlying mechanisms of disease in sickle cell disease (SCD) contribute to a multifaceted nephropathy, commonly manifested as albuminuria. In severe SCD genotypes ( e.g. , Hemoglobin SS [HbSS]), albuminuria and CKD are major predictors of mortality in this population. Therefore, the monitoring and management of renal function is an intrinsic part of comprehensive care in SCD. Management of nephropathy in SCD can be accomplished with SCD-directed therapies and/or CKD-directed therapies. In the past 5 years, novel disease-modifying and palliative therapies have been approved in SCD to target aspects of the disease, such as anemia, inflammation, and vasculopathy. Along with conventional hydroxyurea and chronic transfusion, l -glutamine, crizanlizumab, and voxelotor have all been shown to mitigate some adverse effect of SCD, and their effect on nephropathy is being investigated. CKD-directed therapies such as renin-angiotensin-aldosterone system blockers have long been used in SCD nephropathy; however, more complete long-term studies on benefits are needed. Given the effect of renal disease on survival, further assessment of the mechanisms and efficacy of these SCD-directed or CKD-directed therapeutic agents is essential.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

Quantitative proteomic analysis of SARS-CoV-2 infection of primary human airway ciliated cells and lung epithelial cells demonstrates the effectiveness of SARS-CoV-2 innate immune evasion.

Background: Quantitative proteomics is able to provide a comprehensive, unbiased description of changes to cells caused by viral infection, but interpretation may be complicated by differential changes in infected and uninfected 'bystander' cells, or the use of non-physiological cellular models. Methods: In this paper, we use fluorescence-activated cell sorting (FACS) and quantitative proteomics to analyse cell-autonomous changes caused by authentic SARS-CoV-2 infection of respiratory epithelial cells, the main target of viral infection in vivo. First, we determine the relative abundance of proteins in primary human airway epithelial cells differentiated at the air-liquid interface (basal, secretory and ciliated cells). Next, we specifically characterise changes caused by SARS-CoV-2 infection of ciliated cells. Finally, we compare temporal proteomic changes in infected and uninfected 'bystander' Calu-3 lung epithelial cells and compare infection with B.29 and B.1.1.7 (Alpha) variants. Results: Amongst 5,709 quantified proteins in primary human airway ciliated cells, the abundance of 226 changed significantly in the presence of SARS-CoV-2 infection (q <0.05 and >1.5-fold). Notably, viral replication proceeded without inducing a type-I interferon response. Amongst 6,996 quantified proteins in Calu-3 cells, the abundance of 645 proteins changed significantly in the presence of SARS-CoV-2 infection (q < 0.05 and > 1.5-fold). In contrast to the primary cell model, a clear type I interferon (IFN) response was observed. Nonetheless, induction of IFN-inducible proteins was markedly attenuated in infected cells, compared with uninfected 'bystander' cells. Infection with B.29 and B.1.1.7 (Alpha) variants gave similar results. Conclusions: Taken together, our data provide a detailed proteomic map of changes in SARS-CoV-2-infected respiratory epithelial cells in two widely used, physiologically relevant models of infection. As well as identifying dysregulated cellular proteins and processes, the effectiveness of strategies employed by SARS-CoV-2 to avoid the type I IFN response is illustrated in both models.

The apparent interferon resistance of transmitted HIV-1 is possibly a consequence of enhanced replicative fitness.

HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced 'antiviral state', we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be.

A Human and Rhesus Macaque Interferon-Stimulated Gene Screen Shows That Over-Expression of ARHGEF3/XPLN Inhibits Replication of Hepatitis C Virus and Other Flavivirids.

