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Abstract Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), results in huge economic losses to the world pig industry. MiRNAs have been reported to be involved in regulation of viral infection. In our study, miR-320 was one of 21 common differentially expressed miRNAs of Meishan, Pietrain, and Landrace pig breeds at 9-h post-infection (hpi). Bioinformatics and experiments found that PRRSV replication was inhibited by miR-320 through directly targeting PRRSV ORF6. In addition, the expression of CCAAT enhancer binding protein beta (CEBPB) was also inhibited by miR-320 by targeting the 3ʹ UTR of CEBPB, which significantly promotes PRRSV replication. Intramuscular injection of pEGFP-N1-miR-320 verified that miR-320 significantly inhibited the replication of PRRSV and alleviated the symptoms caused by PRRSV in piglets. Taken together, miR-320 have significant roles in the infection and may be promising therapeutic target for PRRS.

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), results in huge economic losses to the world pig industry. MiRNAs have been reported to be involved in regulation of viral infection. In our study, miR-320 was one of 21 common differentially expressed miRNAs of Meishan, Pietrain, and Landrace pig breeds at 9-h post-infection (hpi). Bioinformatics and experiments found that PRRSV replication was inhibited by miR-320 through directly targeting PRRSV ORF6. In addition, the expression of CCAAT enhancer binding protein beta (CEBPB) was also inhibited by miR-320 by targeting the 3ʹ UTR of CEBPB, which significantly promotes PRRSV replication. Intramuscular injection of pEGFP-N1-miR-320 verified that miR-320 significantly inhibited the replication of PRRSV and alleviated the symptoms caused by PRRSV in piglets. Taken together, miR-320 have significant roles in the infection and may be promising therapeutic target for PRRS. [ABSTRACT FROM AUTHOR]

Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al., Cell Rep. 33, 108234 (2020); L. Miorin et al., Proc. Natl. Acad. Sci. U.S.A. 117, 28344-28354 (2020); and M. Frieman et al., J. Virol. 81, 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surfaced on 31 December, 2019, and was identified as the causative agent of the global COVID-19 pandemic, leading to a pneumonia-like disease. One of its accessory proteins, ORF6, has been found to play a critical role in immune evasion by interacting with KPNA2 to antagonize IFN signaling and production pathways, resulting in the inhibition of IRF3 and STAT1 nuclear translocation. Since various mutations have been observed in ORF6, therefore, a comparative binding, biophysical, and structural analysis was used to reveal how these mutations affect the virus's ability to evade the human immune system. Among the identifiedmutations, the V9F, V24A,W27L, and I33T, were found to have a highly destabilizing effect on the protein structure of ORF6. Additionally, the molecular docking analysis of wildtype andmutant ORF6 and KPNA2 revealed the docking score of - 53.72 kcal/mol for wildtype while, -267.90 kcal/mol, -258.41kcal/mol, -254.51 kcal/mol and -268.79 kcal/mol for V9F, V24A, W27L, and I33T respectively. As compared to the wildtype the V9F showed a stronger binding affinity with KPNA2 which is further verified by the binding free energy (-42.28 kcal/mol) calculation. Furthermore, to halt the binding interface of the ORF6-KPNA2 complex, we used a computational molecular search of potential natural products. A multi-step virtual screening of the African natural database identified the top 5 compounds with best docking scores of -6.40 kcal/mol, -6.10 kcal/mol, -6.09 kcal/mol, -6.06 kcal/mol, and -6.03 kcal/mol for tophit1-5 respectively. Subsequent all-atoms simulations of these top hits revealed consistent dynamics, indicating their stability and their potential to interact effectively with the interface residues. In conclusion, our study represents the first attempt to establish a foundation for understanding the heightened infectivity of new SARS-CoV-2 variants and provides a strong impetus for the development of novel drugs against them. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 belongs to the subgenus Sarbecovirus, which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections. IMPORTANCE The ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections. [ABSTRACT FROM AUTHOR]

