Abstract
Zinc finger antiviral protein (ZAP) is an interferon-inducible host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1 and Ebola virus. ZAP functions as a dimer formed through intermolecular interactions of its N-terminal tails. ZAP binds directly to specific viral mRNAs and inhibits their expression by repressing translation and/or promoting degradation of the target mRNA. ZAP is not a universal antiviral factor, since some viruses grow normally in ZAP-expressing cells. It is not fully understood what determines whether a virus is susceptible to ZAP. We explored the interaction between ZAP and murine gammaherpesvirus 68 (MHV-68), whose life cycle has latent and lytic phases. We previously reported that ZAP inhibits the expression of M2, which is expressed mainly in the latent phase, and regulates MHV-68 latency in cultured cells. Here, we report that ZAP inhibits the expression of ORF64, a tegument protein that is expressed in the lytic phase and is essential for lytic replication. MHV-68 infection induced ZAP expression. However, ZAP did not inhibit lytic replication of MHV-68. We provide evidence showing that the antiviral activity of ZAP is antagonized by MHV-68 RTA, a critical viral transactivator expressed in the lytic phase. We further show that RTA inhibits the antiviral activity of ZAP by disrupting the N-terminal intermolecular interaction of ZAP. Our results provide an example of how a virus can escape ZAP-mediated immunity.
INTRODUCTION
Zinc finger antiviral protein (ZAP) (also named Zc3hav1) is a type I interferon-inducible host factor that specifically inhibits the replication of certain viruses (1–3). Initially, ZAP was recovered as a host factor that confers resistance to the replication of murine leukemia virus (MLV) (4). In addition to MLV, ZAP inhibits the replication of HIV-1 (5), filoviruses (6), and certain members of the alphaviruses, such as Sindbis virus (SINV) (7). ZAP does not induce a general antiviral state, since some viruses, including herpes simplex virus type I and yellow fever virus, grow normally in ZAP-expressing cells (7).
There are four CCCH-type zinc finger motifs in the N-terminal domain of ZAP (4). When the N-terminal 254 amino acids of ZAP are fused with a zeocin resistance gene, the fusion protein (NZAP-Zeo) displays antiviral activity similar to that of the full-length ZAP, highlighting the importance of the domain (4). Structural analyses of the N-terminal domain of ZAP indicate that ZAP functions as a dimer (8). Disruption of the intermolecular interaction mediated by the N-terminal tail abolishes the antiviral activity of ZAP (8).
ZAP binds directly to specific viral mRNAs through the N-terminal domain and recruits cellular mRNA degradation machinery to promote degradation of the target viral mRNA (5, 9–11). ZAP also represses the translation of its target mRNA (12). All the viruses that are sensitive to ZAP contain at least one ZAP-responsive element (ZRE) in their viral mRNAs. The ZRE of MLV was mapped to the 3′ untranslated region (UTR), the ZREs of SINV to multiple fragments (9), the ZRE of Ebola virus to the L domain (6), and the ZREs of HIV-1 to the 5′ UTRs of multiply spliced mRNAs (5). No obvious common motifs or conserved sequences have been identified in these ZREs, but they are all more than 400 nucleotides (nt) long.
Because of the lack of conserved sequences in ZREs, whether a virus is susceptible to ZAP can only be tested experimentally. If a virus is sensitive to ZAP, it is expected to contain a ZRE(s). However, if a virus is not sensitive to ZAP, the lack of a ZRE may not be the only explanation; the antiviral activity of ZAP may be antagonized by viral proteins.
MHV-68 is a gammaherpesvirus, which has a 120-kb-long genome that encodes approximately 80 open reading frames (ORFs) (13). The life cycle of MHV-68 is composed of a latent phase and a lytic phase (14). Latency provides unique advantages for herpesviruses to escape host immune surveillance and to establish life-long persistent infections. However, to maintain viral reservoirs and to be transmitted to other hosts, herpesviruses must be reactivated from latency and enter lytic replication to produce virus progeny. The balance between latency and lytic replication is a critical factor that determines the outcome of infection and pathogenesis.
Replication and transcription activator (RTA), encoded primarily by ORF50, plays a pivotal role in lytic replication of MHV-68 (15). Expression of RTA is necessary and sufficient to trigger reactivation into lytic replication in latently infected cell lines (16–19). RTA activates transcription of multiple genes expressed in the lytic phase (16, 17, 20, 21), some of which are essential for lytic replication (22).
ORF64 is a large tegument protein essential for lytic replication (22, 23). In the N-terminal domain of the protein, there is a ubiquitin-specific cysteine protease (USP) domain (24), which is conserved throughout the herpesvirus family (25). Although the USP activity in MHV-68 does not seem to be involved in the establishment of latency or reactivation, loss of the USP function by a site mutation compromises in vitro virus replication and persistent infection in vivo (26). In addition, ORF64 has been reported to inhibit type I interferon production, suggesting that it may play a role in allowing the virus to evade host immunity (27).
To explore the interaction between ZAP and MHV-68, we screened viral sequences for their sensitivity to ZAP. The initial screening identified M2 as a ZAP-responsive element (28). ZAP inhibits M2 expression by promoting M2 mRNA degradation (28). Furthermore, downregulation of ZAP in a cell line harboring latent MHV-68 led to reactivation of the virus into lytic replication (28), indicating that ZAP regulates the maintenance of MHV-68 latency.
In the present study, we continued the screening and identified MHV-68 ORF64 coding sequence as a target of ZAP. ZAP inhibits the expression of ORF64. However, ZAP had little effect on viral lytic replication. We provide evidence indicating that the antiviral activity of ZAP is antagonized by MHV-68 RTA.
MATERIALS AND METHODS
Plasmid construction.
pGL3-Luc-linker and pMLV-luc have been described previously (5, 9). To map the sequences of MHV-68 that are sensitive to ZAP, fragments of viral genomic DNA covering known open reading frames (ORFs) were PCR amplified from MHV-68 bacterial artificial chromosome (BAC) DNA and cloned into pGL3-Luc-linker downstream of the firefly luciferase coding sequence.
pcDNA3-NZAP254-Flag and pcDNA4-NZAP254-myc, which express the Flag-tagged and myc-tagged N-terminal 254 amino acids of ZAP, respectively, have been described previously (8). To express Flag-tagged ORF64, the coding sequence was PCR amplified and cloned into the expression vector pCMV-Flag (20). To express hemagglutinin (HA)-tagged RTA, the coding sequence was PCR amplified and cloned into the expression vector pCMV-HA.
Cells and viruses.
All cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen). BHK-21 cells have been described previously (9, 29). 293TRex-ZAP cells, which express rat ZAP in a tetracycline-inducible manner, have been described previously (5, 9).
MHV-68 infection and titration on permissive BHK-21 cells have been described previously (15). Briefly, BHK-21 cells were seeded in 12-well plates with 2E5 cells per well 24 h prior to infection. Serial 10-fold dilutions of the virus were used to infect BHK-21 cells. Infections were performed at 37°C for 1 h with rocking every 15 min. Following infection, BHK cells were covered with 5 ml 2× DMEM containing 2% methylcellulose. Plaques were counted 5 or 6 days postinfection. The bacterial artificial chromosome system was used to generate a recombinant MHV-68 that expresses RTA with a Flag tag at the C terminus, using a two-step allelic-exchange protocol in Escherichia coli as described previously (30). Briefly, a fragment spanning the target region and the flanking sequences was cloned into plasmid pGS284, harbored in E. coli GS111, which was used as the donor strain for allelic exchange with the recipient strain GS500 harboring the wild-type MHV-68 BAC. Cointegrates were screened for the desired insertion through two rounds of selection. The Flag tag sequence insertion and its flanking regions were confirmed by sequencing, and the recombinant BACs were further analyzed by enzyme digestion to ensure that there were no undesired mutations, deletions, or insertions in the MHV-68 genome. After identification, the RTA-Flag MHV-68 BAC was transfected into BHK-21 cells to produce virus.
To generate a retroviral vector expressing ORF64_G, the coding sequence was cloned into pBabe-puro (5). VSV-G-pseudotyped transducing retroviruses were generated by cotransfection of HEK293T cells with pVSV-G, which expresses VSV-G; pHIT60, which expresses MLV viral proteins; and the retroviral vectors, as described previously (5).
For all transfection experiments, pRL-TK (Promega), a Renilla luciferase reporter that is not sensitive to ZAP, was included to normalize transfection efficiencies. Luciferase activity was measured at 48 h posttransfection or postinfection with a luciferase assay system (Promega).
Detection of ZAP-associated MHV-68 mRNAs.
293TRex-ZAP cells were infected with MHV-68 for 1 h at a multiplicity of infection (MOI) of 0.5 PFU per cell. At 48 h postinfection, the cells were lysed in RNase-free Passive Lysis Buffer (Promega). The cell lysates were incubated with protein G-Sepharose precoated with anti-myc antibody for 3 h. The beads were washed three times. To recover the RNA associated with ZAP, the beads were incubated with lysis buffer (30 mM HEPES [pH 7.5], 100 mM NaCl, 0.5% NP-40) containing 100 μg/ml of proteinase K and 200 U/ml of RNasin at 37°C for 15 min. Total RNA was extracted using an RNeasy Kit following the manufacturer's instructions (Qiagen). The RNA was reverse transcribed using oligo(dT) as a primer in a 20-μl reaction mixture. Five microliters of the reverse transcription product was PCR amplified using specific primers. The PCR conditions used were 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s (40 cycles for ORF64 and ORF37 and 26 cycles for GAPDH [glyceraldehyde-3-phosphate dehydrogenase]). The sequences of the primers are as follows: ORF64, forward, 5′-TTCCTATATTTTGTAC, and reverse, 5′-TGGCTCGGCCTCCCTGTC; ORF37, forward, 5′-GCCATAATGGGCGTTT, and reverse, 5′-AGTGTCCAGGGAAACTC; GAPDH, forward, 5′-TCACTGCCACCCAGAAGACTGTGG, and reverse, 5′-GGTCCACCACCCTGTTGCTGTAGCC.
Real-time PCR.
mRNA levels were measured by real-time PCR in a Rotor-Gene 6000 (Corbett Life Science) with the following program: (i) 50°C for 2 min, (ii) 95°C for 10 min, (iii) 95°C for 15 s and 60°C for 1 min for 40 cycles, and (iv) 30°C for 1 min. The primers for real-time PCR are as follows: ZAP, forward, 5′-CACATGGAGACGATCGAGAAAG-3′, and reverse, 5′-CTGGCCCTCTCTTCATCTGCTG-3′; ORF64G, forward, 5′-TGAGCACAGGCCATACAGTTC-3, and reverse, 5′-AGCAACCATTTCATGCCCAGT-3′; GAPDH, forward, 5′-TCACTGCCACCCAGAAGACTGTGG-3′, and reverse, 5′-GGTCCACCACCCTGTTGCTGTAGCC-3′.
Statistical analyses.
The data presented are mean values and standard deviations (SD) from three independent experiments. The P values were calculated using the paired Student's t test, and P values of ≤0.05 were considered significant.
RESULTS
Identification of MHV-68 orf64 as a ZAP-responsive sequence.
Genomic-DNA fragments covering MHV-68 ORFs were individually cloned into the reporter vector pGL3-Luc-linker (9), which is barely sensitive to ZAP, downstream of the firefly luciferase coding sequence. These reporters were transfected into 293Trex-ZAP cells, which express rat ZAP in a tetracycline-inducible manner (9). A Renilla luciferase reporter, pRL-TK, which is not inhibited by ZAP, was included to serve as a control for transfection efficiency and sample handling. The sensitivity of a reporter to ZAP was indicated by the fold inhibition, which was calculated as the normalized luciferase activity in mock-treated cells divided by that in tetracycline-treated cells. A fragment derived from SINV (Md) that has been shown to be sensitive to ZAP was used as a positive control (9).
Out of 31 MHV-68 fragments tested (Table 1), a fragment of the ORF64 coding sequence conferred significant sensitivity to the reporter (Fig. 1A and data not shown). We have previously reported that ZAP binds directly to its target RNAs (9). To substantiate that the ORF64 coding sequence is the target of ZAP, we analyzed whether ORF64 mRNA is associated with ZAP in MHV-68-infected ZAP-expressing cells. 293Trex-ZAP cells were infected with MHV-68, followed by treatment of the cells with tetracycline to induce ZAP expression. ZAP in the cell lysate was immunoprecipitated, and the associated RNAs were detected by reverse transcription (RT)-PCR using specific primers. As expected, immunoprecipitation of ZAP coprecipitated ORF64 mRNA (Fig. 1B). The identity of ORF64 mature mRNA was confirmed by sequencing the PCR products and comparing them with the published sequence of MHV-68 (13, 31) (data not shown). In contrast, immunoprecipitation of ZAP failed to coprecipitate MHV-68 ORF37 mRNA or cellular GAPDH mRNA (Fig. 1B).
Table 1.
Sequences of primers for PCR cloning MHV-68 DNA fragments
| Gene name (position in viral genome [nt]) | Forward primer | Reverse primer |
|---|---|---|
| ORF 32 (48295–49554) | 5′-CCGGAATTCATGGATGTATTTAGAATAA-3′ | 5′-CGCGGATCCCAGAATGTATGTTCCTTTAA-3′ |
| ORF 33A(49589–49938) | 5′-CCGGAATTCATGGCAACCAAAGAAGATA-3′ | 5′-CGCGGATCCATAGGTACTGGAATGGCTGA-3′ |
| ORF 33B (49942–50892) | 5′-CCGGAATTCTCCTACAATTCTAGATCC-3′ | 5′-CGCGGATCCTTTATTAGACATGTTATA-3′ |
| ORF 34A(51466–52067) | 5′-CGGAATTCATGGCCACATTAAGT-3′ | 5′-CGCGGATCCTTCACTGAAGTGTCCCC-3′ |
| ORF 34B35 (52069–52882) | 5′-CGGAATTCTCTTTTGCCCATCACC-3′ | 5′-CGCGGATCCTCACCCAATACGCGCGGTA-3′ |
| ORF 36 (52847–54161) | 5′-CCGGAATTCATGGATTACCGACAGTTAC-3′ | 5′-CGCGGATCCTCAAAAAAATCCAGAATA-3′ |
| ORF 39 (55800–56951) | 5′-CCGGAATTCATGCCTGCCCTTAAAGTG-3′ | 5′-CGCGGATCCTTATAGTTCATCTTCTGA-3′ |
| ORF 40 (57047–58879) | 5′-CCGGAATTCATGTGCAGACTTCAATGG-3′ | 5′-CGCGGATCCTTATGATAAATTTAATAAAG-3′ |
| ORF 43A(59632–59881) | 5′-CCGGAATTCAGGAAAGATGATATGTGTTA-3′ | 5′-CGCGGATCCTCATGGTGGCAGCACCTC-3′ |
| ORF 44B (61304–62183) | 5′-CCGGAATTCATGGCACTCTCTGACAGA-3′ | 5′-CGCGGATCCCAGTATCCAGACAGCGC-3′ |
| ORF 54 (71445–71703) | 5′-CCGGAATTCTTATGAGGTCATGGTA-3′ | 5′-CGCGGATCCATGCAGGTCCTGGTCG-3′ |
| ORF 55 (72575–73314) | 5′-CGGAATTCATGAATTGCTGTGGTATC-3′ | 5′-CGCGGATCCTTTATTAGGGTTCCGCAC-3′ |
| ORF 56 (73290–75797) | 5′-CGGAATTCATGGCCAGATACC-3′ | 5′-CGCGGATCCCTACACTCGAGCAT-3′ |
| ORF 57 (75844–77163) | 5′-CGGAATTCATGGCACAGCAGAT-3′ | 5′-CTAGTCTAGATTATTCACACACAAA-3′ |
| ORF 62 (82869–83582) | 5′-CGGAATTCATGGAAGATATGG-3′ | 5′-CGCGGATCCTCAGATAAACTGCC-3′ |
| ORF 63A(83857–85000) | 5′-CGGAATTCATGACACTCTAGTA-3′ | 5′-CGCGGATCCATTCAGACAGAATTTT-3′ |
| ORF 63B (85002–86568) | 5′-CGGAATTCCTACAACACACTG-3′ | 5′-CGCGGATCCTTATGATAGCAGTTT-3′ |
| ORF 64A(86568–88848) | 5′-CGGAATTCATGGCCCTGCCTG-3′ | 5′-CGCGGATCCTGCCTGTGCCAAGACA-3′ |
| ORF 64B (88567–90834) | 5′-CGGAATTC ACAACAATTGTCTTA-3′ | 5′-CGCGGATCC GATCCATTTCAGTC-3′ |
| ORF 64C (90568–92168) | 5′-CTAGTCTAGATGCTGGATGAAATTTT-3′ | 5′-CTAGTCTAGAATGGCAAATATCACAAAT-3′ |
| ORF 64D(91868–92668) | 5′-CGGAATTCCGCTGGTCTGGGGGATATTAT-3′ | 5′-CGGAATTCCTGATCCGCTGAAGATCT-3′ |
| ORF 64E (92108–93941) | 5′-CGGAATTCATTGGATGATGGGGGG-3′ | 5′-CGCGGATCCTTAAATGTAAAGCTG-3′ |
| ORF 64AB (86568–90823) | 5′-CGGAATTCATGGCCCTGCCTGCTTCTT-3′ | 5′-CGCGGATCCGTCACGGTTTTATCGTGGA-3′ |
| ORF65 (93960–94520) | 5′-CGGAATTCATGAATAAAGACAG-3′ | 5′-CGCGGATCCCTACTTTTTCTTTC-3′ |
| ORF 66 (94513–95742) | 5′-CGGAATTCATGGTGATGGGGGCC-3′ | 5′-CGCGGATCCTTATTCATCACCCTTA-3′ |
| ORF 67 (95736–96416) | 5′-CGGAATTCATGGCTAACCAGAAG-3′ | 5′-CGCGGATCCTCACCATACTAGTTTA-3′ |
| ORF 68 (96743–98056) | 5′-CGGAATTCATGTACGTACCATGGTCT-3′ | 5′-CTAGTCTAGATTAGGTGTAAACAGA-3′ |
| ORF 69 (98062–98940) | 5′-CGGAATTCATGCGCTCAACAG-3′ | 5′-CGCGGATCCTTATTGCTGAGAAAG-3′ |
| ORF 75C1 (106068–107203) | 5′-CGGAATTCTGAAAGCCATTTTGA-3′ | 5′-CGCGGATCCCTAATCTCTAGATG-3′ |
| ORF 75C2 (107204–108350) | 5′-CGGAATTCACAGCACAAGAAGCA-3′ | 5′-CGCGGATCCCCATTTCCATTGATTTT-3′ |
| ORF 75C3 (108361–110000) | 5′-CGGAATTCATGGCTAGACACTT-3′ | 5′-CGCGGATCCTTGACTGGAAATCCC-3′ |
Fig 1.
Identification of MHV-68 orf64 as ZAP-responsive sequences. (A) The MHV-68 genomic-DNA fragments indicated were individually cloned into pGL3-Luc-linker downstream of the firefly luciferase coding sequence. The plasmids were transfected into 293TRex-ZAP cells, together with pRL-TK, a Renilla luciferase-expressing reporter that is not affected by ZAP. At 6 h posttransfection, cells were mock treated or treated with tetracycline to induce ZAP expression. At 54 h posttransfection, the cells were lysed and luciferase activities were measured. Firefly luciferase activity was normalized with Renilla luciferase activity. The fold inhibition was calculated as the normalized luciferase activity in mock-treated cells divided by that in tetracycline-treated cells. The data presented are means and SD from three independent experiments. EV, empty vector; SINV-Md, pGL3-Luc-linker reporter containing fragment Md from SINV to serve as a positive control. (B) 293TRex-ZAP cells were infected with MHV68 for 1 h at an MOI of 0.5 PFU per cell, followed by tetracycline (Tet) treatment to induce ZAP expression. At 48 h postinfection, the cells were lysed, and the lysates were incubated with protein G beads with (+) or without (−) anti-myc antibody to precipitate ZAP. The RNA coprecipitated with ZAP was detected by RT-PCR using specific primers. − RT, no reverse transcriptase was added to the reaction mixture. IP, immunoprecipitation.
Mapping ZAP-responsive elements in the ORF64 coding sequence.
The coding sequence of ORF64 is composed of 7,371 nucleotides. To facilitate our analyses, the coding sequence of ORF64 was divided into 5 overlapping fragments. Each fragment was cloned into the pGL3-luc-linker reporter vector to analyze its ability to confer reporter sensitivity to ZAP. Fragments A and B displayed significant activity, while the other three fragments failed to do so (Fig. 2A and B). These results indicate that the ZAP-responsive sequences of orf64 lie in the fragment covering the first 4,267 nucleotides. This 4,267-nt fragment was further divided into three fragments (F, G, and H), which were transiently expressed in HEK293 cells with or without ZAP. The expression of ORF64_G and ORF64_H was dramatically inhibited by ZAP (Fig. 2C). In contrast, the expression of ORF64_F was only marginally affected (Fig. 2C). These results demonstrate that there are at least two ZAP-responsive elements in orf64, with one in the G fragment and the other in the H fragment.
Fig 2.
Mapping orf64 sequences that are sensitive to ZAP. (A) Schematic representation of orf64 sequences. (B) The ORF64 fragments indicated were cloned into pGL3-Luc-linker. The sensitivity of the reporters was assayed as described in the legend to Fig. 1A. The data presented are means and SD from three independent experiments. (C) The ORF64 fragments indicated were cloned into an expression vector, followed by cotransfection with ZAP into HEK293 cells. A plasmid expressing myc-tagged green fluorescent protein (GFP) was included as a control for transfection efficiency and sample handling. At 54 h posttransfection, the cells were lysed and the expression of Flag-tagged ORF64 fragments was detected by Western blotting. (D) The endogenous ZAP mRNA levels in HOS cells stably expressing a control shRNA (Ctrl) or an shRNA against ZAP (ZAPi) were measured by real-time PCR. The relative ZAP mRNA level in HOS-Ctrl cells was set as 100. The data presented are means and SD of three independent experiments. (E and F) Cells were infected with a retroviral vector expressing myc-tagged ORF64_G at an MOI of 1. (E) At 48 h postinfection, ORF64_G mRNA levels were measured by real-time PCR. The relative ORF64_G mRNA level in HOS-Ctrl cells was set as 100. The data presented are means and SD of three independent experiments. (F) The expression of ORF64_G protein was detected by Western blotting.
To test whether endogenous ZAP is able to inhibit ORF64 expression, a retroviral vector expressing ORF64_G was constructed to transduce HOS cells in which endogenous ZAP expression is downregulated by RNA interference (RNAi) (5). The downregulation of the ZAP mRNA level was confirmed by real-time PCR analysis (Fig. 2D). Compared with the control cells, downregulation of ZAP increased the mRNA level of ORF64_G (Fig. 2E) and the expression of the ORF64_G protein (Fig. 2F). Similar results were obtained when the plasmid expressing ORF64_G was cotransfected into HEK293 cells with a plasmid expressing the short hairpin RNA (shRNA) against ZAP (data not shown). These results indicate that downregulation of endogenous ZAP increases ORF64 expression.
MHV-68 infection induces ZAP expression.
Since ORF64 is expressed in the lytic phase and is essential for lytic replication (22), we wondered whether ZAP regulates MHV-68 replication. We first analyzed whether MHV-68 infection affects ZAP expression. HEK293T cells were infected with MHV-68, and ZAP mRNA levels were examined by real-time PCR. The data show that the ZAP mRNA level was increased about 5-fold by MHV-68 infection (Fig. 3).
Fig 3.
MHV-68 infection induces ZAP expression. HEK293T cells were infected with MHV-68 for 1 h at an MOI of 1 PFU per cell. At 24 h postinfection, ZAP mRNA levels were measured by real-time PCR. The relative ZAP mRNA level in mock-infected cells was set as 100. The data presented are means and SD of three independent experiments.
ZAP expression has little effect on MHV-68 lytic replication.
We next analyzed whether ZAP inhibits lytic replication of MHV-68. 293Trex-ZAP cells are fully permissive for MHV-68, where MHV-68 proceeds directly to lytic replication. Cells were infected with MHV-68 and cultured in the absence or presence of tetracycline, which induces ZAP expression. Viral replication was monitored by the numbers of progeny virus in the supernatants, which were measured at various time points by a plaque assay in BHK cells. Surprisingly, the replication of MHV-68 was little affected by tetracycline-induced ZAP expression (Fig. 4A). We further analyzed whether downregulation of endogenous ZAP expression affects MHV-68 replication. ZAP expression was downregulated in HOS cells by stably expressing an shRNA directed against ZAP (5). Consistent with the above-mentioned results, MHV-68 infection increased endogenous ZAP expression (Fig. 4B). The expression of the shRNA against ZAP significantly reduced ZAP mRNA levels in both control cells and MHV-68-infected cells. In line with the above-mentioned results, downregulation of ZAP had little effect on MHV-68 replication (Fig. 4C). These results indicate that ZAP does not inhibit MHV-68 lytic replication.
Fig 4.
ZAP has little effect on MHV-68 lytic replication. (A) 293TRex-ZAP cells were infected with MHV-68 in quadruplicate for 1 h at an MOI of 0.2 PFU per cell. The cells in two wells were mock treated, and those in the other two were treated with tetracycline to induce ZAP expression. The supernatants were collected at the time points indicated. The virus titer in each well was measured in duplicate on BHK-21 cells (top). Tetracycline-induced ZAP expression was confirmed by Western blotting (bottom). The data presented are means ± SD of three independent experiments. d, day(s). (B) HOS cells stably expressing a control shRNA (HOS-Ctrl) or an shRNA against ZAP (HOS-ZAPi) were infected with MHV-68 for 1 h at an MOI of 0.5 PFU per cell. At 24 h postinfection, ZAP mRNA levels were measured by real-time PCR. The relative ZAP mRNA level in mock-infected HOS-ctrl cells was set as 100. The data presented are means and SD of three independent experiments. (C) Supernatants were collected at the time points indicated. The virus titer in each well was measured in duplicate on BHK-21 cells. The data presented are means ± SD of three independent experiments.
MHV-68 RTA suppresses the antiviral activity of ZAP.
We speculated that a possible reason for ZAP's failure to inhibit MHV-68 replication was that the antiviral activity of ZAP is antagonized by a viral protein expressed in the lytic phase. To test this idea, MHV-68 proteins were screened for the ability to inhibit the antiviral activity of ZAP against the pMLV-luc reporter, an MLV-derived reporter that is sensitive to ZAP (4). The plasmids expressing MHV-68 proteins were each cotransfected with pMLV-luc into 293Trex-ZAP cells, and the antiviral activity of ZAP was assayed as indicated by fold inhibition. Out of 19 MHV-68 proteins tested (Table 2), RTA significantly reduced the antiviral activity of ZAP (Fig. 5A and data not shown). The antagonizing activity of RTA against ZAP was dose dependent (Fig. 5B). Notably, RTA did not affect tetracycline-induced ZAP expression in 293Trex cells (Fig. 5C).
Table 2.
MHV-68 proteins tested for activity to antagonize ZAP
| Protein name | Genome location (nt) | Protein |
Possible functiona | |
|---|---|---|---|---|
| No. of amino acids | Mass (kDa) | |||
| ORF6 | 11216–14527 | 1,103 | 122 | Single-stranded DNA binding protein |
| ORF9 | 19217–22297 | 343 | 41.2 | DNA polymerase |
| ORF11 | 23488–24651 | 388 | 46.6 | Virion protein |
| ORF18 | 29917–30768 | 284 | 34.1 | Regulator of late-gene expression |
| ORF21 | 32879–34810 | 644 | 77.3 | Thymidine kinase |
| ORF27 | 45329–46090 | 254 | 30.5 | Glycoprotein |
| ORF33 | 49588–50568 | 327 | 39.2 | Tegument protein |
| ORF38 | 55544–55768 | 75 | 9.0 | Myristylated tegument protein |
| ORF40 | 57046–58875 | 610 | 73.2 | Helicase-primase |
| ORF45 | 63655–64272 | 206 | 24.7 | Tegument protein |
| ORF50 (RTA) | 67907–69373 | 489 | 58.7 | Transcriptional activator |
| ORF52 | 70960–71364 | 135 | 16.2 | Tegument protein |
| ORF56 | 73289–75793 | 835 | 100.2 | Helicase-primase |
| ORF57 | 76662–77159 | 166 | 19.9 | Posttranscriptional regulator |
| ORF59 | 78258–79439 | 394 | 47.3 | Processivity factor |
| ORF63 | 83751–86564 | 938 | 112.6 | Tegument protein |
| ORF67 | 95738–96415 | 226 | 27.1 | Tegument protein |
| ORF75b1 | 113901–111652 | 758 | 91.0 | Tegument protein/FGARAT |
| ORF75a1 | 117904–115883 | 681 | 81.7 | Tegument protein/FGARAT |
FGARAT, N-formylglycinamide ribotide amidotransferase.
Fig 5.
MHV-68 RTA suppresses the antiviral activity of ZAP. (A) 293TRex-ZAP cells were transfected with pMLV-luc and pRL-TK, together with 0.9 μg of a plasmid expressing the MHV-68 ORF indicated. The antiviral activity of ZAP was measured as described in the legend to Fig. 1A. The data presented are means and SD from three independent experiments. (B) RTA inhibits ZAP's antiviral activity in a dose-dependent manner. 293TRex-ZAP cells were transfected with pMLV-luc and pRL-TK, together with the indicated amounts of an RTA-expressing plasmid. The antiviral activity of ZAP was measured as described in the legend to Fig. 1A. The data presented are means and SD from three independent experiments. (C) 293TRex cells were transfected with plasmids expressing myc-tagged ZAP and GFP with or without a plasmid expressing Flag-tagged RTA. At 6 h posttransfection, tetracycline was added to induce ZAP expression. At 48 h posttransfection, the cells were lysed, and expression of ZAP was detected by Western blotting. (D) BHK-21 cells were infected with either the recombinant virus or the wild-type (wt) virus at an MOI of 0.02. The supernatants were harvested at 0, 12, 24, 48, 72, and 96 h postinfection, and viral titers were determined by plaque assays. (E) The same numbers of 293TRex cells were either infected with a recombinant MHV-68 carrying Flag-tagged RTA at the indicated MOI or transfected with the indicated amounts of a plasmid expressing Flag-tagged RTA, together with a fixed amount of a plasmid expressing myc-tagged GFP to serve as a control. At 40 h posttransfection or 36 h postinfection, the cells were lysed, and the expression levels of Flag-tagged RTA were measured by Western blotting. (F) Plasmids expressing Flag-tagged and myc-tagged NZAP254 were cotransfected into HEK293 cells with or without a plasmid expressing HA-tagged RTA. At 48 h posttransfection, cells were lysed in lysis buffer containing RNase A and DNase. Proteins were immunoprecipitated with anti-Flag antibody and Western blotted with the antibodies indicated. (G) Plasmids expressing Flag-tagged and myc-tagged NZAP254 were cotransfected into HEK293 cells. At 6 h posttransfection, cells were mock infected or infected with MHV-68 at an MOI of 1. At 48 h posttransfection, the cells were lysed in lysis buffer containing RNase A and DNase. Proteins were immunoprecipitated with anti-Flag antibody and Western blotted with the antibodies indicated.
Since RTA was overexpressed in the above-described experiments, whether the expression level of RTA required for its antagonizing activity can be achieved in MHV-68 infection is an important question. To address this concern, we used a recombinant replication-competent MHV-68 in which RTA is tagged with a Flag epitope for detection of the protein. This recombinant virus displayed a multiple-step growth curve similar to that of the wild-type virus (Fig. 5D). 293TRex cells were either transfected with different amounts of an RTA-expressing plasmid or infected with the virus at different MOIs, and the expression levels of RTA were compared by Western blotting. The expression level of RTA at an MOI of 0.2 PFU per cell, the MOI used in the lytic replication assay, was comparable to that achieved by transfection of 0.5 μg of the RTA-expressing plasmid (Fig. 5E). These results suggest that the level of transfection-mediated RTA expression is comparable to that in MHV-68 infection.
RTA disrupts the N-terminal intermolecular interactions of ZAP.
Our previous studies revealed that the N-terminal intermolecular interaction of ZAP is required for its antiviral function (8). We wondered whether it is possible that RTA inhibits the antiviral activity of ZAP by interfering with the N-terminal intermolecular interactions of ZAP molecules. To test this idea, the Flag-tagged and myc-tagged N-terminal 254 amino acids of ZAP (NZAP) were coexpressed in HEK293 cells with or without the expression of RTA. The intermolecular interactions of NZAP were analyzed by coimmunoprecipitation assays. Consistent with our previous results, NZAP molecules interacted in the absence of RTA (Fig. 5F). In contrast, in the presence of RTA, the intermolecular interaction of NZAP was almost abolished (Fig. 5F). These results suggest that RTA inhibits the antiviral activity of ZAP by disrupting its N-terminal intermolecular interactions. To further substantiate this notion, we investigated whether the N-terminal intermolecular interactions of ZAP are affected by MHV-68 infection. Plasmids expressing Flag-tagged and myc-tagged NZAP were transfected into HEK293cells, followed by infection with MHV-68 at an MOI of 1. Indeed, the intermolecular interaction between NZAP molecules was dramatically reduced by MHV-68 infection (Fig. 5G). We noticed that MHV-68 infection did not completely disrupt the N-terminal intermolecular interaction as the overexpression of RTA did (compare Fig. 5F and G). A plausible explanation is that in the infection assay, cells were transfected with plasmids expressing NZAP molecules, followed by infection with MHV-68, and thus, some cells expressing NZAP may not be infected by MHV-68. In the transfection assay, plasmids expressing RTA and NZAP molecules were cotransfected, and thus, all the cells expressing NZAP are expected to express RTA.
Mapping the functional domain of RTA that inhibits the antiviral activity of ZAP.
The organization of the functional domains of MHV-68 RTA is similar to that of its homologues in Epstein-Barr virus and human herpesvirus 8, with the DNA binding and dimerization domains at the N terminus and the activation domain in the C-terminal portion (17, 19) (Fig. 6A). To map the domain of MHV-68 RTA that antagonizes ZAP, truncated forms of RTA were constructed (Fig. 6A) and assayed for their ability to inhibit ZAP's antiviral activity against pMLV-luc (Fig. 6B). The C-terminal domain of RTA (RTA_C) displayed ZAP-antagonizing activity comparable to that of the full-length RTA (RTA_FL) (Fig. 6B). In contrast, the N-terminal domain of RTA (RTA_N) displayed little ZAP-antagonizing activity, although the protein was expressed at a level comparable to that of RTA_C (Fig. 6B). Further truncation of RTA_C showed that a fragment covering amino acids 250 to 357 of RTA (RTA_M) displayed ZAP-antagonizing activity comparable to that of the full-length RTA (Fig. 6B). To further demonstrate the ZAP-antagonizing activity of RTA_M, it was coexpressed with ORF64_H in ZAP-expressing cells. As expected, expression of RTA_M antagonized ZAP's activity to inhibit ORF64_H expression (Fig. 6C). In line with the above-mentioned results, RTA_M significantly reduced the N-terminal intermolecular interaction of ZAP (Fig. 6D). Collectively, these results identified RTA_M as a domain sufficient to antagonize the antiviral activity of ZAP.
Fig 6.
Mapping the functional domain of RTA that suppresses the antiviral activity of ZAP. (A) Schematic representation of RTA proteins. The numbers indicate amino acid positions. DZ, the putative dimerization domain. (B) 293TRex-ZAP cells were transfected with pMLV-luc and pRL-TK, together with a construct expressing the truncated forms of RTA indicated. The antiviral activity of ZAP was measured as described in the legend to Fig. 1A (top). The expression of RTA proteins was detected by Western blotting (bottom). The data presented are means and SD from three independent experiments. (C) Plasmids expressing RTA_M and Flag-tagged ORF64_H were cotransfected into 293TRex-ZAP cells, along with a plasmid expressing myc-tagged GFP to serve as an internal control. At 6 h posttransfection, the cells were mock treated or treated with tetracycline to induce ZAP expression. At 54 h posttransfection, the cells were lysed, and expression of ORF64_H was detected by Western blotting. (D) Plasmids expressing Flag-tagged and myc-tagged NZAP254 were cotransfected into HEK293 cells, with or without a plasmid expressing HA-tagged RTA_M. At 48 h posttransfection, the cells were lysed in lysis buffer containing RNase A and DNase. Proteins were immunoprecipitated with anti-Flag antibody and Western blotted with the antibodies indicated.
DISCUSSION
Viral infection induces host immune responses. Type I interferons play critical roles in host innate immunity against viral infection. To establish effective infection, herpesviruses have evolved a variety of mechanisms to counteract interferon-mediated antiviral actions, including blocking the interferon production pathway and antagonizing interferon-induced antiviral effectors (32, 33).
ZAP is an interferon-inducible host antiviral factor (1–3, 34). In this report, we show that MHV-68 infection upregulates ZAP expression and that ZAP inhibits the expression of MHV-68 ORF64. We further demonstrate that the antiviral activity of ZAP is antagonized by RTA (Fig. 5A). Our preliminary results indicated that Kaposi's sarcoma-associated herpesvirus (KSHV) proteins (K8 and KRTA) also inhibit the antiviral activity of ZAP (Y. Xuan and G. Gao, unpublished results), suggesting that such a mechanism may be conserved among gammaherpesviruses. These results provide an additional example of how herpesviruses evade innate immunity. Our results also suggest that ZAP may have a broader antiviral spectrum than it appears to have and is thus an important antiviral effector.
ORF64 is a tegument protein that is essential for MHV-68 replication (22). Because of technical difficulties in expressing full-length ORF64, we analyzed fragments of the ORF64 coding sequence and identified two ZAP-responsive elements (Fig. 2). In addition, ZAP binds to ORF64 mRNA in MHV-68-infected cells (Fig. 1). Based on these results, it is reasonable to assume that ZAP inhibits the expression of ORF64. However, ZAP had little effect on viral lytic replication (Fig. 4). We show here that the antiviral activity of ZAP is antagonized by RTA. Since we analyzed only 19 MHV-68 proteins, determining whether ZAP is also antagonized by other viral proteins awaits further investigation.
RTA is an immediate-early gene expressed in the lytic phase (15, 20, 35). It is required for the transcription of multiple genes and thus plays a pivotal role in the lytic phase of MHV-68 (16, 17, 20, 21). RTA inhibited the antiviral activity of ZAP in a dose-dependent manner. Comparison of transfection-mediated expression and infection-mediated expression of RTA suggests that the expression level of RTA in virus-infected cells would be high enough to inhibit the antiviral activity of ZAP (Fig. 5). The temporal relationship between RTA and ORF64 suggests that ZAP is inhibited before ORF64 expression is initiated, which ensures the later stages of virus replication.
The functional domain of RTA that inhibits ZAP's activity was mapped to a fragment that contains the putative dimerization domain (Fig. 6). RTA and RTA_M disrupted the N-terminal intermolecular interaction of ZAP molecules, which is required for the function of ZAP (Fig. 5 and 6). Based on these results, it would be reasonable to speculate that RTA interacts with ZAP. However, direct interaction between RTA and ZAP was not detected in our coimmunoprecipitation assays (data not shown). One possible explanation for the failure to detect an interaction between RTA and ZAP is that the interaction is too weak to be detected under our experimental conditions. Another possible explanation is that RTA disrupts the N-terminal intermolecular interaction of ZAP via a molecule that has yet to be identified.
ZAP is expressed in multiple tissues in mice as judged by quantitative RT-PCR, with the highest levels in the spleen and lung (data not shown), where MHV-68 replicates (14). These results suggest that the interplay between ZAP and MHV-68 is likely to exist during viral infection of mice. Nonetheless, the in vivo biological relevance of our observation reported here awaits further investigation.
ACKNOWLEDGMENTS
We thank Zhenwei Yang, Institute of Biophysics, CAS, for technical assistance.
This work was supported by grants from the National Science Foundation (81030030 to G. Gao) and the Ministry of Science and Technology (973 Programs 2012CB910203 to G. Gao and 2011CB504805 to H. Deng) of China.
Footnotes
Published ahead of print 19 December 2012
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