Varicella-Zoster Virus Open Reading Frame 10 Is a
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病菌学杂志 2006年第7期
Department of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California
ABSTRACT
The open reading frame 10 (ORF10) of varicella-zoster virus (VZV) encodes a tegument protein that enhances transactivation of VZV genes and has homology to herpes simplex virus type 1 (HSV-1) VP16. While VP16 is essential for HSV replication, ORF10 is dispensable for vaccine OKA (VOKA) growth in vitro. We used parent OKA (POKA) cosmids to delete ORF10, producing POKA10; point mutations that disrupted the acidic activation domain and the putative motif for binding human cellular factor 1 (HCF-1) in ORF10 protein yielded POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF viruses. Deleting ORF10 or mutating these two functional domains had no effect on VZV replication, immediate-early gene transcription, or virion assembly in vitro. However, deleting ORF10 reduced viral titers and the extent of cutaneous lesions significantly in SCIDhu skin xenografts in vivo compared to POKA. Epidermal cells infected with POKA10 had significantly fewer DNA-containing nucleocapsids and complete virions compared to POKA; extensive aggregates of intracytoplasmic viral particles were also observed. Altering the activation or the putative HCF-1 domains of ORF10 protein had no consequences for VZV replication in vivo. Thus, the decreased pathogenic potential of POKA10 in skin could not be attributed to absence of these ORF10 protein functions. In contrast to skin cells, deleting ORF10 did not impair VZV T-cell tropism in vivo, as assessed by infectious virus yields. We conclude that ORF10 protein is required for efficient VZV virion assembly and is a specific determinant of VZV virulence in epidermal and dermal cells in vivo.
INTRODUCTION
Varicella-zoster virus (VZV), the causative agent of varicella (chicken pox) and zoster (shingles), belongs to the Varicellovirus genus within the Alphaherpesvirinae subfamily of Herpesviridae (3, 17, 48). VZV pathogenesis is mediated by the capacity of the virus to infect circulating T cells, skin cells, and sensory ganglia. Studies of VZV have lagged behind investigations of the other human herpesviruses because its growth in vitro is highly cell associated and yields low titers of unstable, cell-free virus. The genomic organization of VZV is closely related to that of herpes simplex virus type 1 (HSV-1), which has allowed predictions about the functions of many VZV genes (12, 13, 29). However, the pathogenesis of VZV and HSV-1 infections differs substantially, implying divergences in viral gene functions. Cosmids derived from VZV genomic DNA have made it possible to perform functional analysis of VZV gene products by deleting open reading frames (ORFs) or introducing targeted mutations into the VZV genome (9). We have used this approach to generate VZV recombinants from the vaccine OKA strain (VOKA) and the low-passage clinical isolate, parent OKA strain (POKA) (28, 42). The purpose of these experiments was to use cosmid mutagenesis to assess the functions of ORF10 in POKA replication in vitro and in the pathogenesis of VZV infection of human skin and T cells in vivo.
VZV ORF10 encodes a protein of 410 amino acids (aa) that has homology with HSV-1 VP16, which is encoded by UL48 (12). Some characteristics of VZV ORF10 have been defined in vitro and indicate that ORF10 protein has some, but not all, of the properties of HSV-1 VP16. Like VP16, ORF10 protein is incorporated into the virion tegument and is therefore presumed to be released when VZV enters cells (20). However, deletion of VP16 abolishes the ability of HSV-1 to grow in cell culture, whereas ORF10 was dispensable for VOKA replication in melanoma cells in vitro (1, 10, 57). ORF10 protein has been shown to influence the transcription of the VZV immediate-early (IE) gene, ORF62, but not ORF61 or ORF4 and ORF63, which encode other IE proteins (35, 38). In contrast, HSV-1 VP16 is a potent transactivator of all HSV-1 IE genes (4, 35, 44, 47). Using a series of GAL4-ORF10 constructs, Moriuchi et al. (37) mapped the ORF10 activation domain to amino acids 5 to 79, and determined that the single phenylalanine at position 28 (Phe28), which resembles the phenylalanine at position 442 (Phe442) in VP16, was essential for the transactivating effects of ORF10 protein.
The mammalian transcriptional coactivator, host cell factor 1 (HCF-1), is strictly required for the induction of transcription by VP16. HCF-1 forms a binary complex with VP16, which subsequently associates with the cellular octamer 1 (Oct-1) protein that binds directly to TAATGARAT motifs, resulting in HSV-1 IE gene expression (23, 24, 27, 43, 58). ORF10 protein, like VP16, has been shown to bind to HCF-1, and ORF10 protein forms a complex with Oct-1 and the TAATGARAT sequence of the IE62 promoter, which suggests that the participation of ORF10 protein in IE62 gene transactivation and the effects of VP16 on HSV-1 IE genes may be similar (36). The association between HCF-1 and VP16 has been mapped to a short tetrapeptide sequence, EHAY, in VP16 (15, 18, 26). This motif is highly conserved among VP16 homologues in herpesviruses, including ORF10 protein, and in human and mouse cellular proteins that bind HCF. Although direct involvement in HCF-1 binding has not been documented, the conserved residues in ORF10 protein that constitute the putative binding site are DHPY at positions 371 to 374 (15).
Although ORF10 has been shown to be dispensable for VZV replication in vitro, the role of ORF10 protein has not been assessed in vivo and the specific contributions of the functional domains of ORF10 protein that have been identified in expression systems have not been examined in the context of the viral genome during VZV replication in vitro or in vivo. The SCIDhu mouse model, in which skin and T-cell xenografts are infected in vivo, provides a unique opportunity to evaluate VZV gene functions in human tissue microenvironments through comparative studies of intact VZV and VZV mutants (25, 32). Experiments in the SCIDhu mouse model are important for determining how VZV gene products contribute to pathogenesis because VZV proteins, such as glycoprotein I, the ORF47 and ORF66 protein kinases, or defined functional domains within these proteins may be dispensable in cultured cells but remain essential for VZV replication in skin, T-cell xenografts, or both in vivo (5, 6, 31, 33, 34, 49).
In the present report, we found that deletion of ORF10 from POKA reduced replication in skin significantly, whereas the contribution of ORF10 protein to VZV T-cell tropism was limited in vivo. In contrast to cultured cells, ORF10 protein was required for producing normal numbers of dense DNA-containing nucleocapsids and efficient formation of complete VZV virions in the cytoplasm of VZV-infected skin cells in vivo. Experiments were also done to determine whether the ORF10 activation or putative HCF-1 binding domains were required for VZV skin tropism. These experiments showed no adverse consequences for pathogenesis when skin xenografts were infected with POKA ORF10 mutants in which phenylalanine at Phe28 was replaced with alanine or serine to abolish ORF10 transactivation activity or when DHPY at amino acids 371 to 374 was changed to DAPA to disrupt the putative HCF-1 binding site. Thus, ORF10 protein appears to be an important determinant of VZV virulence in skin, although not through its domains that enhance IE62 expression. ORF10 protein is not required for VZV infection of T cells in vivo.
MATERIALS AND METHODS
Generation of POKA recombinant viruses with ORF10 deletion or ORF10 mutations. Four overlapping POKA cosmids, pvFsp73, pvSpe14, pvPme2, and pvSpe23, were used to delete ORF10 or to make targeted mutations within ORF10 (42) (Fig. 1A). A 6.7-kb NheI-NheI and a 3.2-kb SacI-EcoRI fragment from pvFsp73 were subcloned into the pT7Blue-3 plasmid (New England Biolabs, Beverly, Mass) (Fig. 1B). To delete the gene, the fragment from nucleotides (nt) 11436 to 12159 was amplified by PCR with primers P1 (5'-GGATTCCCGAAGCGAGCTC-3'), including a SacI restriction site, and P3 (5'-CACTCCATACTTATAGAGTAAAATC-3'), containing the ORF10 start codon. The resulting PCR product was ligated back to the pT7Blue-3 SacI-EcoRI vector, which had been digested with enzyme PmlI (Fig. 1B), to obtain pT7Blue-3 SacI-EcoRI with ORF10 deleted. Then the SacI-EcoRI fragment lacking ORF10 was inserted into the pT7Blue-3 Nhe-NheI plasmid, which was digested by SacI and EcoRI (Fig. 1B), to produce a SacI-NheI fragment without the ORF10 gene. The SacI-NheI ORF10 deletion fragment was used to replace the SacI-EcoRI fragment in the cosmid pvFsp73 (Fig. 1B), removing ORF10 from this cosmid.
