Abstract
The varicella-zoster virus (VZV) genome has unique long (UL) and unique short (US) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. The immediate-early 62 (IE62) protein, encoded by open reading frame 62 (ORF62) and ORF71 in these repeats, is the major VZV transactivating protein. Mutational analyses were done with VZV cosmids generated from parent Oka (pOka), a low-passage clinical isolate, and repair experiments were done with ORF62 from pOka and vaccine Oka (vOka), which is derived from pOka. Transfections using VZV cosmids from which ORF62, ORF71, or the ORF62/71 gene pair was deleted showed that VZV replication required at least one copy of ORF62. The insertion of ORF62 from pOka or vOka into a nonnative site in US allowed VZV replication in cell culture in vitro, although the plaque size and yields of infectious virus were decreased. Targeted mutations in binding sites reported to affect interaction with IE4 protein and a putative ORF9 protein binding site were not lethal. Single deletions of ORF62 or ORF71 from cosmids permitted recovery of infectious virus, but recombination events repaired the defective repeat region in some progeny viruses, as verified by PCR and Southern hybridization. VZV infectivity in skin xenografts in the SCID-hu model required ORF62 expression; mixtures of single-copy recombinant OkaΔ62 (rOkaΔ62) or rOkaΔ71 and repaired rOka generated by recombination of the single-copy deletion mutants were detected in some skin implants. Although insertion of ORF62 into the nonnative site permitted replication in cell culture, ORF62 expression from its native site was necessary for cell-cell spread in differentiated human skin tissues in vivo.
Varicella-zoster virus (VZV) belongs to the alphaherpesvirus subfamily of the Herpesviridae. VZV is the causative agent of varicella, which is characterized by cell-associated viremia and a cutaneous vesicular rash (4). VZV establishes latency in cells within sensory ganglia during primary infection. VZV reactivation from latency results in herpes zoster, a localized skin rash in the distribution of nerves from the affected ganglion. VZV is the first human herpesvirus for which a vaccine has been developed to prevent primary infection (58). This live attenuated varicella vaccine was created by serial passage of a wild-type clinical isolate, the parent Oka (pOka) strain, in guinea pig embryo cells and human fibroblasts to generate the varicella vaccine virus (vOka).
The VZV genome consists of approximately 125 kb and has at least 70 unique open reading frames (ORFs) (52). As is characteristic of herpesviruses, the double-stranded DNA genome has unique long (UL) and unique short (US) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. Three duplicated genes, ORF62/71, ORF63/70, and ORF64/69, are located in repeats at each end of the US segment. The two VZV origins of replication, designated OriS, are also located in these repeat regions. The immediate-early 62 (IE62) protein, encoded by ORF62 and ORF71, is the major VZV transactivating protein and is the homolog of herpes simplex virus type 1 (HSV-1) ICP4. The IE62 protein induces transcription of all viral genes that have thus far been evaluated (19, 25, 27, 40, 48). The IE63 protein, encoded by ORF63 and ORF70, is also an important VZV regulatory protein which modulates transcription from several genes and has some homology to HSV-1 ICP22 (25).
We have used overlapping VZV cosmids to introduce mutations into the viral genome in order to examine the roles of particular ORFs or nucleotide sequences within VZV genes or their promoters in replication in cell culture in vitro (20, 30, 33, 34, 54). In analyses of ORF63/70 and ORF64/69 gene pairs, one copy of ORF63 was necessary and sufficient for VZV replication, whether it was located at the native site, or introduced into a nonnative site between ORF65 and ORF66 in the US region (54). In contrast, deleting both copies of the ORF64/69 gene pair had no effect on production of infectious virus (54).