Evasion and antagonism of host cellular immunity upon SARS-CoV-2 infection provide replication advantage to the virus and contribute to COVID-19 pathogenesis. We explored the ability of different SARS-CoV-2 proteins to antagonize the host’s innate immune system and found that the ORF6 protein mitigated type-I Interferon (IFN) induction and downstream IFN signaling. Our findings also corroborated previous reports that ORF6 blocks the nuclear import of IRF3 and STAT1 to inhibit IFN induction and signaling. Here we show that ORF6 directly interacts with RIG-I and blocks downstream type-I IFN induction and signaling by reducing the levels of K63-linked ubiquitinated RIG-I. This involves ORF6-mediated targeting of E3 ligase TRIM25 for proteasomal degradation, which was also observed during SARS-CoV-2 infection. The type-I IFN antagonistic activity of ORF6 was mapped to its C-terminal cytoplasmic tail, specifically to amino acid residues 52–61. Overall, we provide new insights into how SARS-CoV-2 inhibits type-I IFN induction and signaling through distinct actions of the viral ORF6 protein. [ABSTRACT FROM AUTHOR]

ABSTRACT The accessory protein ORF6 of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key interferon (IFN) antagonist that strongly suppresses the production of primary IFN as well as the expression of IFN-stimulated genes. However, how host cells respond to ORF6 remains largely unknown. Our research of ORF6-binding proteins by pulldown revealed that E3 ligase components such as Cullin 4B (CUL4B), DDB1, and RBX1 are potential ORF6-interacting proteins. Further study found that the substrate recognition receptor PRPF19 interacts with CUL4B, DDB1, and RBX1 to form a CRL4B-based E3 ligase, which catalyzes ORF6 ubiquitination and subsequent degradation. Overexpression of PRPF19 promotes ORF6 degradation, releasing ORF6-mediated IFN inhibition, which inhibits SARS-CoV-2 replication. Moreover, we found that activation of CUL4B by the neddylation inducer etoposide alleviates lung lesions in a SARS-CoV-2 mouse infection model. Therefore, targeting ORF6 for degradation may be an effective therapeutic strategy against SARS-CoV-2 infection.IMPORTANCEThe cellular biological function of the ubiquitin-proteasome pathway as an important modulator for the regulation of many fundamental cellular processes has been greatly appreciated. The critical role of the ubiquitin-proteasome pathway in viral pathogenesis has become increasingly apparent. It is a powerful tool that host cells use to defend against viral infection. Some cellular proteins can function as restriction factors to limit viral infection by ubiquitin-dependent degradation. In this research, we identificated of CUL4B-DDB1-PRPF19 E3 Ubiquitin Ligase Complex can mediate proteasomal degradation of ORF6, leading to inhibition of viral replication. Moreover, the CUL4B activator etoposide alleviates disease development in a mouse infection model, suggesting that this agent or its derivatives may be used to treat infections caused by SARS-CoV-2. We believe that these results will be extremely useful for the scientific and clinic communities in their search for cues and preventive measures to combat the COVID-19 pandemic.