A similar strategy was used to make targeted mutations within ORF10. The amino acid residues that were mutated and the corresponding domains in HSV-1 VP16 are illustrated in Fig. 1C. Three sets of primers, P4 (5'-GCGGTTGTGGACGCAGCTGATGAATCG-3') and P6 (5'-GCGGTTGTGGACGCATCT GATGAATCG-3'), P5 (5'-GCGGTTGTGGACGCATCTGATGAATCG-3') and P7 (5'-CGATTCATCAGATGCGT CCACAACCGC-3'), and P8 (5'-CGAGCGGATGCTCCTGCCG CAAAAGTTG-3') and P9 (5'-CAACTTTTGCGGCAGGAGCATCCGCTCG-3'), were used that are complementary to each other and contain changes in codons to replace Phe28 of ORF10 protein with Ala or Ser and to alter DHPY at ORF10 positions 371 to 374 to DAPA. Three PCR steps were used to make each point mutation. The first PCR was done with P1 and the antisense primer containing the targeted changes in the specific nucleotides (P4, P5, and P9); the second PCR used primer P2 containing the EcoRI site (5'-GGAAGTGCGTAGACGGAATTC-3') and the sense primer that was complementary to its antisense primer (P6, P7, and P8). The resulting PCR products, which are complementary at their junction sites, were taken as templates for the third PCR, done with primers P1 and P2, to generate SacI-EcoRI fragments with targeted mutations. Each of these mutations was inserted into pvFsp73 by ligating the SacI-EcoRI fragment with the mutated codons (Fig. 1C) and the EcoRI-NheI fragment obtained from the pT7Blue-3 NheI-NheI plasmid (Fig. 1B) to the pvFsp73 cosmid that was digested with SacI and NheI (Fig. 1B). The PCR primers were manufactured by OPERON Technologies, Inc. (Huntsville, Alabama). Cosmid mutations were verified by sequencing.
Recombinant viruses. Recombinant viruses, designated POKA10, POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF, were isolated by transfection of human melanoma (MeWo) cells with the mutated pvFsp73 cosmid and the three intact cosmids, pvSpe14, pvPme2, and pvSpe23 (42). Melanoma cells were maintained in tissue culture medium (MEM; Mediatech, Washington, D.C.) supplemented with 10% fetal calf serum (Gemini Bio-Products, Woodland, Calif.), nonessential amino acids, and antibiotics. To confirm the ORF10 deletion and ORF10 mutations, DNA was isolated from melanoma cells or xenograft tissues with DNAzol reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. PCR was performed using Pfu DNA polymerase (Stratagene, La Jolla, Calif.) with primers P1 and P2 (Fig. 1C). The resulting PCR products were electrophoresed on a 1% agarose gel, isolated with QIAquick gel extraction kit 250 (QIAGEN, Inc., Valencia, Calif.), and confirmed by sequencing. Viruses were propagated in human embryonic lung fibroblasts (HELF) and stored at –80° in fetal calf serum with 10% dimethyl sulfoxide for infection of SCIDhu mouse xenografts.
The replication kinetics and peak titers of recombinant viruses were assessed by infectious focus assay. Melanoma cells were seeded in six-well plates and infected with an inoculum of 1 x 104 PFU. Cells were trypsinized on days 1 to 6, and titers were determined as described previously (32).
Northern blots. HELF at 80% confluence were inoculated with 2,000 PFU of each test virus per one T75 flask. Six T75 flasks of HELF were simultaneously infected with each recombinant virus. At 1 to 5 days postinoculation, infected cells were trypsinized and centrifuged and total single-stranded RNAs were extracted with Tri-Reagent (Invitrogen, Carlsbad, Calif.). The RNAs were separated by electrophoresis in formamide-formaldehyde denaturing 1.1% agarose gels in morpholinepropanesulfonic acid (MOPS) buffer and transferred to positively charged nylon membranes (Roche, Inc.). RNA detection on membranes was done with nonradioactive digoxigenin (DIG)-labeled riboprobes; the membranes exposed to DIG-labeled riboprobes were further reacted with anti-DIG-alkaline phosphatase Fab fragment (antibody) and developed with CSPD chemiluminescent substrate (Roche, Inc.). Riboprobes were prepared to detect ORF4, ORF10, ORF61, ORF62, and ORF63 transcripts. (i) The ORF4 probe was made with primers P10 (5'-GCTTCAATTCCAACCAACCGACCC-3') and P11 (5'-CCGCTAACATTTTAATCCACG-3'), including a region of ORF4 from nt 3234 to 4142. (ii) The ORF10 probe was prepared with primers P12 (5'-GGAGTGTAATTTAGGAACCGAAC-3') and P13 (5'-GTATCTCGAGCGGGTGTGCAGATTGAC-3'), covering ORF10 nt 12161 to 12821. (iii) The ORF61 probe was synthesized with primers P14 (5'-CCATGATACCATATTAGCG-3') and P15 (5'-GCCATCCGAGTAAAGGTTGC-3') and included nt 103720 to 104485. (iv) The ORF62 probe was made with primers P16 (5'-CGTCTAAATTCACCCCAGTGC-3') and P17 (5'-CGAAAGGACGTGGTACAATTG-3'), which included the ORF62 region from nt 108283 to 109119. (v) The ORF63 probe was prepared with primers P18 (5'-GGTTCGTCCGATTCATAACG-3') and P19 (5'-CGTC TGGTTCACAAGAATCG-3'), covering the complete ORF63 sequence. The fragments were synthesized by PCR using pvFsp73 or pvSpe23 cosmid DNA as a template; ORF-specific products inserted into pCR4-TOPO clone vector (Invitrogen, Carlsbad, Calif.) were used to synthesize positive-stranded RNA-specific probes by using T7 or Sp6 RNA polymerase (Ambion, Inc.).
Immunofluorescence. Infected HELF were fixed in 2% formaldehyde-0.05% Triton X-100 for 1 h at 8, 12, 20, 24, 36, and 48 h after infection. Cells were washed five times in phosphate-buffered saline (PBS) for 5 min, blocked with 5% normal goat serum, 1x PBS, and 0.02% albumin from bovine serum for 1 h, and incubated overnight at 4°C with murine anti-IE62 monoclonal antibody. Cells were washed 5 times with 1x PBS, and fluorescein isothiocyanate-labeled anti-mouse antibody (Jackson ImmunoResearch, Inc.) was added for 1 h and covered with aluminum foil. After five PBS washes, coverslips were mounted with Vectashield (Vector Laboratories, Inc., Burlingame, Calif.) and stored at 4°C in dark. Imaging was performed with a MultiProbe 2010 laser confocal microscope.
Infection of human xenografts in SCIDhu mice. Skin and thymus/liver xenografts were engrafted in male homozygous C.B-17scid/scid mice (32) using human fetal tissues obtained with informed consent according to federal and state regulations. Animals were cared for according to the guidelines of the Animal Welfare Act PL 94-279 and the Stanford University Administrative Panel on Laboratory Animal Care. VZV recombinant viruses propagated in HELF were used to inoculate xenografts. Infectious virus titers were determined for each inoculum at the time of injection. Skin xenografts were harvested after 10 and 18 days, and T-cell xenografts were harvested at 14 and 24 days. Viral replication was assessed by infectious focus assay.
Immunohistochemistry. Formalin-fixed, paraffin-embedded skin sections (5 μm) were deparaffinized, rehydrated, and treated with antigen retrieval reagent (Vector laboratories). VZV protein was detected with human polyclonal anti-VZV immunoglobulin G, biotinylated goat anti-human secondary antibody, and horseradish peroxidase-conjugated streptavidin (Chemicon IHC Select). Signals were developed with Vector VIP (purple) and counterstained with methyl green (Vector Laboratories, Inc., Burlingame, Calif.).
Transmission electron microscopy. Infected skin xenografts were recovered from SCID-hu mice at day 10 after inoculation and immediately fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (PBS), pH 7.0, for 2 h. The specimens were washed twice in PBS, postfixed with 1% osmium tetroxide (Polysciences, Inc., Warrington, PA) in PBS for 1 h, and after two 10-min washes in double distilled water, specimens were treated with 0.25% uranyl acetate (Polysciences, Inc.) overnight. After 24 h, the specimens were washed with water and dehydrated through a graded series of alcohol and propylene oxide washes. Each sample was infiltrated sequentially with 2:1 and 1:1 propylene oxide-EPON (Resolution Performance Products, Houston, TX) for 4 h, incubated overnight with 100% EPON, transferred to fresh EPON, and embedded and polymerized at 60°C for 24 h. Thin sections were collected on copper grids, stained with uranyl acetate and lead citrate, and viewed using a Phillips CM-12 transmission electron microscope. Melanoma cells were infected with 1000 PFU POKA10 and grown on glass coverslips for 48 h. The cells were fixed for 35 min and fixed and stained using the same procedure.
