Protein S-Nitrosylation of Human Cytomegalovirus pp71 Inhibits Its Ability To Limit STING Antiviral Responses

Abstract

Human Cytomegalovirus (HCMV) is a ubiquitous pathogen that has coevolved with its host and, in doing so, is highly efficient in undermining antiviral responses that limit successful infections. As a result, HCMV infections are highly problematic in individuals with weakened or underdeveloped immune systems, including transplant recipients and newborns. Understanding how HCMV controls the microenvironment of an infected cell so as to favor productive replication is of critical importance. To this end, we took an unbiased proteomics approach to identify the highly reversible, stress-induced, posttranslational modification (PTM) protein S-nitrosylation on viral proteins to determine the biological impact on viral replication. We identified protein S-nitrosylation of 13 viral proteins during infection of highly permissive fibroblasts. One of these proteins, pp71, is critical for efficient viral replication, as it undermines host antiviral responses, including stimulator of interferon genes (STING) activation. By exploiting site-directed mutagenesis of the specific amino acids we identified in pp71 as protein S-nitrosylated, we found this pp71 PTM diminishes its ability to undermine antiviral responses induced by the STING pathway. Our results suggest a model in which protein S-nitrosylation may function as a host response to viral infection that limits viral spread.IMPORTANCE In order for a pathogen to establish a successful infection, it must undermine the host cell responses inhibitory to the pathogen. As such, herpesviruses encode multiple viral proteins that antagonize each host antiviral response, thereby allowing for efficient viral replication. Human Cytomegalovirus encodes several factors that limit host countermeasures to infection, including pp71. Herein, we identified a previously unreported posttranslational modification of pp71, protein S-nitrosylation. Using site-directed mutagenesis, we mutated the specific sites of this modification thereby blocking this pp71 posttranslational modification. In contexts where pp71 is not protein S-nitrosylated, host antiviral response was inhibited. The net result of this posttranslational modification is to render a viral protein with diminished abilities to block host responses to infection. This novel work supports a model in which protein S-nitrosylation may be an additional mechanism in which a cell inhibits a pathogen during the course of infection.

Keywords: HCMV; STING; UL82; cytomegalovirus; human herpesviruses; pp71; protein S-nitrosylation.

FIG 1

HCMV pp71 is protein S-nitrosylated during infection. (A) NuFF-1 cells were infected at a multiplicity of 3 IU/cell with TB40/E-mCherry-UL99eGFP virus; 96 hpi, total lysate was isolated and subjected to a biotin switch assay. Trypsinized proteins were analyzed by mass spectrometry to identify viral peptides that contain a cysteine conjugated to biotin, thereby reveling the site of protein S-nitrosylation. Peptides from 13 viral proteins are shown with the identified sequences. Lowercase “c” denotes the identified biotinylated cysteine. Xcorr*, cross correlation, a measure of the goodness of fit of experimental peptide fragments to theoretical spectra created from the sequence b and y ions; PSM**, frequency of peptide detection; ***, due to partial trypsin digestion, two of the three UL44 peptides and both of the UL25 peptides represent the same identified S-nitrosylated cysteine. Results are from three independent biological replicates. (B) A representative MS/MS spectrum showing the mass shift for the pp71 peptide containing the LxCxD domain. (C) NuFF-1 cells were infected at a multiplicity of 1.0 IU/cell with TB40/E-mCherry-UL99eGFP, and cells were harvested at 96 hpi. After the biotin switch assay, lysates were affinity purified (AP) with avidin beads (top) or immunoprecipitated by pp71-specific antibody (bottom). Purified lysates were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. Proteins were detected using a pp71-specific antibody (top) or antibody against biotin (bottom). n = 3; representative blots are shown. (D) Specificity of the biotin switch reaction was assessed by performing the reaction in the absence of added biotin. NuFF-1 cells were infected at a multiplicity of 3 IU/cell with TB40/E-mCherry-UL99eGFP virus; 96 hpi, total lysate was isolated and subjected to a biotin switch assay in the presence or absence of biotin. Lysates were affinity purified with avidin beads, and then purified lysates were separated by 8% SDS-PAGE and blotted to nitrocellulose membrane (top). A fraction of the lysates (4%) was run in parallel to confirm protein expression (bottom). Proteins were detected by using specific antibodies. A representative blot from three biological repeats is shown.