Abstract SARS-CoV-2 has evolved several strategies to overcome host cell defenses by inducing cell injury to favour its replication. Many viruses have been reported to modulate the intracellular redox balance, affecting the Nuclear factor erythroid 2-Related Factor 2 (NRF2) signaling pathway. Although antioxidant modulation by SARS-CoV-2 infection has already been described, the viral factors involved in modulating the NRF2 pathway are still elusive. Given the antagonistic activity of ORF6 on several cellular pathways, we investigated the role of the viral protein towards NRF2-mediated antioxidant response. The ectopic expression of the wt-ORF6 protein negatively impacts redox cell homeostasis, leading to an increase in ROS production, along with a decrease in NRF2 protein and its downstream controlled genes. Moreover, when investigating the Δ61 mutant, previously described as an inactive nucleopore proteins binding mutant, we prove that the oxidative stress induced by ORF6 is substantially related to its C-terminal domain, speculating that ORF6 mechanism of action is associated with the inhibition of nuclear mRNA export processes. In addition, activation by phosphorylation of the serine residue at position 40 of NRF2 is increased in the cytoplasm of wt-ORF6-expressing cells, supporting the presence of an altered redox state, although NRF2 nuclear translocation is hindered by the viral protein to fully antagonize the cell response. Furthermore, wt-ORF6 leads to phosphorylation of a stress-activated serine/threonine protein kinase, p38 MAPK, suggesting a role of the viral protein in regulating p38 activation. These findings strengthen the important role of oxidative stress in the pathogenesis of SARS-CoV-2 and identify ORF6 as an important viral accessory protein hypothetically involved in modulating the antioxidant response during viral infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surfaced on 31 December, 2019, and was identified as the causative agent of the global COVID-19 pandemic, leading to a pneumonia-like disease. One of its accessory proteins, ORF6, has been found to play a critical role in immune evasion by interacting with KPNA2 to antagonize IFN signaling and production pathways, resulting in the inhibition of IRF3 and STAT1 nuclear translocation. Since various mutations have been observed in ORF6, therefore, a comparative binding, biophysical, and structural analysis was used to reveal how these mutations affect the virus’s ability to evade the human immune system. Among the identified mutations, the V9F, V24A, W27L, and I33T, were found to have a highly destabilizing effect on the protein structure of ORF6. Additionally, the molecular docking analysis of wildtype and mutant ORF6 and KPNA2 revealed the docking score of - 53.72 kcal/mol for wildtype while, -267.90 kcal/mol, -258.41kcal/mol, -254.51 kcal/mol and -268.79 kcal/mol for V9F, V24A, W27L, and I33T respectively. As compared to the wildtype the V9F showed a stronger binding affinity with KPNA2 which is further verified by the binding free energy (-42.28 kcal/mol) calculation. Furthermore, to halt the binding interface of the ORF6-KPNA2 complex, we used a computational molecular search of potential natural products. A multi-step virtual screening of the African natural database identified the top 5 compounds with best docking scores of -6.40 kcal/mol, -6.10 kcal/mol, -6.09 kcal/mol, -6.06 kcal/mol, and -6.03 kcal/mol for tophit1-5 respectively. Subsequent all-atoms simulations of these top hits revealed consistent dynamics, indicating their stability and their potential to interact effectively with the interface residues. In conclusion, our study represents the first attempt to establish a foundation for understanding the heightened infectivity of new SARS-CoV-2 variants and provides a strong impetus for the development of novel drugs against them.

SARS-CoV-2 has evolved several strategies to overcome host cell defenses by inducing cell injury to favour its replication. Many viruses have been reported to modulate the intracellular redox balance, affecting the Nuclear factor erythroid 2-Related Factor 2 (NRF2) signaling pathway. Although antioxidant modulation by SARS-CoV-2 infection has already been described, the viral factors involved in modulating the NRF2 pathway are still elusive. Given the antagonistic activity of ORF6 on several cellular pathways, we investigated the role of the viral protein towards NRF2-mediated antioxidant response. The ectopic expression of the wt-ORF6 protein negatively impacts redox cell homeostasis, leading to an increase in ROS production, along with a decrease in NRF2 protein and its downstream controlled genes. Moreover, when investigating the Δ61 mutant, previously described as an inactive nucleopore proteins binding mutant, we prove that the oxidative stress induced by ORF6 is substantially related to its C-terminal domain, speculating that ORF6 mechanism of action is associated with the inhibition of nuclear mRNA export processes. In addition, activation by phosphorylation of the serine residue at position 40 of NRF2 is increased in the cytoplasm of wt-ORF6-expressing cells, supporting the presence of an altered redox state, although NRF2 nuclear translocation is hindered by the viral protein to fully antagonize the cell response. Furthermore, wt-ORF6 leads to phosphorylation of a stress-activated serine/threonine protein kinase, p38 MAPK, suggesting a role of the viral protein in regulating p38 activation. These findings strengthen the important role of oxidative stress in the pathogenesis of SARS-CoV-2 and identify ORF6 as an important viral accessory protein hypothetically involved in modulating the antioxidant response during viral infection. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve different strategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fide viral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficiently than WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effect on Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-β treatment. However, upon treatment with IFN-β, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficient to antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacy of therapeutics that stimulate innate immune pathways. IMPORTANCE Previous studies identified several SARS-CoV-2 proteins, including ORF6, that antagonize host innate immune responses in the context of overexpression of viral proteins in non-respiratory cells. We set out to determine the role of ORF6 in IFN responses during SARS-CoV-2 infection of respiratory cells. Using a deletion strain, we observed no reduction of infection and no difference in evasion of IFN signaling, with responses limited to bystander cells. Moreover, stimulation of Sendai virus-induced IFN production or IFN-β-stimulated ISG expression was comparable between SARS-CoV-2 virus and SARS-CoV-2 lacking ORF6 virus, suggesting that ORF6 is not sufficient to counteract IFN induction or IFN signaling during viral infection.