When genetic changes were not lethal in vitro, as in the case of the single-copy ORF63 and the dual ORF64/69 deletion mutants, we have evaluated VZV recombinants for infectivity in our SCID-hu mouse model of VZV pathogenesis in vivo (20, 36, 37, 38, 53). Infecting human skin or thymus-liver xenografts in SCID mice permits an assessment of VZV interactions with differentiated human cells located within the intact tissue microenvironment. VZV replication is not influenced by the host immune response, because the animals are immunodeficient. We have found that experiments in the SCID-hu model are necessary to define the contributions of VZV gene products to pathogenesis, because some VZV proteins, such as glycoprotein I and the ORF47 protein kinase, are dispensable in cell culture but are essential for VZV replication in skin and T cells in vivo (37, 38). Evaluation of the effects of mutating the ORF63/70 and ORF64/69 gene pairs showed that the single-copy ORF63 and the dual ORF64/69 deletion mutants retained typical VZV infectivity in skin xenografts (54).
The third repeated gene pair, ORF62/71, is of particular interest for mutational analysis because it encodes the IE62 protein, which is the primary viral transactivating protein and a component of the VZV virion tegument. The IE62 protein exhibits functional characteristics resembling those of HSV ICP4, which it complements, and has some similarities to HSV VP16 (19, 27, 28, 40, 44, 49, 59). IE62 protein substitutes for ICP4 function in transient-expression assays (14, 15) and in HSV mutants from which ICP4 has been deleted (10). IE62 protein differs from ICP4 in being a relatively abundant tegument protein, whereas much less ICP4 is associated with HSV-1 virions (63). The IE62 protein (1,310 amino acids [aa]) is the largest gene product of the duplicated genes; it contains an N-terminal activation domain, a DNA binding domain, a nuclear localization signal, and regions that bind viral and cellular proteins (6, 26, 29, 32, 60, 62). By analogy with its homologs, IE62 protein consists of five regions (region I, aa 1 to 460; region II, aa 460 to 630; region III, aa 630 to 750; region IV, aa 750 to 1170; and region V, aa 1170 to 1310). Region I contains the transcriptional activation domain (aa 1 to 90), region II is involved in DNA binding and dimerization, and region III has a nuclear localization signal. Phosphorylation of IE62 protein, mediated by the ORF47 protein kinase in vitro, appears to activate IE62 protein and induce its nuclear translocation (43, 44). Negative regulation of IE62 protein, associated with nuclear exclusion at late times after infection, is achieved by ORF66, the second VZV serine/threonine kinase (28). Using highly purified recombinant IE62 protein, the Ruyechan and Hay laboratories have shown that it binds to IE4 and ORF9 proteins (29, 55, 56).
The objective of these experiments was to examine the contributions of ORF62/71 to VZV replication in vitro and in skin xenografts in vivo. Mutational analyses were done with VZV cosmids generated from pOka, a low-passage clinical isolate (44a), and repair experiments were done with ORF62 from both pOka and vOka, because it has been suggested that sequence differences in ORF62 may be related to the attenuation of vOka (16). Targeted mutations were also made in IE4 protein binding sites that were mapped in vitro (55, 56), and in a putative ORF9 protein binding site (W. Ruyechan and J. Hay, unpublished observations). These experiments demonstrated that VZV replication required at least one copy of ORF62. Further, while insertion of ORF62 into the nonnative site permitted replication in cell culture, ORF62 expression from its native site was essential for cell-cell spread in differentiated human skin tissues in vivo.
MATERIALS AND METHODS
Cosmids and plasmids.
Four overlapping fragments of genomic DNA from pOka were introduced into SuperCos 1 cosmid vectors (Stratagene, La Jolla, Calif.) by using methods reported for vOka (22, 30). Deletion of an AvrII site at SuperCos 1 nucleotide (nt) 3359 produced a unique AvrII site at VZV nt 112956 (Fig. 1).
FIG. 1.