RESULTS
Generation of POKA recombinants with ORF10 mutations. Transfection of the pvFsp73 cosmid, from which ORF10 had been deleted (Fig. 1B), with the other three POKA cosmids, pvSpe14, pvPme2, and pvSpe23, into melanoma cells yielded infectious virus, as expected (10). The resulting virus, POKA10, was propagated in melanoma cells and HELF. Transfection of pvFsp73 cosmids, in which ORF10 had been mutated by substitutions replacing Phe28 with alanine or serine to disrupt the ORF10 activation domain (37), along with the other three intact cosmids, also yielded infectious viruses, designated POKA10-Phe28Ala and POKA10-Phe28Ser (Fig. 1C). POKA10-mHCF was derived from transfections with pvFsp73 in which the putative HCF-1 binding motif of ORF10 was disrupted to evaluate the effects on ORF10 protein-mediated activation of VZV IE gene expression (15). The ORF10 deletion and the presence of the ORF10 targeted mutations were confirmed in the VZV recombinants by sequencing (data not shown).
To verify that ORF10 transcripts were not made in cells infected with POKA10 and to show that ORF10 transcription was maintained in cells infected with POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF, total single-stranded RNAs were recovered from infected cells for Northern hybridization. As expected, no ORF10 transcripts were detected in POKA10-infected cells by Northern blotting with the RNA probe specific for ORF10; ORF10 transcripts were detected in cells infected with the three ORF10 point mutants at levels comparable to those in POKA-infected cells (Fig. 1D).
Growth of POKA ORF10 mutants in vitro. The kinetics of replication of POKA, POKA10, POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF were evaluated for 6 days in melanoma cells. The growth of POKA and the POKA ORF10 mutants was similar. POKA titers were 4.0 x 105 PFU/ml at day 2 and 4.5 x 105 PFU/ml at day 3, which was slightly higher than those of POKA ORF10 mutants, which were 2.0 x 105 PFU/ml at day 2 and 3.0 x 105 PFU/ml at day 3, but the differences were not statistically significant (P > 0.05). All titers were approximately 5.0 x 105 PFU/ml at day 4 and remained equivalent at days 5 and 6 after inoculation. The morphologies of plaques produced by POKA10 and POKA ORF10 point mutants were indistinguishable from POKA in melanoma cells and HELF (data not shown).
Effects of ORF10 deletion or mutations in ORF10 protein on VZV IE gene transcription in vitro. HELF were infected with POKA, POKA10, POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF. Plaques appeared in monolayers infected with all viruses at 3 days postinoculation, and most HELF were depleted and detached from the flask by 6 days. Northern blot analysis of HELF inoculated with the POKA ORF10 deletion and ORF10 point mutation recombinants showed that the kinetics of accumulation of ORF4, ORF61, ORF62, and ORF63 mRNAs were similar to that observed in HELF infected with POKA (Fig. 2); these experiments were repeated four times with reproducible results. No transcripts were detected from mock-infected HELF (data not shown). The accumulation of ORF4, ORF62, and ORF63 transcripts was detected beginning 2 days after infection and continuing through the 6-day time course (Fig. 2: rows 1, 3 and 4); ORF61 mRNA from all samples was detected beginning 1 day after infection (Fig. 2, row 2). The ORF4 probe showed hybridization with the major 1.8-kb and the minor 3.0-kb forms of ORF4 mRNA, as has been described previously in analyses of ORF4-related mRNAs in cells infected with VZV Scott strain (21). The ORF63 probe revealed two RNA species of about 0.9 and 1.8 kb in size, corresponding to ORF63-associated transcripts reported in cells infected with the VZV Ellen strain but not the VZV Scott strain (22). The probe specific for ORF61 revealed one transcript of about 1.5 kb and a fainter 0.7-kb product that may result from abortive transcription or rapid degradation of ORF61 RNAs. Two transcripts were detected with the ORF62 probe; one transcript was about 4.1 kb, which is consistent with the expected size of ORF62 transcripts, and the other was 5.5 kb, which was also observed by with the ORF61 probe (data not shown), which may represent read-through mRNAs originating from the ORF62 start site and terminating at the ORF61 stop site.
Pattern of IE62 distribution in cells infected with POKA and POKA ORF10 mutants in vitro. Since IE62 and ORF10 proteins both function as regulatory and tegument proteins, we investigated whether deletion of ORF10 or disruption of ORF10 activation and putative HCF functional domains influenced the intracellular localization of IE62 in VZV-infected HELF over a time course of 8 to 48 h after infection with POKA or POKA ORF10 mutants (Fig. 3). As was observed in POKA-infected cells, IE62 was exclusively in the nuclei at 8 h in cells infected with POKA10 and POKA ORF10 point mutants; this pattern was also observed at 12 h (data not shown). IE62 was detected in the cytoplasm but remained predominantly nuclear by 20 h in cells infected with POKA and POKA ORF10 mutants. At 24 h, IE62 from all recombinant viruses was observed in more than 50% cells that were examined, and extensive cell fusion had occurred by 36 h postinoculation (data not shown). By 48 h, most cells were infected and had cytopathic changes associated with cytoplasmic accumulation of IE62 in HELF infected with POKA and POKA ORF10 mutants.
Effects of ORF10 deletion or mutations in ORF10 protein on VZV infection of skin xenografts in vivo. To determine the possible roles played by ORF10 protein during VZV infection in vivo, skin xenografts were inoculated with POKA10, POKA10-Phe28Ala, POKA10-Phe28Ser, POKA10-mHCF, and POKA. The inoculum titers were similar, as determined by infectious center assay (Fig. 4). The growth of POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF was equivalent to POKA, with titers of 1 x 104 to 2 x 104 PFU per implant at 10 and 18 days (Fig. 4). In contrast, the replication of POKA10 was decreased and delayed significantly compared to POKA and the ORF10 protein point mutants (P < 0.05). On day 10, infectious virus was recovered from 6 of 8 xenografts inoculated with POKA10 and the mean titer for positive xenografts was 1 x 103 PFU per implant, which was 10-fold less than the other viruses (Fig. 4). On day 18, only 4 of 8 implants inoculated with POKA10 yielded infectious virus; the mean titer of virus in positive xenografts decreased to 1.5 x 102 PFU, which was 100-fold lower than that of POKA10-Phe28Ala, POKA10-Phe28Ser, POKA10-mHCF, and POKA (Fig. 4). The expected sequences were confirmed for POKA and all ORF10 mutants recovered from skin xenografts (data not shown).
The pattern of skin lesions formed in skin xenografts is another measure of the relative pathogenic potential of POKA and VZV mutants. Inoculation with POKA, POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF resulted in cutaneous lesions by day 10. Infection was associated with disruption of the keratinized outer layer of skin and spread of the virus into the dermis (Fig. 5). In contrast, POKA10 infection progressed much more slowly, forming small lesions restricted to the epidermal layer only at day 10 (Fig. 5, panel B1). By 18 days after POKA inoculation, extensive skin lesions were observed at the epidermal surfaces, and large segments of dermal-epidermal junctions that form the basement membrane were destroyed (Fig. 5, panel A2). POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF also generated large necrotic lesions, and the basement membranes were damaged by day 18, as observed with POKA infection (Fig. 5). POKA10 lesions remained small and confined to the epidermis without penetration of the basement membrane at day 18 (Fig. 5, panel B2).
Effects of ORF10 deletion on VZV virion formation in vitro and in skin in vivo. We next examined VZV virion morphogenesis in melanoma cells and skin xenografts infected with POKA or POKA10 by electron microscopy. POKA10-infected melanoma cells showed no changes compared with POKA in the stages of nucleocapsid assembly, capsid budding at the inner nuclear membrane, and intracellular VZV virion maturation, and many complete enveloped particles and incomplete particles lined the outer surfaces of the infected cells (Fig. 6). In contrast, when four electron microscopy (EM) fields from skin xenograft specimens that had been obtained 10 days after infection with either POKA or POKA10 were examined, the total number of virions, including nucleocapsids and cytoplasmic virus particles, in POKA10-infected skin cells was 198 compared to 253 in POKA-infected specimens, representing a reduction of approximately 23%. Skin xenografts infected with POKA10 also had accumulations of unenveloped capsids in the cytoplasm in association with aggregates of tegument-like proteins (Fig. 7, panel B1), which were not found in POKA-infected skin xenografts (Fig. 7, panel A1). Particles within these aggregates were counted in determining the total number of virions in POKA10-infected skin cells. However, in spite of these aberrant capsid accumulation structures, some complete virions with the same morphology as POKA virions (Fig. 7, panel A2) were detected in epidermal cells after infection with POKA10 (Fig. 7, panel B2). In addition, more POKA10 nucleocapsids appeared to be empty compared to POKA (Fig. 7, panels A3 and B3). When equivalent numbers of nucleocapsids in POKA- and POKA10-infected cells were examined, only about 23% of POKA10 nucleocapsids (46/201) appeared to contain DNA compared to 73% of POKA nucleocapsids (142/194). The percentage of mature complete virions in the cytoplasm of POKA10-infected skin cells was also only 25% (26/105) compared to 69% (75/109) in POKA-infected skin cells.