Lipid droplets (LDs) form inter‐organelle contacts with the endoplasmic reticulum (ER) that promote their biogenesis, while LD contacts with mitochondria enhance β‐oxidation of contained fatty acids. Viruses have been shown to take advantage of lipid droplets to promote viral production, but it remains unclear whether they also modulate the interactions between LDs and other organelles. Here, we showed that coronavirus ORF6 protein targets LDs and is localized to the mitochondria‐LD and ER‐LD contact sites, where it regulates LD biogenesis and lipolysis. At the molecular level, we find that ORF6 inserts into the LD lipid monolayer via its two amphipathic helices. ORF6 further interacts with ER membrane proteins BAP31 and USE1 to mediate ER‐LDs contact formation. Additionally, ORF6 interacts with the SAM complex in the mitochondrial outer membrane to link mitochondria to LDs. In doing so, ORF6 promotes cellular lipolysis and LD biogenesis to reprogram host cell lipid flux and facilitate viral production. Synopsis: Viruses, including SARS‐CoV‐2, target lipid droplets to enhance viral replication. This study identifies SARS‐CoV‐2 protein ORF6 as a regulator of lipid droplet interactions with the ER and mitochondria. ORF6 localizes to lipid droplets and inserts into the lipid monolayer via its two amphipathic helices.ORF6 reprograms cellular lipid metabolism to enhance lipolysis.ORF6 promotes ER‐lipid droplet contact formation and lipid droplet biogenesis via an interaction with ER membrane proteins BAP31 and USE1.ORF6 links lipid droplets to mitochondria by binding to the mitochondrial outer membrane SAM complex, leading to increased ATP production. [ABSTRACT FROM AUTHOR]

Molecular mechanisms of Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named "Rainbow-KSHV," that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication.IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic, induces an unbalanced immune response in the host. For instance, the production of type I interferon (IFN) and the response to it, which act as a front-line defense against virus invasion, are inhibited during SARS-CoV-2 infection. In addition, tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, is upregulated in COVID-19 patients with severe symptoms. Studies on the closely related betacoronavirus, SARS-CoV, showed that viral proteins such as Nsp1, Orf6 and nucleocapsid protein inhibit IFN-β production and responses at multiple steps. Given the conservation of these proteins between SARS-CoV and SARS-CoV-2, it is not surprising that SARS-CoV-2 deploys similar immune evasion strategies. Here, we carried out a screen to examine the role of individual SARS-CoV-2 proteins in regulating innate immune signaling, such as the activation of transcription factors IRF3 and NF-κB and the response to type I and type II IFN. In addition to established roles of SARS-CoV-2 proteins, we report that SARS-CoV-2 proteins Nsp6 and Orf8 inhibit the type I IFN response but at different stages. Orf6 blocks the translocation of STAT1 and STAT2 into the nucleus, whereas ORF8 inhibits the pathway in the nucleus after STAT1/2 translocation. SARS-CoV-2 Orf6 also suppresses IRF3 activation and TNF-α-induced NF-κB activation.

