Protein-S-nitrosylation of human cytomegalovirus pp65 reduces its ability to undermine cGAS

Abstract

Post-translational modifications (PTMs) are key regulators of various processes important for cell survival. These modifications are critical for dealing with stress conditions, such as those observed in disease states, and during infections with various pathogens. We previously reported that during infection of primary dermal fibroblasts, multiple human cytomegalovirus (HCMV)-encoded proteins were post-translationally modified by the addition of a nitric oxide group to cysteine residues, a modification called protein-S-nitrosylation. For example, tegument protein pp71 is nitrosylated, diminishing its ability to inhibit STING, a protein necessary for DNA virus immune response. Herein, we report that an additional HCMV tegument protein, pp65, responsible for the inhibition of cGAS is also modified by protein-S-nitrosylation on two cysteine residues. Utilizing site-directed mutagenesis to generate recombinant viruses that encode a pp65 that cannot be protein-S-nitrosylated, we evaluated the impact of this PTM on viral replication and how the virus impacts the cGAS/STING pathway. We report that the nitrosylation of pp65 negatively impacts its ability to block cGAS enzymatic functions. pp65 protein-S-nitrosylation mutants demonstrated a decrease in cGAS/STING-induced IRF3 and TBK1 phosphorylation. Additionally, we observed a reduction in IFN-β1 secretion in NuFF-1 cells expressing a nitrosylation-resistant pp65. We report that HCMV expressing a protein-S-nitrosylation-deficient pp65 is resistant to the activation of cGAS in the infection of primary dermal fibroblasts. Our work suggests that nitrosylation of viral proteins may serve as a broadly neutralizing mechanism in HCMV infection.

Importance: Post-translational modifications (PTM) are utilized by host cells to limit an invading pathogen's ability to establish a productive infection. A potent PTM, called protein-S-nitrosylation, has anti-bacterial and anti-viral properties. Increasing protein-S-nitrosylation with the addition of nitric oxide donor compounds reduced HCMV replication in fibroblasts and epithelial cells. We previously reported that protein-S-nitrosylation of HCMV pp71 limits its ability to inhibit STING. Herein, we report that the protein-S-nitrosylation of HCMV pp65 impacts its ability to limit cGAS activity, an additional protein important in regulating interferon response. Therapeutically, patients provided nitric oxide by inhalation reduced viral replication in coronavirus disease 2019, influenza, and even impacted bacterial growth within patients' lungs. It is thought that an increase in free nitric oxide increases the frequency of nitrosylated proteins. Understanding how protein-S-nitrosylation regulates a common DNA virus like HCMV will provide insights into the development of broadly neutralizing therapeutics in drug-resistant viral infections.

Keywords: HCMV; PTM; STING; cGAS; herpesviruses; pp65; protein-S-nitrosylation.

Fig 1

HCMV pp65 is protein-S-nitrosylated in infection of primary dermal fibroblasts. (A) Proteomic analysis of HCMV-infected fibroblast lysates 96 hpi identified specific sites of pp65 that are protein-S-nitrosylated (40). (B) NuFF-1 cells were infected at an MOI of 1 for 24, 48, and 72 hpi, and 100 µg of protein was subjected to a biotin switch, and then affinity purified with streptavidin beads. Biotinylated protein was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with primary antibody to pp65. A representative result of three biological repeats is shown. (C) Band intensities for pp65 expression in the input, −biotin switch, and +biotin switch groups were measured for panel B, and all samples were normalized to actin. n = 3.

Fig 2

Nitrosylation-deficient pp65 expressing HCMV replicates with similar kinetics to WT virus. (A) Table representing the mutations made in all HCMV mutant viruses. WT virus is denoted as C276/C306, SM1-pp65 C276S, SM2-pp65 C306S, and DM-pp65 C276S/C306S. (B) NuFF-1 cells were infected at an MOI of 1 with WT, DM, or Revertant HCMV, and the supernatant was collected on days 0, 3, 5, and 7 dpi. Viral titers were measured by TCID50 with mCherry expression. The inoculum was saved to determine equal PFU upon infection of NuFF-1 cells. (C) NuFF-1 cells were infected at an MOI of 0.1 with WT-pp65, DM-pp65, and Rev-pp65, and cell-free virus was collected at days 0, 3, 6, 9, 12, and 15 dpi. Viral titers were measured by TCID50 with mCherry expression. Inoculum was saved to determine equal PFU upon infection of NuFF-1 cells. (D) NuFF-1 cells were infected with WT or DM pp65 at an MOI of 1, and cell lysates were collected at 0, 24, 48, and 72 hpi. Protein lysates were immunoblotted with primary antibody to pp65 and actin. A representative western of three biological replicates is shown with quantification of all three experiments shown to the right. (E) NuFF-1 cells were infected at an MOI of 1 with WT or DM-pp65 HCMV for 6 h. Protein lysates were immunoblotted with primary antibody to pp65 and actin. (B–E) n = 3.

