Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins

Abstract

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

Keywords: cytomegalovirus; herpesviruses; interferons; zinc finger antiviral protein.

FIG 1

HCMV replication in HFF cells containing CRISPR. (A) Uninfected cell lysates were prepared for Western blotting. Each CRISPR-containing cell line used is indicated above the figure. Proteins recognized by the antibodies used in the experiment are indicated to the right of the figure. The positions of molecular weight markers (kDa) are indicated to the left of the figure. Relative band intensity (band intensity relative to β-actin signal in the same lane) was analyzed using ImageJ, and fold knockdown of proteins compared to CRISPR-Luc is reported in the text. The data are representative of two independent experiments. (B and C) Cell lines shown in panel A were infected at a multiplicity of infection of 1. Virus in infected cell supernatant was collected at 96 h postinfection, and viral titer was determined by titration of virus supernatant on HFF cells. Virus and cell line used is shown below each figure. (i) Titer in plaque-forming units/mL (PFU/mL) of each experiment. (ii) Fold increase in virus titer in each cell line compared to virus titer from HFF cells containing CRISPR inhibiting Luciferase expression. In each figure, data are representative of three independent experiments (black data points) and presented as average (block) and standard deviation (error bars) of the data. Statistical relevance was examined using a Student's t test. ns, not significant; *, P < 0.05; **, P < 0.01.

FIG 2

HCMV replication in HFF cells treated with siRNAs inhibiting ZAP expression. (A) Schematic of HFF cells were treated with Ctrl, ZAP-L siRNA, or ZAP-S siRNAs infected with HCMV. At the time points indicated in the figure (hours postinfection [h.p.i.]), viral supernatant was collected and lysates were prepared for Western blotting from infected cells treated with siRNA. (B) Western blotting of cell lysates from uninfected and infected cells. Each condition used is shown above the figures. Proteins recognized by the antibodies used in each experiment are indicated to the right of each figure (ZAP-L [101 kDa] or ZAP-S [78 kDa]). The positions of molecular weight markers (kDa) are indicated to the left of each figure. Relative band intensity (band intensity relative to β-actin signal in the same lane) was analyzed using ImageJ, and fold knockdown of proteins compared to the same protein in Ctrl siRNA-treated cells is reported in the text. The data are representative of two independent experiments. (C) Viral titer of virus in cell supernatant harvested at 96 h postinfection from the infected cells analyzed in panel B determined by titration of virus supernatant on HFF cells. Data shown are the means and standard deviations of data from three independent experiments. Viral titer is expressed in plaque-forming units/mL (PFU/mL). The data are representative of three independent experiments (black data points) and presented as average (block) and standard deviation (error bars) of the data. Statistical relevance was examined using a Student's t test. ns, not significant; *, P < 0.05.

FIG 3

ZAP protein and RNA expression in HFF cells infected with AD169 or Merlin(R1111). (A and B) HFF cells were infected at a multiplicity of infection of 1 with either AD169 or Merlin(R1111). Cell lysates were prepared for Western blotting at the time points (hours postinfection [h.p.i.]) indicated above the figure. (C) Cell lysates were prepared for Western blotting from uninfected cells at the time of treatment (0 h.p.i.), from HFF cells treated with complete media or complete media containing α-IFN (–α-IFN and +α-IFN, respectively) for 24 h, and from HFF cells infected with either AD169 or Merlin(R1111) (24 h postinfection). (D) HFF cells were infected at a multiplicity of infection of 1 with either AD169 or Merlin(R1111) and then treated with either DMSO or Ruxolitinib (Ruxo.). (E) HFF cells were infected with HCMV or Sendai virus (Cantell) at a multiplicity of infection of 1 or a dilution of 1:50, respectively. Cell lysates were prepared for Western blotting at the time points (hours postinfection [h.p.i.]) indicated above the figure. (F) HFF cells were infected at a multiplicity of infection of 1 with either AD169 or Merlin(R1111). Cells were prepared for preparation of RNA at the time points (hours postinfection [h.p.i.]) indicated above the figure. RNA was prepared from cells, and in each sample the number of copies of ZAP mRNA (encoding both ZAP-S and ZAP-L) and cellular GAPDH mRNA was assayed using quantitative PCR. For each reaction, the 2^(-deltaCT) value was calculated. Relative abundance of ZAP mRNA to GAPDH mRNA was calculated, and values from infected cells were normalized to the values from uninfected cells. The data are representative of three independent experiments (black data points) and presented as average (block) and standard deviation (error bars) of the data. The red horizontal bar indicates a value of 1, the data from uninfected cells. (G and H) HFF cells were infected at a multiplicity of infection of 1 with Merlin(R1111). Infected cells were treated as shown in Figure G and described in the text. In Figure H, cell lysates from the experiments described in Figure G were prepared for Western blotting at the time points (hours postinfection [h.p.i.]) indicated above the figure. In all Western blotting figures, proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular weight markers (kDa) are indicated to the left of each figure. In those experiments, uninfected cells harvested at the time of infection are shown as 0 h.p.i.