Intrinsically disordered proteins (IDPs) often contain proline residues, which undergo cis/trans isomerisation. While molecular dynamics (MD) simulations have the potential to fully characterise the proline cis and trans sub-ensembles, they are limited by the slow timescales of isomerisation and force field inaccuracies. Nuclear magnetic resonance (NMR) spectroscopy can report on ensemble-averaged observables for both the cis-proline and trans-proline states, but a full atomistic characterisation of these conformers is challenging. Given the importance of proline cis/trans isomerisation for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle X-ray scattering (SAXS) to characterise the two sub-ensembles of the ORF6 C-terminal region (ORF6 CTR ) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimised for disordered proteins. Each simulation was run for an accumulated time of 180-220 μs until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamic simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamic simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6 CTR are extremely dynamic and that interdisciplinary approaches combining both multi-scale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterised by either approach in isolation., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds nuclear pore component Nup98--Rae1 to inhibit nuclear translocation of activated STAT1 and IRF3 transcription factors. However, this direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6--Nup98 interaction domain robustly blocked nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral membrane protein, as opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was isolated from the oro/pharyngeal swabs of two Italian COVID-19 patients, physicians in a COVID-19 division hospital, with different courses of the disease. The complete genome sequences show that the two isolates belong to the B1.1 lineage, but contain a nucleotide mutation in the ORF6, leading to a stop codon and to the deletion of 6 amino acids in the C terminus. This deletion was unique, compared to the currently available sequences deposited in the GISAID and GenBank database. It did not affect the in vitro viral replication, neither the neutralizing activities of the patients' antibodies. Based on homology analysis with other Coronaviruses, the two isolated lacked the ORF6 aminoacidic portion responsible for the inhibition of the antiviral Interferon (IFN)-based host response. IFN seems to have a dual role of in SARS-CoV-2 infected patients: not only antiviral activity, but also a detrimental role in case of excessive production. A deletion in the SARS-CoV-2 ORF6 protein might have a specific, still unknown role in the viral pathogenesis.

During routine molecular surveillance of SARS-CoV-2 performed at the National Reference Center of Respiratory Viruses (Lyon, France) (n = 229 sequences collected February–April 2020), two frameshifting deletions were detected in the open reading frame 6, at the same position (27267). While a 26-nucleotide deletion variant (D26) was only found in one nasopharyngeal sample in March 2020, the 34-nucleotide deletion (D34) was found within a single geriatric hospital unit in 5/9 patients and one health care worker in April 2020. Phylogeny analysis strongly suggested a nosocomial transmission of D34, with potential fecal transmission, as also identified in a stool sample. No difference in disease severity was observed between patients hospitalized in the geriatric unit infected with WT or D34. In vitro D26 and D34 characterization revealed comparable replication kinetics with the wild-type (WT), but differential host immune responses. While interferon-stimulated genes were similarly upregulated after infection with WT and ORF6 variants, the latter specifically induced overexpression of 9 genes coding for inflammatory cytokines in the NF-kB pathway, including CCL2/MCP1, PTX3, and TNFα, for which high plasma levels have been associated with severe COVID-19. Our findings emphasize the need to monitor the occurrence of ORF6 deletions and assess their impact on the host immune response.

ABSTRACTThe Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.

The accessory protein Orf6 is uniquely expressed in sarbecoviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is an ongoing pandemic. SARS-CoV-2 Orf6 antagonizes host interferon signaling by inhibition of mRNA nuclear export through its interactions with the ribonucleic acid export 1 (Rae1)–nucleoporin 98 (Nup98) complex. Here, we confirmed the direct tight binding of Orf6 to the Rae1-Nup98 complex, which competitively inhibits RNA binding. We determined the crystal structures of both SARS-CoV-2 and SARS-CoV-1 Orf6 C-termini in complex with the Rae1–Nup98 heterodimer. In each structure, SARS-CoV Orf6 occupies the same potential mRNA-binding groove of the Rae1–Nup98 complex, comparable to the previously reported structures of other viral proteins complexed with Rae1-Nup98, indicating that the Rae1–Nup98 complex is a common target for different viruses to impair the nuclear export pathway. Structural analysis and biochemical studies highlight the critical role of the highly conserved methionine (M58) of SARS-CoVs Orf6. Altogether our data unravel a mechanistic understanding of SARS-CoVs Orf6 targeting the mRNA-binding site of the Rae1–Nup98 complex to compete with the nuclear export of host mRNA, which further emphasizes that Orf6 is a critical virulence factor of SARS-CoVs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic, responsible for millions of deaths globally. Even with effective vaccines, SARS-CoV-2 will likely maintain a hold in the human population through gaps in efficacy, percent vaccinated, and arising new strains. Therefore, understanding how SARS-CoV-2 causes widespread tissue damage and the development of targeted pharmacological treatments will be critical in fighting this virus and preparing for future outbreaks. Herein, we summarize the progress made thus far by using in vitro or in vivo models to investigate individual SARS-CoV-2 proteins and their pathogenic mechanisms. We have grouped the SARS-CoV-2 proteins into three categories: host entry, self-acting, and host interacting. This review focuses on the self-acting and host-interacting SARS-CoV-2 proteins and summarizes current knowledge on how these proteins promote virus replication and disrupt host systems, as well as drugs that target the virus and virus interacting host proteins. Encouragingly, many of these drugs are currently in clinical trials for the treatment of COVID-19. Future coronavirus outbreaks will most likely be caused by new virus strains that evade vaccine protection through mutations in entry proteins. Therefore, study of individual self-acting and host-interacting SARS-CoV-2 proteins for targeted therapeutic interventions is not only essential for fighting COVID-19 but also valuable against future coronavirus outbreaks. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARSCoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, 26, 27a, 27b, and 28 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (DORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of DORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the DORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while DORF7a, DORF7b, and DORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. [ABSTRACT FROM AUTHOR]