Schema of cosmid mutagenesis. The upper section depicts the VZV IRS -US -TRS region of the genome containing the coding regions of ORF62 to ORF71, and the unique AvrII site in the US region is indicated. The arrows indicate the orientations of the genes. The nucleotide numbers of the start and stop sites of the ORFs that were deleted (ORF62 and ORF71), as well as the nucleotide numbers of the relevant adjacent ORFs, are given. The designations of the cosmids that were generated are given on the left. Hatched boxes indicate the ORFs that were deleted from the cosmid. The insertions and orientations of vOka ORF62 and pOka ORF62 at the nonnative AvrII site are shown.
Deletions of ORF62, ORF71, and ORF62/71.
ORF62 is carried on VZV nt 105164 to 109096, with the gene being in the complementary orientation, located in pvSpe23. Primers were designed to amplify VZV genomic DNA flanking ORF62 by using Elongase enzyme mix (Invitrogen, Inc., Carlsbad, Calif.). These PCR products were then ligated to delete ORF62 before being reintroduced into the cosmid. The 5′ primer 62N was designed to bind to nt 100583 to 100602 and thus to contain an NheI restriction site (nt 100586) unique in pvSpe23. Primer sequences are shown in Table 1. The 3′ primer 71SA was designed to bind to nt 105144 to 105166, with the incorporation of an EcoRI site. PCR with these two primers resulted in a 4,584-bp product. This product was digested with NheI and EcoRI and ligated into the pIRES2-EGFP vector (Clontech, Palo Alto, Calif.), which had been digested with the same enzymes. The 5′ primer 71SS was designed to bind to nt 109107 to 109132, with the incorporation of an EcoRI site. The 3′ primer 62A was designed to bind to nt 112956 to 112975 and thus to contain an AvrII restriction site (nt 112956) unique in pvSpe23. PCR with these two primers resulted in a 3,869-bp product. This product was cloned into pCR4-TOPO (Invitrogen, Inc.) and digested out with EcoRI before ligation into the single EcoRI site in the pIRES2-EGFP vector, which already contained the first PCR product. Colonies were screened for correct orientation of the insert. The two PCR products ligated together by a single EcoRI site were then digested out by using the unique NheI and AvrII sites and ligated into the 25.5-kb pvSpe23 fragment produced by digestion with NheI and AvrII. Colonies were screened with a range of restriction enzymes to ensure correct orientation of the fragment, yielding pvSpe23ΔORF62.
TABLE 1.
Summary of primers used for cloning and PCR
Primer | Sequence (5′ to 3′)a | nt in VZVb |
---|---|---|
62N | AACGCTAGCCCATGTGCATG | 100583-100602 |
71SA | TGACGGAATTCCCTCCTTTTCTCc | 105144-105166, 124937-124959 |
71SS | TGAATTCGACGTACCCGAGTTTTCC | 109107-109132, 120971-120996 |
62A | TGGATTTGATTGTTCCTAGG | 112956-112975 |
MARV-L | GAAAATTTTCCGGTTTAAGGCGTTTC | NAc |
MARV-U | TTTGCGTTTGCGTGTATGGA | 113454-113473 |
S245AF | CCCGCTCAGGGAAAGGCCCCGAAGAAAAAG | 108350-108379 |
S245AR | CTTTTTCTTCGGGGCCTTTCCCTGAGCGGG | 108350-108379 |
T250AF | GCCCGAAGAAAAAGGCTTTGAAGGTTAAGG | 108334-108363 |
T250AR | CCTTAACCTTCAAAGCCTTTTTCTTCGGGC | 108334-108363 |
A28PF | ATGGACCTGTTGGACCCGGCCGCCGCGGCC | 109001-109030 |
A28PR | GGCCGCGGCGGCCGGGTCCAACAGGTCCAT | 109001-109030 |
Primer 1 | CGCTCACGAGAAAAGGAGGG | 105137-105156, 124947-124966 |
Primer 2 | CCGGAACGTCACCACTTCTA | 117499-117518 |
Primer 3 | CCGCGAGCGCAACCAAATAA | 112661-112680 |
Primer 4 | AATGACGGCTCAGAAAAACCATCG | 104171-104194 |
Primer 5 | CGGTCGGTGGCGCTGTATG | 124466-124484 |
Primer 6 | GCCGGGCGACATTTCAACTG | 113255-113274 |
Underlined nucleotides are mutated sequences.