FIG 2

S-Nitrosylation-defective viruses have similar growth kinetics to that of WT virus. (A) NuFF-1 cells were infected at a multiplicity of 1 IU/cell with TB40/E-mCherry-UL99eGFP virus (WT) or a recombinant virus containing three-point mutations in pp71 ORF at amino acids C34S, C94S, and C218S (pp71-TM). Medium containing cell-free virus was collected over the indicated time course, and virus yields were determined by TCID₅₀ assay on NuFF-1 cells. Ino, inoculum; LOD, limit of detection. Samples were analyzed in triplicates. Error bars represent the standard deviations of the replicates. (B) Total de novo expression of wild-type pp71 or pp71-TM was monitored by immunoblotting. NuFF-1 cells were infected at a multiplicity of 1 IU/cell with WT or pp71-TM virus. Cells were harvested at the indicated time points after infection, and the proteins were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. Cellular tubulin levels served as a control for equal protein loading. n = 3, representative blots are shown.

FIG 3

Protein S-nitrosylation-defective pp71 is expressed with wild-type kinetics and incorporated into tegument. (A) NuFF-1 cells were infected at a multiplicity of 1 IU/cell with WT virus. Cells were harvested at the indicated time points after infection, and 4% (20 μg) protein lysates were separated by SDS-PAGE and blotted to nitrocellulose membrane (top, total lysate); the remaining 96% (0.5 mg) of protein lysate was used in a biotin switch assay, affinity purified on streptavidin beads (AP), and then separated by SDS-PAGE and transferred to a nitrocellulose membrane (bottom, AP/WB). Cellular tubulin levels served as a control for equal protein loading. n = 3; representative blots are shown. (B) NuFF-1 cells were infected at a multiplicity of 1 IU/cell with WT virus, a recombinant virus containing single point mutation in pp71 ORF at amino acid C218S (pp71-C218S), or a revertant of that mutant (pp71-C218Srev). Medium containing cell-free virus was collected over the indicated time course, and virus yields were determined by TCID₅₀ assay on NuFF-1 cells. Ino, inoculum. Samples were analyzed in triplicates. Error bars represent the standard deviations of the replicates. A representative analysis of three independent biological replicates is shown. (C) MRC5 cells were infected at a multiplicity of 30 IU/cell with the TB40/E-mCherry-UL99eGFP virus, pp71-C218S, or pp71-TM. Cells were harvested 6 hpi to detect pp71 and pp65 delivery into newly infected cells. Total cell lysates were separated by 8% SDS-PAGE and analyzed by Western blotting. Cellular tubulin levels served as a control for equal protein loading. n = 3; representative blots are shown.

FIG 4

pp71 interacts with STING in vitro and colocalizes with STING during infection. (A) HEK293 cells were cotransfected with pCDNA-STING-HA (STING-HA) and pCMSeGFP-FLAG-pp71-WT (FLAG-pp71-WT) or pCMSeGFP-FLAG-pp71-C218S (FLAG-pp71-C218S). After 72 h, total cell lysates were collected. Equal amounts of total cell lysates were incubated with α-HA (A) or α-FLAG affinity beads (B). The bound complexes were washed, eluted, separated by 8% SDS-PAGE, and immunoblotted (IB) with an α-FLAG (A) or an α-HA antibody (B). n = 3; representative blots are shown.

FIG 5

pp71 does not alter the protein expression levels of STING at early or late times during lytic infection. MRC5 cells were infected at a multiplicity of 1 IU/cell with WT or pp71-C218S virus. Infected cells were harvested over 48 h prior to de novo pp71 expression (A) or after 5 days, when de novo pp71 is expressed (B). Total protein (20 μg) was separated by SDS-PAGE and transferred to a nitrocellulose membrane. Cellular tubulin is shown as a loading control. n = 3; representative blots are shown.

FIG 6

Activation of the STING pathway inhibits DNA replication and viral production of pp71-WT and pp71-C218Srev but not pp71-C218S virus. (A) MRC5 cells were treated with indicated concentrations of 2′,3′-cGAMP; 48 h later cell viability was assessed by quantifying the metabolic activity of the treated cells using an NADH-dependent cellular oxidoreductase enzyme assay (MTT assay). MTT conversion results were normalized to the vehicle-treated conditions, and graphed results are shown as percent cell viability. Results are shown as the means from three independent biological replicates. (B) MRC5 cells were treated with indicated concentrations of 2′,3′-cGAMP, and 24 h later, total RNA was isolated. IFN-β1 transcripts were profiled by RT-qPCR using transcript-specific primers and plotted as threshold cycle (ΔΔC_T) relative to the control transcript, GAPDH. Results are shown as mean fold change in the mRNA levels. (C) MRC5 cells were infected with either WT, pp71-C218S, or pp71-C218Srev virus (MOI = 1) in the presence of 10 μM 2′,3′-cGAMP. DNA was isolated over the indicated days, and genome copy number was measured by qPCR using eGFP-specific primers to detect viral genomes and normalized to human mdm2. (D) Parallel cultures were treated as for panel C, and medium was collected over the indicated days. Cell-free virus accumulation was quantified by TCID₅₀ analysis. Ino, inoculum. (n = 3). *, P < 0.01 as determined by Student's t test; NS, not significant.

FIG 7

STING activation impacts viral RNA accumulation in WT virus- and pp71-C218Srev-infected cells but not pp71-C218S-infected cultures. MRC5 cells infected with either WT, pp71-C218S, or pp71-C218Srev (MOI = 1) in the absence or presence of 10 μM 2′,3′-cGAMP (added at time of infection). Total RNA was isolated from the cultures over the indicated times. Transcripts IE (UL122), E (UL54), and L (UL32) were profiled by RT-qPCR using transcript-specific primers and normalized to cellular GAPDH. Results are shown as mean fold change in the mRNA levels for each of three different kinetic classes of viral transcripts within cells infected by the listed viruses in the presence of vehicle (A) or 10 μM 2′,3′-cGAMP (B). AU, arbitrary units. (n = 3) *, P < 0.01 as determined by Student's t test; NS, not significant.

FIG 8

S-nitrosylation of the LxCxD domain of pp71 impacts 2′,3′-cGAMP induced IFN-β1 and IL-1β transcription. MRC-5 cells were mock infected or infected with WT, pp71-C218S, or pp71-C218Srev virus (MOI = 1) in the presence of 10 μM 2′,3′-cGAMP. Total RNA was isolated at 8 or 24 hpi. IFN-β1 and IL-1β transcripts were profiled by RT-qPCR using transcript-specific primers and plotted as ΔΔC_T relative to the control transcript, GAPDH. Results are shown as mean fold change in the mRNA levels of IFN-β1 and IL-1β in the cells. (n = 3) *, P < 0.01 as determined by Student's t test; NS, not significant.

FIG 9

S-Nitrosylation of pp71 is independent of viral infection and impacts STING-induced transcription in the absence of additional HCMV factors. (A) Recombinant retroviruses, pLXSN-pp71-WT or pLXSN-pp71-C218S, were used to transduce MRC5 cells that were then selected in G418 for two cell doublings yielding the parental MRC5 cells (M5), M5-pp71^WT, and M5-pp71^C218S. Cell lysates were isolated, and total protein (20 μg) was separated by SDS-PAGE and then transferred to a nitrocellulose membrane. Immunoblotting was performed with α-pp71 antibody and α-tubulin antibody, which served as a loading control; n = 3; representative blots are shown. (B) Lysates from M5 and M5-pp71^WT were harvested and used in a biotin switch assay. Lysates were either affinity purified (AP) with avidin beads or immunoprecipitated (IP) with pp71 antibody. Proteins were separated by SDS-PAGE and then transferred to nitrocellulose membrane. Protein S-nitrosylated pp71 was detected by α-biotin or α-pp71 antibody. n = 3; representative blots are shown. (C) 2′,3′-cGAMP (10 μM) was added to M5-pp71^WT and M5-pp71^C218S, and 24 h later, total RNA was isolated. IFN-β1 and IL-1β transcripts were profiled by RT-qPCR using transcript-specific primers and plotted as ΔΔC_T relative to the control transcript, GAPDH. Results are shown as mean fold change in the mRNA levels of IFN-β1 and IL-1β in M5-pp71^WT and M5-pp71^C218S cells (n = 3). *, P < 0.01 as determined by Student's t test.