Summary: The presence of an ORF6 gene distinguishes sarbecoviruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 from other betacoronaviruses. Here we show that ORF6 inhibits induction of innate immune signaling, including upregulation of type I interferon (IFN) upon viral infection as well as type I and III IFN signaling. Intriguingly, ORF6 proteins from SARS-CoV-2 lineages are more efficient antagonists of innate immunity than their orthologs from SARS-CoV lineages. Mutational analyses identified residues E46 and Q56 as important determinants of the antagonistic activity of SARS-CoV-2 ORF6. Moreover, we show that the anti-innate immune activity of ORF6 depends on its C-terminal region and that ORF6 inhibits nuclear translocation of IRF3. Finally, we identify naturally occurring frameshift/nonsense mutations that result in an inactivating truncation of ORF6 in approximately 0.2% of SARS-CoV-2 isolates. Our findings suggest that ORF6 contributes to the poor IFN activation observed in individuals with coronavirus disease 2019 (COVID-19).

The accessory protein ORF6 of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key interferon (IFN) antagonist that strongly suppresses the production of primary IFN as well as the expression of IFN-stimulated genes. However, how host cells respond to ORF6 remains largely unknown. Our research of ORF6-binding proteins by pulldown revealed that E3 ligase components such as Cullin 4B (CUL4B), DDB1, and RBX1 are potential ORF6-interacting proteins. Further study found that the substrate recognition receptor PRPF19 interacts with CUL4B, DDB1, and RBX1 to form a CRL4B-based E3 ligase, which catalyzes ORF6 ubiquitination and subsequent degradation. Overexpression of PRPF19 promotes ORF6 degradation, releasing ORF6-mediated IFN inhibition, which inhibits SARS-CoV-2 replication. Moreover, we found that activation of CUL4B by the neddylation inducer etoposide alleviates lung lesions in a SARS-CoV-2 mouse infection model. Therefore, targeting ORF6 for degradation may be an effective therapeutic strategy against SARS-CoV-2 infection.IMPORTANCEThe cellular biological function of the ubiquitin-proteasome pathway as an important modulator for the regulation of many fundamental cellular processes has been greatly appreciated. The critical role of the ubiquitin-proteasome pathway in viral pathogenesis has become increasingly apparent. It is a powerful tool that host cells use to defend against viral infection. Some cellular proteins can function as restriction factors to limit viral infection by ubiquitin-dependent degradation. In this research, we identificated of CUL4B-DDB1-PRPF19 E3 Ubiquitin Ligase Complex can mediate proteasomal degradation of ORF6, leading to inhibition of viral replication. Moreover, the CUL4B activator etoposide alleviates disease development in a mouse infection model, suggesting that this agent or its derivatives may be used to treat infections caused by SARS-CoV-2. We believe that these results will be extremely useful for the scientific and clinic communities in their search for cues and preventive measures to combat the COVID-19 pandemic., Competing Interests: The authors declare no conflict of interest.

