SARS-CoV-2 Mac1 is an essential virulence factor

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in this set of proteins is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and interferon-stimulated gene (ISG) expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.National Institutes of Health (NIH) grant P20GM103648 (RC) National Institutes of Health (NIH) grant 2P01AI060699 (LE) National Institutes of Health (NIH) grant P20GM113117 (ARF) National Institutes of Health (NIH) grant K22AI134993 (ARF) National Institutes of Health (NIH) grant R35GM138029 (ARF) National Science Foundation (NSF) grant 2135167 (RLU) University of Kansas General Research Fund (GRF) and Start-up funds (ARF) NIH Graduate Training at the Biology-Chemistry Interface grant T32GM132061 (CMK) University of Kansas College of Liberal Arts and Sciences Graduate Research Fellowship (CMK) Government of Spain (PID2019-107001RB-I00 AEI/FEDER, UE) LE European Commission (H2020-SC1-2019, ISOLDA Project nº 848166-2) LEN

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity

International audienceMiddle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b. Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface

The cellular redox environment alters antigen presentation

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5-10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.This work was supported, in whole or in part, by National Institutes of Health Grant R01 NS036592. This work was also supported by an infrastructure grant (Grant LE100100036) from the Australian Research Council (ARC) and a project grant from the Juvenile Diabetes Research Foundation (17-2012-134)

Anti-PDL-1 antibody treatment increases the proportion of IL-2, IFN-γ and TNF-α producing SSIEFARL specific effector CD8 T cells.

<p>Mice from anti-PDL-1 or isotype treated groups were euthanized on day 6 post infection. Single cell suspensions obtained from PLN and spleen tissue were stained intra-cellularly for pro-inflammatory cytokines after brief <i>in vitro</i> stimulation with SSIEFARL peptide (10 µg/ml). The representative FACS plots in A, B and C demonstrates the proportion of IFN-γ, TNF-α and IL-2 secreting CD8 T cells, respectively, in PLN and spleen tissue derived from isotype and anti-PDL-1 treated mouse. Scatter plots in panel A, B and C show mouse to mouse variation in the frequency of IFN-γ, TNF-α and IL-2 producing CD8 T cell in PLN and spleen tissue of isotype and anti-PDL-1 treated groups of mice. Data shown is derived from two similar experiments with four mice per group in each experiment. Statistical significance was determined by unpaired student’s t test where *p<0.05 and **p<0.01 are considered statistically significant.</p

Kinetics of PDL-1 expression on CD11c+ dendritic cells derived from popliteal lymph node (PLN) of HSV-1 infected mice.

<p>A, A representative histogram overlay, obtained from gating on CD11c+ cells, denotes the expression of PDL-1 on CD11c+ dendritic cells in PLN at different time-points post-infection. Naïve represents level of PDL-1 expression on CD11c+ DCs in the PLN of uninfected mice. B, Bar graph shows mean ± S.D. of PDL-1 mean fluorescence intensity (MFI) on CD11c+ DCs in naive and infected mice at different time-points post-infection. Data shown is derived from four mice at each time-point.</p

Anti-PDL-1 antibody treatment increases the frequency and absolute numbers of SSIEFARL specific CD8 T cell in PLN and spleen tissue of HSV-1 infected mice.

<p>Anti-PDL-1 or Isotype matched antibody was intra-peritoneally administered on -1 and 3 days post footpad HSV-1 infection. Both groups of mice were euthanized on day 6 post-infection and single cell suspensions from PLN and spleen tissue were cell surface stained with gB_498–505 tetramer to detect immunodominant SSIEFARL peptide specific CD8 T cells. The representative FACS plots are gated on CD8 T cells and demonstrate the proportion of SSIEFARL specific CD8 T cells in isotype and anti-PDL-1 treated mice in PLN and spleen, respectively. The scatter plots represent average percentage and the bar diagram shows the average absolute numbers of SSIEFARL specific CD8 T cells in PLN and spleen tissue of isotype and anti-PDL-1 antibody treated groups of mice. Data shown is the representative of two independent experiments including 4mice/experiment. Statistical significance was determined by unpaired students t test with *P<0.05, **P<0.01 and ***P<0.0001 were considered statistically significant.</p

Granzyme B secreting potential of PD-1^high and PD-1^medium CD8 T cells in spleen during lytic phase of HSV-1 infection.

<p><i>Ex-vivo</i> cytotoxicity of splenic PD-1^high and PD-1^medium CD8 T cells on day 6 post-infection was compared after stimulating with SSIEFARL peptide as described in the methods. A, The representative FACS plot denotes the ratio of PD-1^high (2.88), PD-1^medium (7.54) and PD-1^negative (88.6) splenic CD8 T cells on day 6 post-infection. The frequencies of cell surface expressed CD107a/b by PD-1^high and PD-1^medium CD8 T cells are shown in second column. B, The bar diagram shows the mean ± S.D. of the frequency of cell surface expressing CD107a/b PD-1^high and PD-1^medium CD8 T cells from five mice. Statistical significance was determined with unpaired student’s t test where ***p<0.0001 were considered statistically significant.</p

Anti-PDL-1 treatment increases the cytotoxic potential of HSV-1 specific effector CD8 T cell in the PLN and spleen tissue during lytic phase of HSV-1 infection.

<p>Mice from isotype and anti-PDL1 treated groups were euthanized on day 6 post-infection. Single cell suspensions obtained from PLN and spleen tissue were <i>ex-vivo</i> stimulated with SSIEFARL peptide in the presence of fluorochrome conjugated anti-CD107a/b antibodies followed by intra-cellular staining for granzyme B molecule. A, Representative FACS plots and scatter plots denotes the proportion of granzyme B expressing CD8 T cells in PLN and spleen tissue of control and anti-PDL1 treated groups on day 6 post-infection. B, Histogram FACS plots, derived from gated CD8 T cell, and the bar diagram demonstrate the frequency of CD8 T cells expressing membrane bound CD107a/b in PLN and spleen tissue of control and anti-PDL1 treated groups of mice. Data shown is derived from two similar experiments with four mice per group in each experiment. Statistical significance was determined by unpaired two-tailed student’s t test where *p<0.05 and **p<0.01 are considered statistically significant.</p