Effects of ORF10 deletion on VZV infection of T-cell xenografts in vivo. To assess POKA10 replication in T cells, we inoculated T-cell xenografts with POKA and POKA10. POKA10 exhibited a growth pattern that was similar to that of POKA in T-cell xenografts (Fig. 8). The inoculum titers were 3.5 x 104 PFU for POKA and 5.6 x 104 PFU for POKA10. The yields of infectious virus from POKA- and POKA10-infected T-cell xenografts were indistinguishable at 14 days; titers were 4.0 x 103 PFU and 4.5 x 103 PFU, respectively. POKA and POKA10 infectious virus yields also declined with similar kinetics, decreasing to approximately 4 x 10 PFU at day 24. The depletion of T cells at this time point was equivalent, based on immunohistochemistry analysis of the POKA- and POKA10-infected T-cell xenografts (data not shown), and similar to the pattern that is observed consistently in T-cell xenografts infected with POKA and other low-passage clinical isolates of VZV (33, 34).
DISCUSSION
These experiments document the importance of ORF10 protein for VZV pathogenesis, showing that this gene product is a virulence determinant in human skin in vivo in the SCIDhu mouse model. Decreased production of infectious virus in the absence of ORF10 protein was associated with impaired formation of VZV virions in epidermal cells in vivo. This effect was evident at several steps in VZV virion assembly, including reduced DNA encapsidation, trapping of DNA-containing capsids in cytoplasmic aggregates, and diminished numbers of complete virions in the cytoplasm. These changes were not evident in cultured cells. The analysis of the POKA10 mutant in the SCIDhu mouse model revealed a differential requirement for ORF10 protein in skin and T-cell xenografts in vivo. The growth of POKA10 was indistinguishable from that of POKA in T cells.
The contribution of ORF10 protein to VZV replication differs significantly from VP16, its HSV-1 homolog. VP16 is a potent transcriptional activator and is required for HSV-1 growth in cell culture (1, 57), whereas ORF10 can be deleted from VOKA (10), and its nonessential role was confirmed by our POKA10 experiments. Our VZV mutants, POKA10-Phe28Ala and POKA10-Phe28Ser, were designed to disrupt the activation domain that has been shown to mediate the transactivating effects of ORF10 protein on the ORF62 promoter in transient expression systems (35). Mutations that inactivated the corresponding transcriptional activation function of VP16 reduced the levels of HSV-1 IE gene expression and viral replication substantially in vitro and in heterologous murine systems in vivo. However, these VP16 mutants could be propagated in tissue culture to some extent, and most were infectious in vivo, demonstrating that the activation function of VP16 was not absolutely essential for HSV-1 replication (2, 50, 51, 52, 55, 56). When introduced into the VZV genome, mutations of the functionally related acidic activation domain of ORF10 protein had no effect on transcription of ORF62 or putative VZV regulatory genes that are downstream targets of IE62, including ORF4, ORF10, ORF61, and ORF63, in cultured cells. VZV virion morphogenesis was also intact in POKA10-Phe28Ala- and POKA10-Phe28Ser-infected melanoma cells. The failure of these mutations in the acidic activation domain to affect VZV gene transcription and replication in vitro was expected, given observations with POKA10 and VOKA from which ORF10 was deleted (10). However, altering the activation domain of ORF10 protein also had no impact on VZV virulence in human epidermal and dermal cells in vivo, compared to POKA. Thus, while the capacity of VP16 to transactivate HSV-1 IE genes cannot be fully compensated by any other viral transactivating proteins, even in highly permissive cultured cells, VZV does not require the activation function of ORF10 protein. In VZV, the major IE transactivator, IE62, has the capacity to induce its own transcription independently and regulates transcription of all other VZV genes that have been evaluated (14, 19, 20, 40, 45, 46). Therefore, the autoregulatory function of IE62 and its induction of other IE proteins appears to be sufficient in the absence of an intact activation domain in ORF10 protein in vitro and, most importantly, in differentiated human skin cells in vivo. Since deleting ORF10 had no effect on VZV T-cell infection, this activation function can also be presumed to be dispensable for T-cell tropism.
The cellular factor HCF-1 is required for VP16-mediated activation and the regulated transcription of HSV-1 IE genes (24, 27, 58). Similarly, HCF-1 is also critical for induction of the IE62 promoter by IE62 and ORF10 protein in reporter constructs or VZV-infected cells (36, 41). However, disrupting the conserved DHPY motif that is presumed to mediate ORF10 protein binding to HCF-1, in the context of the viral genome in our POKA10-mHCF mutant, did not alter VZV replication or transcription of regulatory genes in vitro. In contrast, mutations that disrupt the capacity of VP16 to form complexes with HCF-1 and Oct-1 and the TAATGARAT element reduce HSV growth (2). Altering the putative HCF-1 binding motif did not affect the pathogenesis of VZV skin infection, as determined by yields of infectious virus and the histopathologic appearance of cutaneous lesions. Our observations suggest that, while ORF10 protein has a motif that is closely related to the HCF-1 binding domain in VP16 and the ORF10/HCF-1 complex enhances ORF62 expression, HCF-1 interaction with this putative binding site in ORF10 is incidental for VZV infection in vitro and in skin in vivo. As observed with the ORF10 activation domain mutants, VZV IE62, as a potent single viral transactivator, has the potential to rescue the loss of the ORF10/HCF-1 interaction in VZV-infected epidermal and dermal cells. In addition, the fact that removing the complete ORF10 gene from POKA did not affect VZV T-cell tropism suggests that viral gene regulatory effects resulting from the formation of Oct-1/ORF10/HCF-1 complexes are redundant for VZV replication in T cells.
Although the targeted mutations in functional domains of ORF10 protein did not alter VZV infection in skin xenografts, the complete deletion of ORF10 was associated with substantially impaired replication and cell-cell spread of VZV in vivo. VZV plaque formation can be fully preserved in cultured cells infected with VZV mutants even when virion assembly is severely defective, as we demonstrated with our VZV ORF47 kinase-null mutants (6). However, the EM analysis of melanoma cells infected with POKA and POKA10 revealed no consequences of ORF10 deletion on VZV capsid formation or subsequent steps in virion maturation in vitro. In contrast, examining these processes in skin cells infected with POKA10 in vivo demonstrated significant deficiencies in total virus particles produced, in the proportion of dense capsids in nuclei, and in relative numbers of complete virus particles in the cytoplasm. When VP16 and pseudorabies (PrV) UL48 protein, which is an ORF10 homologue, were disrupted or absent, unenveloped capsids also accumulated in the cytoplasm of infected cells (16, 30, 39). Interestingly, comparing POKA10 with the corresponding HSV-1 and PrV mutants suggests a hierarchy in the requirement for this gene product in the alphaherpesviruses (16, 30, 39). The complete absence of VP16 in HSV-1 prevents the production of infectious progeny virus, indicating that as a tegument protein, VP16 plays a more important role than ORF10 protein in virion assembly. Deleting PrV UL48 yields replication-competent mutants, but these PrV mutants have impaired growth and marked deficiencies in virion morphogenesis in vitro (16, 30). Deficiencies in virion formation were observed only when VZV lacking ORF10 was challenged to replicate in skin cells within the intact tissue microenvironment in vivo. Although some complete virus particles were observed, POKA10 infection of skin cells was associated with the appearance of many capsid-less particles and cytoplasmic aggregates of apparently tegumented capsids that were similar to those observed with PrV glycoprotein deletion mutants (7, 8, 30). Since ORF10 protein is a tegument component, tegumentation of capsids might be altered, but it is not clear why viral DNA encapsidation would be impaired in the absence of ORF10 protein. However, the EM analysis of POKA10 in skin cells suggests that ORF10 protein facilitates DNA insertion into capsids. Investigations of HSV-1 VP16 mutants have also shown inefficient DNA encapsidation for unexplained reasons (39, 57). Overall, defective POKA10 virion morphogenesis appears to account for low yields of infectious virus from skin xenografts and the observation that cutaneous lesions, which reflect cell-cell spread of VZV, were much smaller.
In summary, these experiments show that ORF10 protein is required for efficient viral replication, virion formation, and cell-cell spread in human skin in vivo. However, these functions do not appear to depend on preservation of the acidic activation domain or the putative HCF-1 binding motif that facilitate IE62 activation. The requirement for ORF10 protein in skin appears to be related to a role in virion assembly. Given these observations, it will be of particular interest to determine whether these ORF10 protein domains are important for VZV neurotropism. Whereas ORF10 protein was not required for replication in T cells, VZV ORF10 protein is a virulence determinant in skin in vivo.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI053846) and the Astellas USA Foundation.
We thank Nafisa Ghori and Anne Schaap for valuable technical assistance and Chia-Chi Ku for helpful discussions.