Natural hepatitis C virus (HCV) infection is restricted to humans, whereas other primates such as rhesus macaques are non-permissive for infection. To identify human and rhesus macaque genes that differ or share the ability to inhibit HCV replication, we conducted a medium-throughput screen of lentivirus-expressed host genes that disrupt replication of HCV subgenomic replicon RNA expressing secreted Gaussia luciferase. A combined total of >800 interferon-stimulated genes (ISGs) were screened. Our findings confirmed established anti-HCV ISGs, such as IRF1, PKR and DDX60. Novel species−specific inhibitors were also identified and independently validated. Using a cell-based system that recapitulates productive HCV infection, we identified that over-expression of the 'Rho Guanine Nucleotide Exchange Factor 3' gene (ARHGEF3) from both species inhibits full-length virus replication. Additionally, replication of two mosquito-borne flaviviruses, yellow fever virus (YFV) and Zika virus (ZIKV), were also reduced in cell lines over-expressing ARHGEF3 compared to controls. In conclusion, we ascribe novel antiviral activity to the cellular gene ARHGEF3 that inhibits replication of HCV and other important human viral pathogens belonging to the Flaviviridae, and which is conserved between humans and rhesus macaques.

The Structural and Spectral Features of Light-Harvesting Complex II Proteoliposomes Mimic Those of Native Thylakoid Membranes.

The major photosystem II light-harvesting antenna (LHCII) is the most abundant membrane protein in nature and plays an indispensable role in light harvesting and photoprotection in the plant thylakoid. Here, we show that "pseudothylakoid characteristics" can be observed in artificial LHCII membranes. In our proteoliposomal system, at high LHCII densities, the liposomes become stacked, mimicking the in vivo thylakoid grana membranes. Furthermore, an unexpected, unstructured emission peak at ∼730 nm appears, similar in appearance to photosystem I emission, but with a clear excimeric character that has never been previously reported. These states correlate with the increasing density of LHCII in the membrane and a decrease in its average fluorescence lifetime. The appearance of these low-energy states can also occur in natural plant membrane structures, which has unique consequences for the interpretation of the spectroscopic and physiological properties of the photosynthetic membrane.

SARS-CoV-2 host-shutoff impacts innate NK cell functions, but antibody-dependent NK activity is strongly activated through non-spike antibodies.

The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.

TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction.

Ebola virus (EBOV) causes highly pathogenic disease in primates. Through screening a library of human interferon-stimulated genes (ISGs), we identified TRIM25 as a potent inhibitor of EBOV transcription-and-replication-competent virus-like particle (trVLP) propagation. TRIM25 overexpression inhibited the accumulation of viral genomic and messenger RNAs independently of the RNA sensor RIG-I or secondary proinflammatory gene expression. Deletion of TRIM25 strongly attenuated the sensitivity of trVLPs to inhibition by type-I interferon. The antiviral activity of TRIM25 required ZAP and the effect of type-I interferon was modulated by the CpG dinucleotide content of the viral genome. We find that TRIM25 interacts with the EBOV vRNP, resulting in its autoubiquitination and ubiquitination of the viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry and leads to dissociation of NP from the vRNA. We propose that TRIM25 targets the EBOV vRNP, exposing CpG-rich viral RNA species to restriction by ZAP.

Quantifying the long-term interplay between photoprotection and repair mechanisms sustaining photosystem II activity.

The photosystem II reaction centre (RCII) protein subunit D1 is the main target of light-induced damage in the thylakoid membrane. As such, it is constantly replaced with newly synthesised proteins, in a process dubbed the 'D1 repair cycle'. The mechanism of relief of excitation energy pressure on RCII, non-photochemical quenching (NPQ), is activated to prevent damage. The contribution of the D1 repair cycle and NPQ in preserving the photochemical efficiency of RCII is currently unclear. In this work, we seek to (1) quantify the relative long-term effectiveness of photoprotection offered by NPQ and the D1 repair cycle, and (2) determine the fraction of sustained decrease in RCII activity that is due to long-term protective processes. We found that while under short-term, sunfleck-mimicking illumination, NPQ is substantially more effective in preserving RCII activity than the D1 repair cycle (Plant. Cell Environ.41, 1098-1112, 2018). Under prolonged constant illumination, its contribution is less pronounced, accounting only for up to 30% of RCII protection, while D1 repair assumes a predominant role. Exposure to a wide range of light intensities yields comparable results, highlighting the crucial role of a constant and rapid D1 turnover for the maintenance of RCII efficiency. The interplay between NPQ and D1 repair cycle is crucial to grant complete phototolerance to plants under low and moderate light intensities, and limit damage to photosystem II under high light. Additionally, we disentangled and quantified the contribution of a slowly reversible NPQ component that does not impair RCII activity, and is therefore protective.