Fig 3

Nitrosylation-deficient pp65 is resistant to G3-YSD treatment. (A) WST assay of NuFF-1 cells transfected with G3-YSD 24 h after transfection. NuFF-1 cells were transfected for 6 h, with media removed, and 24 h later subjected to the WST assay to measure mitochondrial activity. WST-1 solution was added to each well and incubated for 2 h at 37°C. Following this, the absorbance of each well was measured at 420 nm. 10% Triton X-100 was used as a control to observe cell death within the assay. (B) NuFF-1 cells were transfected with G3-YSD for 6 h with indicated concentrations, and then infected with WT HCMV 24 h later at an MOI of 0.1. Viral titers were measured by TCID50 assay 7 days post-infection and reported as PFU/mL. (C) NuFF-1 cells were transfected with 0.4, 0.8, and 1.6 µg of G3-YSD for 6 h, and then 24 h later infected with WT or DM-pp65 at an MOI of 0.1. All viral titers were measured 7 days post-infection by TCID50 assay. (D) NuFF-1 cells were transfected with 0.8 μg of G3-YSD for 2 h, and then infected with WT-pp65 HCMV or DM-pp65 HCMV at an MOI of 1.0. 24 hpi protein lysates were collected and immunoblotted with primary antibody to IE1, and then incubated with a secondary anti-mouse antibody. Densitometry of IE1 expression is included for comparisons of IE1 expressions from the western blots. (A–) n = 3, P < 0.01**, P < 0.001***, and P < 0.0001****.

Fig 4

Expression levels of cGAS are unaffected by infection by WT and DM HCMV. NuFF-1 cells were infected with HCMV WT or DM-pp65 at an MOI of 1, and cell lysates were collected at 12, 24, 48, and 72 hpi. Protein lysates were immunoblotted with primary antibody to pp65 and GAPDH. n = 3.

Fig 5

HCMV pp65 mutants interact with cGAS in stable cell lines and infection. (A) NuFF-1 parental and pp65 WT expressing NuFF-1 cells were lysed using the protein-S-nitrosylation detection kit. Following cell lysis, 100 µg of protein cell lysate underwent the "biotin switch" reaction following the manufacturer’s instructions. Following the biotin switch, all reactions were affinity purified with avidin-coated beads. Protein was then separated to 8% SDS-PAGE and blotted with primary antibody specific to pp65. (B) Stable cell lines expressing WT pp65 (C276/C306), SM1-pp65 (C276S), SM2-pp65 (C306S), and DM-pp65 (C276S/C306S) were lysed with Co-IP lysis buffer and incubated with anti-mouse dynabeads. Protein was separated by 8% SDS-PAGE, and 5 µg of input lysate was used to confirm expression of the pp65 protein. The membrane was immunoblotted with pp65 antibody to confirm pulldown from interacting with cGAS. Actin was used to confirm equal loading of input. (C) NuFF-1 cells were infected at an MOI of 3 for 72 h with WT and all pp65 nitrosylation-deficient mutants. Cells were lysed with Co-IP lysis buffer, and protein was incubated with cGAS-coupled dynabeads overnight at 4°C. Next, 5 µg of input was separated to confirm equal expression of pp65 in each reaction. Isolated proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with pp65 antibody to confirm pulldown from interacting with cGAS. Actin was used to confirm equal loading of input. (A–C) n = 3.

Fig 6

The presence of DM-pp65 in NuFF-1 cells decreases the phosphorylation of pIRF3 and TBK1 after G3-YSD treatment. (A) NuFF-1–1 cells were transfected with 0.1 to 0.8 ug of G3-YSD. Cell lysates were collected after 4 h post-transfection, and protein lysates were separated by 8% SDS-PAGE. Membranes were then immunoblotted with pIRF3 or IRF3 antibody in two separate blots. (B) NuFF-1 and stable cells were transfected with 0.2 µg of G3-YSD for 4 h, and then cell lysates were collected and processed. Blots were probed with anti-IRF3 and pIRF3. (C) NuFF-1 parental and stable cells were transfected with 0.2 µg of G3-YSD or vehicle for 2 h, and cell lysates were collected and processed. Protein lysates were immunoblotted with anti-TBK1 and pTBK1, and then multiplexed with fluorescent secondary antibodies. (D) NuFF-1 parental and stable cell lines were transfected with 0.2 µg of G3-YSD for 8 h, and then supernatant was collected. Supernatant was then analyzed for IFN-β1 secretion by ELISA. OD readings were read at 450 nm. (A–D) n = 3, P < 0.05*, and P < 0.01**.

Fig 7

The nitrosylation of pp65 is an antiviral mechanism limiting its ability to inhibit cGAS. Our data suggest that when nitrosylation of pp65 is blocked, it has a better ability to antagonize cGAS, as observed by a decrease in phosphorylation of IRF3 and TBK1 in the presence of the DM-pp65 mutant protein. This decrease in IRF3 phosphorylation led to a decrease in IFN-β1 secretion. We report that DM-pp65 is resistant to cGAS activation in NuFF-1 cells, resulting in increased replication during STING activation compared to WT virus. Created in BioRender. Cox, J. (2025) https://BioRender.com/b57t258.