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ABSTRACT
The open reading frame 10 (ORF10) of varicella-zoster virus (VZV) encodes a tegument protein that enhances transactivation of VZV genes and has homology to herpes simplex virus type 1 (HSV-1) VP16. While VP16 is essential for HSV replication, ORF10 is dispensable for vaccine OKA (VOKA) growth in vitro. We used parent OKA (POKA) cosmids to delete ORF10, producing POKA10; point mutations that disrupted the acidic activation domain and the putative motif for binding human cellular factor 1 (HCF-1) in ORF10 protein yielded POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF viruses. Deleting ORF10 or mutating these two functional domains had no effect on VZV replication, immediate-early gene transcription, or virion assembly in vitro. However, deleting ORF10 reduced viral titers and the extent of cutaneous lesions significantly in SCIDhu skin xenografts in vivo compared to POKA. Epidermal cells infected with POKA10 had significantly fewer DNA-containing nucleocapsids and complete virions compared to POKA; extensive aggregates of intracytoplasmic viral particles were also observed. Altering the activation or the putative HCF-1 domains of ORF10 protein had no consequences for VZV replication in vivo. Thus, the decreased pathogenic potential of POKA10 in skin could not be attributed to absence of these ORF10 protein functions. In contrast to skin cells, deleting ORF10 did not impair VZV T-cell tropism in vivo, as assessed by infectious virus yields. We conclude that ORF10 protein is required for efficient VZV virion assembly and is a specific determinant of VZV virulence in epidermal and dermal cells in vivo.
INTRODUCTION
Varicella-zoster virus (VZV), the causative agent of varicella (chicken pox) and zoster (shingles), belongs to the Varicellovirus genus within the Alphaherpesvirinae subfamily of Herpesviridae (3, 17, 48). VZV pathogenesis is mediated by the capacity of the virus to infect circulating T cells, skin cells, and sensory ganglia. Studies of VZV have lagged behind investigations of the other human herpesviruses because its growth in vitro is highly cell associated and yields low titers of unstable, cell-free virus. The genomic organization of VZV is closely related to that of herpes simplex virus type 1 (HSV-1), which has allowed predictions about the functions of many VZV genes (12, 13, 29). However, the pathogenesis of VZV and HSV-1 infections differs substantially, implying divergences in viral gene functions. Cosmids derived from VZV genomic DNA have made it possible to perform functional analysis of VZV gene products by deleting open reading frames (ORFs) or introducing targeted mutations into the VZV genome (9). We have used this approach to generate VZV recombinants from the vaccine OKA strain (VOKA) and the low-passage clinical isolate, parent OKA strain (POKA) (28, 42). The purpose of these experiments was to use cosmid mutagenesis to assess the functions of ORF10 in POKA replication in vitro and in the pathogenesis of VZV infection of human skin and T cells in vivo.
VZV ORF10 encodes a protein of 410 amino acids (aa) that has homology with HSV-1 VP16, which is encoded by UL48 (12). Some characteristics of VZV ORF10 have been defined in vitro and indicate that ORF10 protein has some, but not all, of the properties of HSV-1 VP16. Like VP16, ORF10 protein is incorporated into the virion tegument and is therefore presumed to be released when VZV enters cells (20). However, deletion of VP16 abolishes the ability of HSV-1 to grow in cell culture, whereas ORF10 was dispensable for VOKA replication in melanoma cells in vitro (1, 10, 57). ORF10 protein has been shown to influence the transcription of the VZV immediate-early (IE) gene, ORF62, but not ORF61 or ORF4 and ORF63, which encode other IE proteins (35, 38). In contrast, HSV-1 VP16 is a potent transactivator of all HSV-1 IE genes (4, 35, 44, 47). Using a series of GAL4-ORF10 constructs, Moriuchi et al. (37) mapped the ORF10 activation domain to amino acids 5 to 79, and determined that the single phenylalanine at position 28 (Phe28), which resembles the phenylalanine at position 442 (Phe442) in VP16, was essential for the transactivating effects of ORF10 protein.
The mammalian transcriptional coactivator, host cell factor 1 (HCF-1), is strictly required for the induction of transcription by VP16. HCF-1 forms a binary complex with VP16, which subsequently associates with the cellular octamer 1 (Oct-1) protein that binds directly to TAATGARAT motifs, resulting in HSV-1 IE gene expression (23, 24, 27, 43, 58). ORF10 protein, like VP16, has been shown to bind to HCF-1, and ORF10 protein forms a complex with Oct-1 and the TAATGARAT sequence of the IE62 promoter, which suggests that the participation of ORF10 protein in IE62 gene transactivation and the effects of VP16 on HSV-1 IE genes may be similar (36). The association between HCF-1 and VP16 has been mapped to a short tetrapeptide sequence, EHAY, in VP16 (15, 18, 26). This motif is highly conserved among VP16 homologues in herpesviruses, including ORF10 protein, and in human and mouse cellular proteins that bind HCF. Although direct involvement in HCF-1 binding has not been documented, the conserved residues in ORF10 protein that constitute the putative binding site are DHPY at positions 371 to 374 (15).
Although ORF10 has been shown to be dispensable for VZV replication in vitro, the role of ORF10 protein has not been assessed in vivo and the specific contributions of the functional domains of ORF10 protein that have been identified in expression systems have not been examined in the context of the viral genome during VZV replication in vitro or in vivo. The SCIDhu mouse model, in which skin and T-cell xenografts are infected in vivo, provides a unique opportunity to evaluate VZV gene functions in human tissue microenvironments through comparative studies of intact VZV and VZV mutants (25, 32). Experiments in the SCIDhu mouse model are important for determining how VZV gene products contribute to pathogenesis because VZV proteins, such as glycoprotein I, the ORF47 and ORF66 protein kinases, or defined functional domains within these proteins may be dispensable in cultured cells but remain essential for VZV replication in skin, T-cell xenografts, or both in vivo (5, 6, 31, 33, 34, 49).
In the present report, we found that deletion of ORF10 from POKA reduced replication in skin significantly, whereas the contribution of ORF10 protein to VZV T-cell tropism was limited in vivo. In contrast to cultured cells, ORF10 protein was required for producing normal numbers of dense DNA-containing nucleocapsids and efficient formation of complete VZV virions in the cytoplasm of VZV-infected skin cells in vivo. Experiments were also done to determine whether the ORF10 activation or putative HCF-1 binding domains were required for VZV skin tropism. These experiments showed no adverse consequences for pathogenesis when skin xenografts were infected with POKA ORF10 mutants in which phenylalanine at Phe28 was replaced with alanine or serine to abolish ORF10 transactivation activity or when DHPY at amino acids 371 to 374 was changed to DAPA to disrupt the putative HCF-1 binding site. Thus, ORF10 protein appears to be an important determinant of VZV virulence in skin, although not through its domains that enhance IE62 expression. ORF10 protein is not required for VZV infection of T cells in vivo.
MATERIALS AND METHODS
Generation of POKA recombinant viruses with ORF10 deletion or ORF10 mutations. Four overlapping POKA cosmids, pvFsp73, pvSpe14, pvPme2, and pvSpe23, were used to delete ORF10 or to make targeted mutations within ORF10 (42) (Fig. 1A). A 6.7-kb NheI-NheI and a 3.2-kb SacI-EcoRI fragment from pvFsp73 were subcloned into the pT7Blue-3 plasmid (New England Biolabs, Beverly, Mass) (Fig. 1B). To delete the gene, the fragment from nucleotides (nt) 11436 to 12159 was amplified by PCR with primers P1 (5'-GGATTCCCGAAGCGAGCTC-3'), including a SacI restriction site, and P3 (5'-CACTCCATACTTATAGAGTAAAATC-3'), containing the ORF10 start codon. The resulting PCR product was ligated back to the pT7Blue-3 SacI-EcoRI vector, which had been digested with enzyme PmlI (Fig. 1B), to obtain pT7Blue-3 SacI-EcoRI with ORF10 deleted. Then the SacI-EcoRI fragment lacking ORF10 was inserted into the pT7Blue-3 Nhe-NheI plasmid, which was digested by SacI and EcoRI (Fig. 1B), to produce a SacI-NheI fragment without the ORF10 gene. The SacI-NheI ORF10 deletion fragment was used to replace the SacI-EcoRI fragment in the cosmid pvFsp73 (Fig. 1B), removing ORF10 from this cosmid.
A similar strategy was used to make targeted mutations within ORF10. The amino acid residues that were mutated and the corresponding domains in HSV-1 VP16 are illustrated in Fig. 1C. Three sets of primers, P4 (5'-GCGGTTGTGGACGCAGCTGATGAATCG-3') and P6 (5'-GCGGTTGTGGACGCATCT GATGAATCG-3'), P5 (5'-GCGGTTGTGGACGCATCTGATGAATCG-3') and P7 (5'-CGATTCATCAGATGCGT CCACAACCGC-3'), and P8 (5'-CGAGCGGATGCTCCTGCCG CAAAAGTTG-3') and P9 (5'-CAACTTTTGCGGCAGGAGCATCCGCTCG-3'), were used that are complementary to each other and contain changes in codons to replace Phe28 of ORF10 protein with Ala or Ser and to alter DHPY at ORF10 positions 371 to 374 to DAPA. Three PCR steps were used to make each point mutation. The first PCR was done with P1 and the antisense primer containing the targeted changes in the specific nucleotides (P4, P5, and P9); the second PCR used primer P2 containing the EcoRI site (5'-GGAAGTGCGTAGACGGAATTC-3') and the sense primer that was complementary to its antisense primer (P6, P7, and P8). The resulting PCR products, which are complementary at their junction sites, were taken as templates for the third PCR, done with primers P1 and P2, to generate SacI-EcoRI fragments with targeted mutations. Each of these mutations was inserted into pvFsp73 by ligating the SacI-EcoRI fragment with the mutated codons (Fig. 1C) and the EcoRI-NheI fragment obtained from the pT7Blue-3 NheI-NheI plasmid (Fig. 1B) to the pvFsp73 cosmid that was digested with SacI and NheI (Fig. 1B). The PCR primers were manufactured by OPERON Technologies, Inc. (Huntsville, Alabama). Cosmid mutations were verified by sequencing.
Recombinant viruses. Recombinant viruses, designated POKA10, POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF, were isolated by transfection of human melanoma (MeWo) cells with the mutated pvFsp73 cosmid and the three intact cosmids, pvSpe14, pvPme2, and pvSpe23 (42). Melanoma cells were maintained in tissue culture medium (MEM; Mediatech, Washington, D.C.) supplemented with 10% fetal calf serum (Gemini Bio-Products, Woodland, Calif.), nonessential amino acids, and antibiotics. To confirm the ORF10 deletion and ORF10 mutations, DNA was isolated from melanoma cells or xenograft tissues with DNAzol reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. PCR was performed using Pfu DNA polymerase (Stratagene, La Jolla, Calif.) with primers P1 and P2 (Fig. 1C). The resulting PCR products were electrophoresed on a 1% agarose gel, isolated with QIAquick gel extraction kit 250 (QIAGEN, Inc., Valencia, Calif.), and confirmed by sequencing. Viruses were propagated in human embryonic lung fibroblasts (HELF) and stored at –80° in fetal calf serum with 10% dimethyl sulfoxide for infection of SCIDhu mouse xenografts.
The replication kinetics and peak titers of recombinant viruses were assessed by infectious focus assay. Melanoma cells were seeded in six-well plates and infected with an inoculum of 1 x 104 PFU. Cells were trypsinized on days 1 to 6, and titers were determined as described previously (32).
Northern blots. HELF at 80% confluence were inoculated with 2,000 PFU of each test virus per one T75 flask. Six T75 flasks of HELF were simultaneously infected with each recombinant virus. At 1 to 5 days postinoculation, infected cells were trypsinized and centrifuged and total single-stranded RNAs were extracted with Tri-Reagent (Invitrogen, Carlsbad, Calif.). The RNAs were separated by electrophoresis in formamide-formaldehyde denaturing 1.1% agarose gels in morpholinepropanesulfonic acid (MOPS) buffer and transferred to positively charged nylon membranes (Roche, Inc.). RNA detection on membranes was done with nonradioactive digoxigenin (DIG)-labeled riboprobes; the membranes exposed to DIG-labeled riboprobes were further reacted with anti-DIG-alkaline phosphatase Fab fragment (antibody) and developed with CSPD chemiluminescent substrate (Roche, Inc.). Riboprobes were prepared to detect ORF4, ORF10, ORF61, ORF62, and ORF63 transcripts. (i) The ORF4 probe was made with primers P10 (5'-GCTTCAATTCCAACCAACCGACCC-3') and P11 (5'-CCGCTAACATTTTAATCCACG-3'), including a region of ORF4 from nt 3234 to 4142. (ii) The ORF10 probe was prepared with primers P12 (5'-GGAGTGTAATTTAGGAACCGAAC-3') and P13 (5'-GTATCTCGAGCGGGTGTGCAGATTGAC-3'), covering ORF10 nt 12161 to 12821. (iii) The ORF61 probe was synthesized with primers P14 (5'-CCATGATACCATATTAGCG-3') and P15 (5'-GCCATCCGAGTAAAGGTTGC-3') and included nt 103720 to 104485. (iv) The ORF62 probe was made with primers P16 (5'-CGTCTAAATTCACCCCAGTGC-3') and P17 (5'-CGAAAGGACGTGGTACAATTG-3'), which included the ORF62 region from nt 108283 to 109119. (v) The ORF63 probe was prepared with primers P18 (5'-GGTTCGTCCGATTCATAACG-3') and P19 (5'-CGTC TGGTTCACAAGAATCG-3'), covering the complete ORF63 sequence. The fragments were synthesized by PCR using pvFsp73 or pvSpe23 cosmid DNA as a template; ORF-specific products inserted into pCR4-TOPO clone vector (Invitrogen, Carlsbad, Calif.) were used to synthesize positive-stranded RNA-specific probes by using T7 or Sp6 RNA polymerase (Ambion, Inc.).
Immunofluorescence. Infected HELF were fixed in 2% formaldehyde-0.05% Triton X-100 for 1 h at 8, 12, 20, 24, 36, and 48 h after infection. Cells were washed five times in phosphate-buffered saline (PBS) for 5 min, blocked with 5% normal goat serum, 1x PBS, and 0.02% albumin from bovine serum for 1 h, and incubated overnight at 4°C with murine anti-IE62 monoclonal antibody. Cells were washed 5 times with 1x PBS, and fluorescein isothiocyanate-labeled anti-mouse antibody (Jackson ImmunoResearch, Inc.) was added for 1 h and covered with aluminum foil. After five PBS washes, coverslips were mounted with Vectashield (Vector Laboratories, Inc., Burlingame, Calif.) and stored at 4°C in dark. Imaging was performed with a MultiProbe 2010 laser confocal microscope.
Infection of human xenografts in SCIDhu mice. Skin and thymus/liver xenografts were engrafted in male homozygous C.B-17scid/scid mice (32) using human fetal tissues obtained with informed consent according to federal and state regulations. Animals were cared for according to the guidelines of the Animal Welfare Act PL 94-279 and the Stanford University Administrative Panel on Laboratory Animal Care. VZV recombinant viruses propagated in HELF were used to inoculate xenografts. Infectious virus titers were determined for each inoculum at the time of injection. Skin xenografts were harvested after 10 and 18 days, and T-cell xenografts were harvested at 14 and 24 days. Viral replication was assessed by infectious focus assay.
Immunohistochemistry. Formalin-fixed, paraffin-embedded skin sections (5 μm) were deparaffinized, rehydrated, and treated with antigen retrieval reagent (Vector laboratories). VZV protein was detected with human polyclonal anti-VZV immunoglobulin G, biotinylated goat anti-human secondary antibody, and horseradish peroxidase-conjugated streptavidin (Chemicon IHC Select). Signals were developed with Vector VIP (purple) and counterstained with methyl green (Vector Laboratories, Inc., Burlingame, Calif.).
Transmission electron microscopy. Infected skin xenografts were recovered from SCID-hu mice at day 10 after inoculation and immediately fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (PBS), pH 7.0, for 2 h. The specimens were washed twice in PBS, postfixed with 1% osmium tetroxide (Polysciences, Inc., Warrington, PA) in PBS for 1 h, and after two 10-min washes in double distilled water, specimens were treated with 0.25% uranyl acetate (Polysciences, Inc.) overnight. After 24 h, the specimens were washed with water and dehydrated through a graded series of alcohol and propylene oxide washes. Each sample was infiltrated sequentially with 2:1 and 1:1 propylene oxide-EPON (Resolution Performance Products, Houston, TX) for 4 h, incubated overnight with 100% EPON, transferred to fresh EPON, and embedded and polymerized at 60°C for 24 h. Thin sections were collected on copper grids, stained with uranyl acetate and lead citrate, and viewed using a Phillips CM-12 transmission electron microscope. Melanoma cells were infected with 1000 PFU POKA10 and grown on glass coverslips for 48 h. The cells were fixed for 35 min and fixed and stained using the same procedure.