Nucleotide number in parent Oka strain.
NA, not applicable because it anneals to vector sequence.
ORF71 is carried on nt 121007 to 124939. Due to the repeat regions of VZV, the 5′ primer 71SA also binds to VZV nt 124937 to 124959 with the incorporation of an EcoRI site. The 3′ primer MARV-L was designed to bind to the cosmid vector. PCR with these two primers yielded a 443-bp product which contained an AscI site in the cosmid vector. This product was ligated into pCR4-TOPO and digested out with NotI and EcoRI before ligation into the pFastBac1 plasmid (Invitrogen, Inc.), which was previously digested with NotI and EcoRI. The 5′ primer MARV-U was designed to bind to nt 113454 to 113473. Due to the repeat regions, the 3′ primer 71SS binds to nt 120971 to 120996 and to nt 109107 to 109132, with the incorporation of an EcoRI site. PCR with MARV-U and 71SS results in a 7,543-bp product that contains an SgrAI restriction site (nt 117458) unique in pvSpe23. This product was digested with EcoRI, and the 3.9-kb fragment was cloned into the unique EcoRI site in the pFastBac1 plasmid containing the 443-bp product. The two PCR products ligated together by a single EcoRI site were digested out by using the unique SgrAI and AscI sites and ligated into the 29.5-kb pvSpe23 fragment produced by digestion with SgrAI and partial digestion with AscI. Colonies were screened with restriction enzymes to verify construction of pvSpe23ΔORF71.
To delete both ORF62 and ORF71, the cosmid containing the single deletion of ORF71 was digested with NheI and AvrII, and the 17.8-kb fragment was ligated with the 8.5-kb ORF62 deletion fragment from the pIRES2-EGFP vector. Colonies were screened with restriction enzymes to identify pvSpe23Δ ORF62/71.
Insertion of a single copy of ORF62/71 into pvSpe23.
The vOka and pOka cosmids containing ORF71 (pvSpe21 and pvSpe23) were digested with HpaI and EcoRI, releasing a 5,817-bp fragment from each cosmid, which contained 40 bp of the SuperCos 1 cosmid, the ORF71 coding sequence, and 1,658 bp of upstream sequence. This sequence represents the complete intragenic region between ORF70 and ORF71 (1,588 bp) and 70 bp of the ORF70 coding region and is assumed to contain the ORF71 promoter. These fragments were ligated into the pIRES2-EGFP vector, which had been digested with EcoRI and SmaI. The 40 bp of SuperCos 1 sequence contains a unique AscI site which, if not removed, would affect the AscI digestion step prior to transfection of melanoma cells. The AscI site was removed by digesting the vectors with EcoRI and AscI, blunt ending with T4 DNA polymerase, and religating. These vectors, pIRES2-vOka62 and pIRES2-pOka62, were then digested with NheI and AvrII, and the 6-kb fragment was ligated into the unique AvrII site in the pvSpe23ΔORF62/71 cosmid; these steps yielded pvSpe23ΔORF62/71(vORF62@Avr) and pvSpe23ΔORF62/71(pORF62@Avr).The plasmid pIRES2-pOka62, containing the 6-kb fragment of the pOka ORF62 gene, was used for introduction of point mutations into ORF62; mutations were made with the QuikChange XL site-directed mutagenesis kit (Stratagene, Inc.). Primer sets were designed to disrupt the reported ORF4 binding site in IE62 by introducing mutation S245A or T250A (29, 55, 56). The primers were S245AF and S245AR or T250AF and T250AR, respectively. The putative ORF9 protein binding site has been localized to the N terminus of IE62 protein (56; Ruyechan and Hay, unpublished observations). Primers A28PF and A28PRwere used to introduce the mutation A28P into this region.