Molecular mechanisms of Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named "Rainbow-KSHV," that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication. [ABSTRACT FROM AUTHOR]

Background/Purpose: Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins including ORF6 inhibit Type I interferon (IFN) signaling. Methods: This study identified SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries, and examined the association of ORF6–host factor interaction with Type I IFN antagonism. After the fifth round of biopanning with Escherichia coli-synthesized ORF6-His tagged protein, the relative binding affinity of phage clones to ORF6 was determined using direct enzyme-linked immunosorbent assay. Results: The highest affinity clone to ORF6 displayed the C-terminal domain of NPIPB3 (nuclear pore complex interacting protein family, member B3; also named as phosphatidylinositol-3-kinase-related kinase SMG-1 isoform 1 homolog). The coimmunoprecipitation assay demonstrated the direct binding of ORF6 to the C-terminal domain of NPIPB3 in vitro. Confocal imaging revealed a close colocalization of SARS-CoV ORF6 protein with NPIPB3 in human promonocytes. The dual luciferase reporter assay showed that the C-terminal domain of NPIPB3 attenuated the antagonistic activity of SARS-CoV ORF6 on IFN-β-induced ISRE (IFN stimulated response element)-responsive firefly luciferase activity. In addition, confocal imaging and Western blotting assays revealed that the increases in STAT-1 nuclear translocation and phosphorylation occurred in the transfected cells expressing both genes of ORF6 and NPIPB3, but not in the ORF6-expressing cells in response to IFN-β. Conclusion: The overexpression of NPIPB3 restored the IFN-β responses in SARS-CoV ORF6 expressing cells, indicating that the interaction of SARS CoV ORF6 and NPIPB3 reduced Type I IFN antagonism by SARS-CoV ORF6.

Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al ., Cell Rep. 33 , 108234 (2020); L. Miorin et al ., Proc. Natl. Acad. Sci. U.S.A. 117 , 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.et al ., J. Virol. 81 , 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport., Competing Interests: Competing interests statement:The authors declare no competing interest.

SARS-CoV-2 belongs to the subgenus Sarbecovirus , which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections.IMPORTANCEThe ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections., Competing Interests: The authors declare no conflict of interest.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2. [Display omitted] • SARS-CoV-2 ORF6 antagonizes IFN-induced signaling during infection • ORF6 binds Nup98-Rae1 and selectively inhibits nucleocytoplasmic trafficking • ORF6 expression contributes to SARS-CoV-2 pathogenesis • The D61L polymorphism disrupts ORF6 protein functions at the NPC Kehrer, Cupic et al. dissect the role of ORF6 in the host response to SARS-CoV-2 infection. ORF6 is an innate immune antagonist that binds to Nup98-Rae1 and suppresses host gene expression by selectively inhibiting nucleocytoplasmic trafficking. ORF6 loss-of-function mutations result in SARS-CoV-2 attenuation both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve different strategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fide viral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficiently than WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effect on Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-β treatment. However, upon treatment with IFN-β, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficient to antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacy of therapeutics that stimulate innate immune pathways. IMPORTANCE Previous studies identified several SARS-CoV-2 proteins, including ORF6, that antagonize host innate immune responses in the context of overexpression of viral proteins in non-respiratory cells. We set out to determine the role of ORF6 in IFN responses during SARS-CoV-2 infection of respiratory cells. Using a deletion strain, we observed no reduction of infection and no difference in evasion of IFN signaling, with responses limited to bystander cells. Moreover, stimulation of Sendai virus-induced IFN production or IFN-β-stimulated ISG expression was comparable between SARS-CoV-2 virus and SARS-CoV-2 lacking ORF6 virus, suggesting that ORF6 is not sufficient to counteract IFN induction or IFN signaling during viral infection., Competing Interests: The authors declare no conflict of interest.

In the present study, the function of a novel ORF6 gene in the PCV2 genome was determined and functionally analyzed in vitro. ORF6 expression was demonstrated by indirect immunofluorescence in PCV2-infected cells. The antibody against ORF6 was detected in PCV2-infected pigs. The start codon of ORF6 was mutated and an infectious clone was used to create an ORF6-deficient mutant virus. Viral DNA replication curves and immunofluorescence analysis indicated that ORF6 is unnecessary for viral replication and ORF6 deletion reduces viral DNA replication in PK-15 cells. The activities of caspases 3 and 8 in ORF6-deficient virus-infected cells were significantly different from those in wild-type virus-infected cells. The ORF6 protein can increase the expression of IFN-β, TNF-α, IL-1b, IL-10, and IL-12p40. These results demonstrated that the newly discovered ORF6 protein may be involved in caspases regulation and the expression of multiple cytokines in PCV2-infected cells. The functions of this gene in viral pathogenesis remain to be further elucidated. [ABSTRACT FROM AUTHOR]

• The BA.1, BA.2 and BA.5 present mutations on antagonistic proteins to IFN-β. • Omicron BA.2 did not exert an in vitro downregulation of IFN-β secretion. • The in vitro reduced IFN-β secretion by BA.2 correlate with the ORF6 D61L mutation. • The recombinant mutated ORF6 protein fails to inhibit IFN-β production in vitro. • Post-transcriptional events can be involved in innate immunity escape by BA.1. Although most of the attention was focused on the characterization of changes in the Spike protein among variants of SARS-CoV-2 virus, mutations outside the Spike region are likely to contribute to virus pathogenesis, virus adaptation and escape to the immune system. Phylogenetic analysis of SARS-CoV-2 Omicron strains reveals that several virus sub-lineages could be distinguished, from BA.1 up to BA.5. Regarding BA.1, BA.2 and BA.5, several mutations concern viral proteins with antagonistic activity to the innate immune system, such as NSP1 (S 135 R), which is involved in mRNAs translation, exhibiting a general shutdown in cellular protein synthesis. Additionally, mutations and/or deletions in the ORF6 protein (D 61 L) and in the nucleoprotein N (P 13 L, D 31-33 ERS, P 151 S, R 203 K, G 204 R and S 413 R) have been reported, although the impact of such mutations on protein function has not been further studied. The aim of this study was to better investigate the innate immunity modulation by different Omicron sub-lineages, in the attempt to identify viral proteins that may affect virus fitness and pathogenicity. Our data demonstrated that, in agreement with a reduced Omicron replication in Calu-3 human lung epithelial cells compared to the Wuhan-1 strain, a lower secretion of interferon beta (IFN-β) from cells was observed in all sub-lineages, except for BA.2. This evidence might be correlated with the presence of a mutation within the ORF6 protein (D 61 L), which is strikingly associated to the antagonistic function of the viral protein, since additional mutations in viral proteins acting as interferon antagonist were not detected or did not show significant influence. Indeed, the recombinant mutated ORF6 protein failed to inhibit IFN-β production in vitro. Furthermore, we found an induction of IFN-β transcription in BA.1 infected cells, that was not correlated with the cytokine release at 72 h post-infection, suggesting that post-transcriptional events can be involved in controlling the innate immunity. [ABSTRACT FROM AUTHOR]

One of the unique features of herpesvirus infection is latent infection following an initial exposure, which is characterized by viral genome persistence in a small fraction of cells within the latently infected tissue. Investigation of the mechanisms of herpesvirus latency has been very challenging in tissues with only a small fraction of cells that are latently infected. Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is an important and deadly pathogen of koi and common carp, Cyprinus carpio . Acute infection can cause up to 100% mortality in exposed fish, and fish that survive the infection become latently infected. KHV becomes latent in a small percentage of B lymphocytes and can reactivate under stressful conditions. During latency, KHV ORF6 transcript is expressed in the latently infected B lymphocytes. In order to study KHV latent infection in cells that are only latently infected, a nanoflare probe specific to ORF6 RNA was used to separate KHV latently infected cells from total peripheral white blood cells (WBC). Using the ORF6 nanoflare probe, less than 1% of peripheral WBC was isolated from KHV latently infected koi. When this enriched population of WBC was examined by real-time PCR specific for KHV, it was estimated that about 1–2 copies of viral genome persists in the sorted cells. In addition, KHV ORF6 transcript was shown to be the major transcript expressed during latency by RNA-seq analysis. This study demonstrated that an RNA nanoflare probe could be used to enrich latently infected cells, which can subsequently be used to investigate the molecular mechanisms of KHV latency. [ABSTRACT FROM AUTHOR]