RESULTS
Generation of POKA recombinants with ORF10 mutations. Transfection of the pvFsp73 cosmid, from which ORF10 had been deleted (Fig. 1B), with the other three POKA cosmids, pvSpe14, pvPme2, and pvSpe23, into melanoma cells yielded infectious virus, as expected (10). The resulting virus, POKA10, was propagated in melanoma cells and HELF. Transfection of pvFsp73 cosmids, in which ORF10 had been mutated by substitutions replacing Phe28 with alanine or serine to disrupt the ORF10 activation domain (37), along with the other three intact cosmids, also yielded infectious viruses, designated POKA10-Phe28Ala and POKA10-Phe28Ser (Fig. 1C). POKA10-mHCF was derived from transfections with pvFsp73 in which the putative HCF-1 binding motif of ORF10 was disrupted to evaluate the effects on ORF10 protein-mediated activation of VZV IE gene expression (15). The ORF10 deletion and the presence of the ORF10 targeted mutations were confirmed in the VZV recombinants by sequencing (data not shown).
To verify that ORF10 transcripts were not made in cells infected with POKA10 and to show that ORF10 transcription was maintained in cells infected with POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF, total single-stranded RNAs were recovered from infected cells for Northern hybridization. As expected, no ORF10 transcripts were detected in POKA10-infected cells by Northern blotting with the RNA probe specific for ORF10; ORF10 transcripts were detected in cells infected with the three ORF10 point mutants at levels comparable to those in POKA-infected cells (Fig. 1D).
Growth of POKA ORF10 mutants in vitro. The kinetics of replication of POKA, POKA10, POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF were evaluated for 6 days in melanoma cells. The growth of POKA and the POKA ORF10 mutants was similar. POKA titers were 4.0 x 105 PFU/ml at day 2 and 4.5 x 105 PFU/ml at day 3, which was slightly higher than those of POKA ORF10 mutants, which were 2.0 x 105 PFU/ml at day 2 and 3.0 x 105 PFU/ml at day 3, but the differences were not statistically significant (P > 0.05). All titers were approximately 5.0 x 105 PFU/ml at day 4 and remained equivalent at days 5 and 6 after inoculation. The morphologies of plaques produced by POKA10 and POKA ORF10 point mutants were indistinguishable from POKA in melanoma cells and HELF (data not shown).
Effects of ORF10 deletion or mutations in ORF10 protein on VZV IE gene transcription in vitro. HELF were infected with POKA, POKA10, POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF. Plaques appeared in monolayers infected with all viruses at 3 days postinoculation, and most HELF were depleted and detached from the flask by 6 days. Northern blot analysis of HELF inoculated with the POKA ORF10 deletion and ORF10 point mutation recombinants showed that the kinetics of accumulation of ORF4, ORF61, ORF62, and ORF63 mRNAs were similar to that observed in HELF infected with POKA (Fig. 2); these experiments were repeated four times with reproducible results. No transcripts were detected from mock-infected HELF (data not shown). The accumulation of ORF4, ORF62, and ORF63 transcripts was detected beginning 2 days after infection and continuing through the 6-day time course (Fig. 2: rows 1, 3 and 4); ORF61 mRNA from all samples was detected beginning 1 day after infection (Fig. 2, row 2). The ORF4 probe showed hybridization with the major 1.8-kb and the minor 3.0-kb forms of ORF4 mRNA, as has been described previously in analyses of ORF4-related mRNAs in cells infected with VZV Scott strain (21). The ORF63 probe revealed two RNA species of about 0.9 and 1.8 kb in size, corresponding to ORF63-associated transcripts reported in cells infected with the VZV Ellen strain but not the VZV Scott strain (22). The probe specific for ORF61 revealed one transcript of about 1.5 kb and a fainter 0.7-kb product that may result from abortive transcription or rapid degradation of ORF61 RNAs. Two transcripts were detected with the ORF62 probe; one transcript was about 4.1 kb, which is consistent with the expected size of ORF62 transcripts, and the other was 5.5 kb, which was also observed by with the ORF61 probe (data not shown), which may represent read-through mRNAs originating from the ORF62 start site and terminating at the ORF61 stop site.
Pattern of IE62 distribution in cells infected with POKA and POKA ORF10 mutants in vitro. Since IE62 and ORF10 proteins both function as regulatory and tegument proteins, we investigated whether deletion of ORF10 or disruption of ORF10 activation and putative HCF functional domains influenced the intracellular localization of IE62 in VZV-infected HELF over a time course of 8 to 48 h after infection with POKA or POKA ORF10 mutants (Fig. 3). As was observed in POKA-infected cells, IE62 was exclusively in the nuclei at 8 h in cells infected with POKA10 and POKA ORF10 point mutants; this pattern was also observed at 12 h (data not shown). IE62 was detected in the cytoplasm but remained predominantly nuclear by 20 h in cells infected with POKA and POKA ORF10 mutants. At 24 h, IE62 from all recombinant viruses was observed in more than 50% cells that were examined, and extensive cell fusion had occurred by 36 h postinoculation (data not shown). By 48 h, most cells were infected and had cytopathic changes associated with cytoplasmic accumulation of IE62 in HELF infected with POKA and POKA ORF10 mutants.
Effects of ORF10 deletion or mutations in ORF10 protein on VZV infection of skin xenografts in vivo. To determine the possible roles played by ORF10 protein during VZV infection in vivo, skin xenografts were inoculated with POKA10, POKA10-Phe28Ala, POKA10-Phe28Ser, POKA10-mHCF, and POKA. The inoculum titers were similar, as determined by infectious center assay (Fig. 4). The growth of POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF was equivalent to POKA, with titers of 1 x 104 to 2 x 104 PFU per implant at 10 and 18 days (Fig. 4). In contrast, the replication of POKA10 was decreased and delayed significantly compared to POKA and the ORF10 protein point mutants (P < 0.05). On day 10, infectious virus was recovered from 6 of 8 xenografts inoculated with POKA10 and the mean titer for positive xenografts was 1 x 103 PFU per implant, which was 10-fold less than the other viruses (Fig. 4). On day 18, only 4 of 8 implants inoculated with POKA10 yielded infectious virus; the mean titer of virus in positive xenografts decreased to 1.5 x 102 PFU, which was 100-fold lower than that of POKA10-Phe28Ala, POKA10-Phe28Ser, POKA10-mHCF, and POKA (Fig. 4). The expected sequences were confirmed for POKA and all ORF10 mutants recovered from skin xenografts (data not shown).
The pattern of skin lesions formed in skin xenografts is another measure of the relative pathogenic potential of POKA and VZV mutants. Inoculation with POKA, POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF resulted in cutaneous lesions by day 10. Infection was associated with disruption of the keratinized outer layer of skin and spread of the virus into the dermis (Fig. 5). In contrast, POKA10 infection progressed much more slowly, forming small lesions restricted to the epidermal layer only at day 10 (Fig. 5, panel B1). By 18 days after POKA inoculation, extensive skin lesions were observed at the epidermal surfaces, and large segments of dermal-epidermal junctions that form the basement membrane were destroyed (Fig. 5, panel A2). POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF also generated large necrotic lesions, and the basement membranes were damaged by day 18, as observed with POKA infection (Fig. 5). POKA10 lesions remained small and confined to the epidermis without penetration of the basement membrane at day 18 (Fig. 5, panel B2).
Effects of ORF10 deletion on VZV virion formation in vitro and in skin in vivo. We next examined VZV virion morphogenesis in melanoma cells and skin xenografts infected with POKA or POKA10 by electron microscopy. POKA10-infected melanoma cells showed no changes compared with POKA in the stages of nucleocapsid assembly, capsid budding at the inner nuclear membrane, and intracellular VZV virion maturation, and many complete enveloped particles and incomplete particles lined the outer surfaces of the infected cells (Fig. 6). In contrast, when four electron microscopy (EM) fields from skin xenograft specimens that had been obtained 10 days after infection with either POKA or POKA10 were examined, the total number of virions, including nucleocapsids and cytoplasmic virus particles, in POKA10-infected skin cells was 198 compared to 253 in POKA-infected specimens, representing a reduction of approximately 23%. Skin xenografts infected with POKA10 also had accumulations of unenveloped capsids in the cytoplasm in association with aggregates of tegument-like proteins (Fig. 7, panel B1), which were not found in POKA-infected skin xenografts (Fig. 7, panel A1). Particles within these aggregates were counted in determining the total number of virions in POKA10-infected skin cells. However, in spite of these aberrant capsid accumulation structures, some complete virions with the same morphology as POKA virions (Fig. 7, panel A2) were detected in epidermal cells after infection with POKA10 (Fig. 7, panel B2). In addition, more POKA10 nucleocapsids appeared to be empty compared to POKA (Fig. 7, panels A3 and B3). When equivalent numbers of nucleocapsids in POKA- and POKA10-infected cells were examined, only about 23% of POKA10 nucleocapsids (46/201) appeared to contain DNA compared to 73% of POKA nucleocapsids (142/194). The percentage of mature complete virions in the cytoplasm of POKA10-infected skin cells was also only 25% (26/105) compared to 69% (75/109) in POKA-infected skin cells.