FIG 4

ZAP expression in HFF cells infected with Merlin mutants. (A) Schematic of the genomes of Merlin GFP reporter viruses. The major deletion in the AD169 genome is shown at the top (UL, unique long region; US, unique short region; rectangles, inverted repeat regions flanking UL and US). The relevant features of the Merlin(R1278) genome are indicated below AD169 and include the gene UL32 protein fused to GFP and the deletions in genes UL16 and UL18 (solid black lines). The deletions in three Merlin mutants, including Merlin (R1293), are indicated. (B) The number of peptides from ZAP proteins found at 72 h postinfection in cells infected with the viruses indicated in the figure. Data taken from reference Nightingale et al. (41). (C) HFF cells were infected with Merlin(R1278) or Merlin(R1293) at a multiplicity of infection of 1. Cell lysates were prepared for Western blotting at 72 h postinfection. Uninfected cells harvested at the time of infection are shown as 0 h.p.i. Proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular weight markers (kDa) are indicated to the left of each figure. Relative band intensity (band intensity relative to β-actin signal in the same lane) was analyzed using ImageJ, and fold knockdown of ZAP-L at 72 h.p.i. is reported in the text. The data are representative of two independent experiments. (D) CRISPR cell lines were infected at a multiplicity of infection of 1 with viruses indicated in the figure. Virus in infected cell supernatant was collected at 96 h postinfection, and viral titer was determined by titration of virus supernatant on HFF cells. Virus and cell line used are shown below each figure (i) Titer in plaque-forming units/mL (PFU/mL) of each experiment. (ii) Data shown are the fold increase in virus titer in HFF cells containing CRISPR inhibiting ZAP expression compared to virus titer from HFF cells containing CRISPR inhibiting Luciferase expression. In each figure, data are representative of three independent experiments (black data points) and presented as average (block) and standard deviation (error bars) of the data. Statistical relevance was examined used a Student's t test. ns, not significant; *, P < 0.05.

FIG 5

HCMV protein expression in infected cells. (A) Schematic of HCMV gene expression with relevant proteins grouped into kinetic classes. (B to E) HFF cells containing CRISPR inhibiting expression of either Luciferase (Luc) or ZAP were infected with either AD169 or Merlin(R1111) at a multiplicity of infection of 1. Cell lysates were prepared for Western blotting at each time point indicated above the figure (hours postinfection [h.p.i.]). Uninfected cells harvested at the time of infection are shown as 0 h.p.i. Proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular weight markers (kDa) are indicated to the left of each figure. The data are representative of two independent experiments.

FIG 6

CpG and UpA dinucleotide content of AD169, Merlin, and TB40/E mRNAs. The plots show observed versus expected (obs/exp) dinucleotide ratios in relation to the numbers of CpG or UpA dinucleotides per kilobase of RNA, with each dot representing an annotated viral RNA. The conditions are indicated at the top of each panel. The red dotted line in each figure indicates an obs/exp of 1, and the identities of selected outlying RNAs are marked. IE1 is indicated in panels A, C, and E. In each figure, the identities of the RNAs with the greatest CpG or UpA content are indicated.

FIG 7

HCMV protein expression in CAV and CRV from HFF cells infected with AD169 or Merlin(R1111). (A) HFF containing CRISPR inhibiting expression of either Luciferase (Luc) or ZAP were infected with either AD169 or Merlin(R1111) at a multiplicity of infection of 1. At 96 h postinfection (h.p.i.), cell lysates were prepared for Western blotting and cell supernatant was concentrated by centrifugation, then prepared for Western blotting. Proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular weight markers (kDa) are indicated to the left of each figure. Relative band intensity (band intensity relative to β-actin signal in the same lane) was analyzed using ImageJ, and fold change of UL86 protein in supernatant is reported in the text. The data are representative of two independent experiments. (B and C) CRISPR cells were infected at a multiplicity of infection of 1. At 96 h postinfection, virus in supernatant (cell release virus [CRV]) was harvested and cell-associated virus (CAV) was collected in tissue culture media by sonification. CRV and CAV titer was determined by titration of virus on HFF cells. Virus and cell line used is shown below each figure. (B) Titer in plaque-forming units/mL (PFU/mL) of each experiment. (C) Data shown is the fold increase in virus titer in each cell line compared to virus titer from HFF cells containing CRISPR inhibiting Luciferase expression. In each figure, data are representative of three independent experiments (black data points) and presented as average (block) and standard deviation (error bars) of the data. Statistical relevance was examined used a Student's t test. *, P < 0.05; **, P < 0.01.