Melanoma cell transfection and virus isolation.
Cosmid DNA was prepared and purified as described previously (30, 54). Cosmids were digested with AscI and mixed in water to a final concentration of 100 ng of pvFsp73, pvSpe14, or pvPme2 per μl or 50 ng of pvSpe23 per μ l (30). Transfections were done with human melanoma cells, using 30 μ l of the cosmid mix in 31.5 μ l of 2 M CaCl2 in water and HEPES-buffered saline. Transfected cells were kept in tissue culture medium at 37°C for 3 days, trypsinized, and transferred to a 75-cm2 flask; plaques appeared at 6 to 10 days after transfection with intact cosmids. Cells transfected with mutant cosmids were passed at a 1:3 ratio every 3 to 4 days. Infectious virus was propagated in melanoma cells, and growth kinetics were assessed as described previously (36). A doxycycline-inducible gE-expressing melanoma cell line, designated the Met-gE cell line, was established as described in a report on experiments to investigate effects of deleting VZV ORF68, encoding gE (33).
PCR and sequencing.
Cosmid DNA was purified with Qiagen columns, and recombinant virus DNA was recovered from infected cells by using the DNeasy tissue kit (Qiagen Inc.). PCR was performed with Elongase enzyme mix (Invitrogen, Inc.) or Platinum Taq DNA polymerase High Fidelity (Invitrogen, Inc.) together with a PCR cosolvent, PCRx Enhancer Solution (Invitrogen, Inc.). Primers 1 and 2 were used to assess deletion of ORF71, primers 3 and 4 were used to assess deletion of ORF62, and primers 5 and 6 were used to analyze the insertion at the unique AvrII site (Table 1).
Virus-infected-cell DNA was amplified and isolated by using the Qiagen gel extraction kit, or PCR products were cloned directly into the pCR4-TOPO cloning vector (Invitrogen, Inc.). Sequence analysis was carried out at the Stanford University Protein and Nucleic Acid Facility. VZV genomic DNA containing ORF62 and ORF71 from the vOka pvSpe21 cosmid had been previously subcloned into pLITMUS vectors to generate ORF63 and ORF70 deletion mutants (54). After digestion with Nhe I and Avr II, a 12.3-kb fragment of pOka pvSpe23 DNA containing ORF62 was subcloned into the unique NheI site of pIRES2-EGFP. ORF71 was subcloned from pvSpe23 as described for the repaired mutants. ORF62 and ORF71 were sequenced, in both directions, directly from these subclones, using a total of 18 oligonucleotide primers.
Southern hybridization.
A probe to assess deletion of ORF62 (300 bp) was designed to anneal just before the unique AvrII site in the US . The 62 probe was prepared by PCR with primers 5′-ACGTTATATATCCCAAGGCA-3′ and 5′-GGATTTCTATGGCCGGACAA-3′. A probe to assess deletion of ORF71 (517 bp) was designed to anneal in the ORF68 coding region just after the unique EcoRI site in the US region. The 71 probe was prepared with primers 5′-CATTTACCTCGCCACATTTA-3′ and 5′-GGAAGGCCAGCGTAATACAT-3′. After the complete digestion of DNA with EcoRI, a DNA sample was loaded on an agarose gel, transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.), and immobilized by using Stratalinker (Stratagene, Inc.), Southern hybridization was done at 55°C with probes labeled with alkaline phosphatase (AlkPhos direct labeling kit; Amersham Pharmacia Biotech, Inc.), with chemiluminescence detection.
Western blotting.
Melanoma cells or skin xenograft tissue extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 4 to 15% gradient gel (Bio-Rad Laboratories, Hercules, Calif.) and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, Mass.); rabbit anti-ORF62 antibody and anti-ORF4 antibody were kindly provided by Paul R. Kinchington, University of Pittsburgh, