Effect of cannabidiol on apoptosis and cellular interferon and interferon-stimulated gene responses to the SARS-CoV-2 genes ORF8, ORF10 and M protein

Aims: To study effects on cellular innate immune responses to novel genes ORF8 and ORF10, and the more conserved Membrane protein (M protein) from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, either alone, or in combination with cannabidiol (CBD). Main Methods: HEK293 cells were transfected with a control plasmid, or plasmids expressing ORF8, ORF10, or M protein, and assayed for cell number and markers of apoptosis at 24 h, and expression of interferon and interferon-stimulated genes at 14 h. Key findings: A significant reduction in cell number, and increase in early and late apoptosis, was found after 24 h in cells where expression of viral genes was combined with 1-2 μM CBD treatment, but not in control-transfected cells treated with CBD, or in cells expressing viral genes but treated only with vehicle. CBD (2 μM) augmented expression of IFNγ, IFNλ1 and IFNλ2/3, as well as the 2'-5'-oligoadenylate synthetase (OAS) family members OAS1, OAS2, OAS3, and OASL, in cells expressing ORF8, ORF10, and M protein. CBD also augmented expression of these genes in control cells not expressing viral genes, without enhancing apoptosis. Significance: Our results demonstrate a poor ability of HEK293 cells to respond to SARS-CoV-2 genes alone, but suggest an augmented innate anti-viral response to these genes in the presence of CBD. Furthermore, our results indicate that CBD may prime components of the innate immune system, increasing readiness to respond to viral infection without activating apoptosis, and therefore could be studied for potential in prophylaxis.

An M protein coiled coil unfurls and exposes its hydrophobic core to capture LL-37

Surface-associated, coiled-coil M proteins of Streptococcus pyogenes (Strep A) disable human immunity through interaction with select proteins. However, coiled coils lack features typical of protein-protein interaction sites, and it is therefore challenging to understand how M proteins achieve specific binding, for example, with the human antimicrobial peptide LL-37, which results in its neutralization. The crystal structure of a complex of LL-37 with M87 protein, an antigenic variant from a strain that is an emerging threat, revealed a novel interaction mode. The M87 coiled coil unfurled and asymmetrically exposed its hydrophobic core to capture LL-37. A single LL-37 molecule bound M87 in the crystal, but in solution recruited additional LL-37 molecules, consistent with a protein trap neutralization mechanism. The interaction mode visualized crystallographically was verified to contribute significantly to LL-37 resistance in an M87 Strep A strain, and was identified to be conserved in a number of other M protein types that are prevalent in human populations. Our results provide specific detail for therapeutic inhibition of LL-37 neutralization by M proteins.

Distinct serotypes of streptococcal M proteins mediate fibrinogen-dependent platelet activation and pro-inflammatory effects

Sepsis is a life-threatening complication of infection that is characterised by a dysregulated inflammatory state and disturbed hemostasis. Platelets are the main regulators of hemostasis, and they also respond to inflammation. The human pathogen Streptococcus pyogenes can cause local infection that may progress to sepsis. There are more than 200 different serotypes of S. pyogenes defined according to sequence variations in the M protein. The M1 serotype is among ten serotypes that are predominant in invasive infection. M1 protein can be released from the surface and has previously been shown to generate platelet, neutrophil and monocyte activation. The platelet dependent pro-inflammatory effects of other serotypes of M protein associated with invasive infection (M3, M5, M28, M49 and M89) is now investigated using a combination of multiparameter flow cytometry, ELISA, aggregometry and quantitative mass spectrometry. We demonstrate that only M1-, M3- and M5 protein serotypes can bind fibrinogen in plasma and mediate fibrinogen and IgG dependent platelet activation and aggregation, release of granule proteins, upregulation of CD62P to the platelet surface, and complex formation with neutrophils and monocytes. Neutrophil and monocyte activation, determined as upregulation of surface CD11b, is also mediated by M1-, M3- and M5 protein serotypes, while M28-, M49- or M89 proteins failed to mediate activation of platelets or leukocytes. Collectively, our findings reveal novel aspects of the immunomodulatory role of fibrinogen acquisition and platelet activation during streptococcal infections.

Multiple RNA virus matrix proteins interact with SLD5 to manipulate host cell cycle

The matrix protein of many enveloped RNA viruses regulates multiple stages of viral life cycle and has the characteristics of nucleocytoplasmic shuttling. We have previously demonstrated that matrix protein 1 (M1) of an RNA virus, influenza virus, blocks host cell cycle progression by interacting with SLD5, a member of the GINS complex, which is required for normal cell cycle progression. In this study, we found that M protein of several other RNA viruses, including VSV, SeV and HIV, interacted with SLD5. Furthermore, VSV/SeV infection and M protein of VSV/SeV/HIV induced cell cycle arrest at G0/G1 phase. Importantly, overexpression of SLD5 partially rescued the cell cycle arrest by VSV/SeV infection and VSV M protein. In addition, SLD5 suppressed VSV replication in vitro and in vivo, and enhanced type Ⅰ interferon signalling. Taken together, our results suggest that targeting SLD5 by M protein might be a common strategy used by multiple enveloped RNA viruses to block host cell cycle. Our findings provide new mechanistic insights for virus to manipulate cell cycle progression by hijacking host replication factor SLD5 during infection.

Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target

ABSTRACTSARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and a PLA assay. In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase of PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

Design of M protein immunogens to elicit broadly reactive antibodies against Streptococcus pyogenes

M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (Strep A) are immunodominant targets of opsonizing antibodies. However, the antigenic sequence variability of the M protein into >220 M types has limited its utility as a vaccine immunogen, as antibody recognition is usually type-specific. At present no vaccine against Strep A exists. Unlike type-specific antibodies, C4BP binds type-promiscuously to M proteins. We recently showed that this was due to a three-dimensional (3D) pattern of amino acids that is conserved in numerous M types. We hypothesized that M protein immunogens biased towards the 3D pattern and away from variable sequences would evoke a broadly protective response. We show here that an immunogen containing only 34 amino acids of M2 protein retained C4BP-binding and was sufficient to evoke antibodies that were cross-reactive and opsonophagocytic against multiple M types. These proof-of-principle experiments provide significant evidence that an essential Strep A virulence trait (i.e., C4BP binding) can be targeted in the design of an immunogen that evokes a broadly protective response.

Establishment of a peptide-based enzyme-linked immunosorbent assay for detecting antibodies against PRRSV M protein

Background Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the swine industry globally. Evaluation of antibody responses and neutralizing antibody titers is the most effective method for vaccine evaluation. In this study, the B cell line epitopes of PRRSV M protein were predicted, and two peptide ELISA assays were established (M-A110-129 ELISA, M-A148-174 ELISA) to detect antibodies against PRRSV M protein. Field serum samples collected from pig farms were used to validate the peptide ELISA and compare it with an indirect immunofluorescence assay. Results The sensitivity and specificity of M-A110-129 ELISA and M-A148-174 ELISA were (111/125) 88.80%, (69/70) 98.57% and (122/125) 97.60%, (70/70) 100%, relative to indirect immunofluorescence assay. This peptide ELISA could detect antibodies against different genotypes of PRRSV including type 1 PRRSV, classical PRRSV, HP-PRRSV, and NADC30 like PRRSV, but not antibodies against other common swine viruses. The results of ROC analysis showed that the area under the curve (AUC) of the M-A110-129 ELISA and M-A148-174 ELISA were 0.967 and 0.996, respectively. Compared the concordance of results using two peptide ELISA assays, the IDEXX PRRSV X3 Ab ELISA and a virus neutralization test, were assessed using a series of 147 sera from pigs vaccinated with the NADC30-like PRRSV inactivated vaccine. The M-A148-174 ELISA had the best consistency, with a Cohen’s kappa coefficient of 0.8772. The concordance rates of the Hipra PRRSV ELISA kit, M-A110-129 ELISA and M-A148-174 ELISA in the field seropositive detection results were 91.08, 86.32 and 95.35%, relative to indirect immunofluorescence assay. Conclusions In summary, compared with M-A110-129 ELISA, the PRRSV M-A148-174 ELISA is of value for detecting antibodies against PRRSV and the evaluation of the NADC30-like PRRSV inactivated vaccine, but the advantage is insufficient in serological early diagnosis.