Effects of ORF10 deletion on VZV infection of T-cell xenografts in vivo. To assess POKA10 replication in T cells, we inoculated T-cell xenografts with POKA and POKA10. POKA10 exhibited a growth pattern that was similar to that of POKA in T-cell xenografts (Fig. 8). The inoculum titers were 3.5 x 104 PFU for POKA and 5.6 x 104 PFU for POKA10. The yields of infectious virus from POKA- and POKA10-infected T-cell xenografts were indistinguishable at 14 days; titers were 4.0 x 103 PFU and 4.5 x 103 PFU, respectively. POKA and POKA10 infectious virus yields also declined with similar kinetics, decreasing to approximately 4 x 10 PFU at day 24. The depletion of T cells at this time point was equivalent, based on immunohistochemistry analysis of the POKA- and POKA10-infected T-cell xenografts (data not shown), and similar to the pattern that is observed consistently in T-cell xenografts infected with POKA and other low-passage clinical isolates of VZV (33, 34).
DISCUSSION
These experiments document the importance of ORF10 protein for VZV pathogenesis, showing that this gene product is a virulence determinant in human skin in vivo in the SCIDhu mouse model. Decreased production of infectious virus in the absence of ORF10 protein was associated with impaired formation of VZV virions in epidermal cells in vivo. This effect was evident at several steps in VZV virion assembly, including reduced DNA encapsidation, trapping of DNA-containing capsids in cytoplasmic aggregates, and diminished numbers of complete virions in the cytoplasm. These changes were not evident in cultured cells. The analysis of the POKA10 mutant in the SCIDhu mouse model revealed a differential requirement for ORF10 protein in skin and T-cell xenografts in vivo. The growth of POKA10 was indistinguishable from that of POKA in T cells.
The contribution of ORF10 protein to VZV replication differs significantly from VP16, its HSV-1 homolog. VP16 is a potent transcriptional activator and is required for HSV-1 growth in cell culture (1, 57), whereas ORF10 can be deleted from VOKA (10), and its nonessential role was confirmed by our POKA10 experiments. Our VZV mutants, POKA10-Phe28Ala and POKA10-Phe28Ser, were designed to disrupt the activation domain that has been shown to mediate the transactivating effects of ORF10 protein on the ORF62 promoter in transient expression systems (35). Mutations that inactivated the corresponding transcriptional activation function of VP16 reduced the levels of HSV-1 IE gene expression and viral replication substantially in vitro and in heterologous murine systems in vivo. However, these VP16 mutants could be propagated in tissue culture to some extent, and most were infectious in vivo, demonstrating that the activation function of VP16 was not absolutely essential for HSV-1 replication (2, 50, 51, 52, 55, 56). When introduced into the VZV genome, mutations of the functionally related acidic activation domain of ORF10 protein had no effect on transcription of ORF62 or putative VZV regulatory genes that are downstream targets of IE62, including ORF4, ORF10, ORF61, and ORF63, in cultured cells. VZV virion morphogenesis was also intact in POKA10-Phe28Ala- and POKA10-Phe28Ser-infected melanoma cells. The failure of these mutations in the acidic activation domain to affect VZV gene transcription and replication in vitro was expected, given observations with POKA10 and VOKA from which ORF10 was deleted (10). However, altering the activation domain of ORF10 protein also had no impact on VZV virulence in human epidermal and dermal cells in vivo, compared to POKA. Thus, while the capacity of VP16 to transactivate HSV-1 IE genes cannot be fully compensated by any other viral transactivating proteins, even in highly permissive cultured cells, VZV does not require the activation function of ORF10 protein. In VZV, the major IE transactivator, IE62, has the capacity to induce its own transcription independently and regulates transcription of all other VZV genes that have been evaluated (14, 19, 20, 40, 45, 46). Therefore, the autoregulatory function of IE62 and its induction of other IE proteins appears to be sufficient in the absence of an intact activation domain in ORF10 protein in vitro and, most importantly, in differentiated human skin cells in vivo. Since deleting ORF10 had no effect on VZV T-cell infection, this activation function can also be presumed to be dispensable for T-cell tropism.
The cellular factor HCF-1 is required for VP16-mediated activation and the regulated transcription of HSV-1 IE genes (24, 27, 58). Similarly, HCF-1 is also critical for induction of the IE62 promoter by IE62 and ORF10 protein in reporter constructs or VZV-infected cells (36, 41). However, disrupting the conserved DHPY motif that is presumed to mediate ORF10 protein binding to HCF-1, in the context of the viral genome in our POKA10-mHCF mutant, did not alter VZV replication or transcription of regulatory genes in vitro. In contrast, mutations that disrupt the capacity of VP16 to form complexes with HCF-1 and Oct-1 and the TAATGARAT element reduce HSV growth (2). Altering the putative HCF-1 binding motif did not affect the pathogenesis of VZV skin infection, as determined by yields of infectious virus and the histopathologic appearance of cutaneous lesions. Our observations suggest that, while ORF10 protein has a motif that is closely related to the HCF-1 binding domain in VP16 and the ORF10/HCF-1 complex enhances ORF62 expression, HCF-1 interaction with this putative binding site in ORF10 is incidental for VZV infection in vitro and in skin in vivo. As observed with the ORF10 activation domain mutants, VZV IE62, as a potent single viral transactivator, has the potential to rescue the loss of the ORF10/HCF-1 interaction in VZV-infected epidermal and dermal cells. In addition, the fact that removing the complete ORF10 gene from POKA did not affect VZV T-cell tropism suggests that viral gene regulatory effects resulting from the formation of Oct-1/ORF10/HCF-1 complexes are redundant for VZV replication in T cells.
Although the targeted mutations in functional domains of ORF10 protein did not alter VZV infection in skin xenografts, the complete deletion of ORF10 was associated with substantially impaired replication and cell-cell spread of VZV in vivo. VZV plaque formation can be fully preserved in cultured cells infected with VZV mutants even when virion assembly is severely defective, as we demonstrated with our VZV ORF47 kinase-null mutants (6). However, the EM analysis of melanoma cells infected with POKA and POKA10 revealed no consequences of ORF10 deletion on VZV capsid formation or subsequent steps in virion maturation in vitro. In contrast, examining these processes in skin cells infected with POKA10 in vivo demonstrated significant deficiencies in total virus particles produced, in the proportion of dense capsids in nuclei, and in relative numbers of complete virus particles in the cytoplasm. When VP16 and pseudorabies (PrV) UL48 protein, which is an ORF10 homologue, were disrupted or absent, unenveloped capsids also accumulated in the cytoplasm of infected cells (16, 30, 39). Interestingly, comparing POKA10 with the corresponding HSV-1 and PrV mutants suggests a hierarchy in the requirement for this gene product in the alphaherpesviruses (16, 30, 39). The complete absence of VP16 in HSV-1 prevents the production of infectious progeny virus, indicating that as a tegument protein, VP16 plays a more important role than ORF10 protein in virion assembly. Deleting PrV UL48 yields replication-competent mutants, but these PrV mutants have impaired growth and marked deficiencies in virion morphogenesis in vitro (16, 30). Deficiencies in virion formation were observed only when VZV lacking ORF10 was challenged to replicate in skin cells within the intact tissue microenvironment in vivo. Although some complete virus particles were observed, POKA10 infection of skin cells was associated with the appearance of many capsid-less particles and cytoplasmic aggregates of apparently tegumented capsids that were similar to those observed with PrV glycoprotein deletion mutants (7, 8, 30). Since ORF10 protein is a tegument component, tegumentation of capsids might be altered, but it is not clear why viral DNA encapsidation would be impaired in the absence of ORF10 protein. However, the EM analysis of POKA10 in skin cells suggests that ORF10 protein facilitates DNA insertion into capsids. Investigations of HSV-1 VP16 mutants have also shown inefficient DNA encapsidation for unexplained reasons (39, 57). Overall, defective POKA10 virion morphogenesis appears to account for low yields of infectious virus from skin xenografts and the observation that cutaneous lesions, which reflect cell-cell spread of VZV, were much smaller.
In summary, these experiments show that ORF10 protein is required for efficient viral replication, virion formation, and cell-cell spread in human skin in vivo. However, these functions do not appear to depend on preservation of the acidic activation domain or the putative HCF-1 binding motif that facilitate IE62 activation. The requirement for ORF10 protein in skin appears to be related to a role in virion assembly. Given these observations, it will be of particular interest to determine whether these ORF10 protein domains are important for VZV neurotropism. Whereas ORF10 protein was not required for replication in T cells, VZV ORF10 protein is a virulence determinant in skin in vivo.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI053846) and the Astellas USA Foundation.
We thank Nafisa Ghori and Anne Schaap for valuable technical assistance and Chia-Chi Ku for helpful discussions.
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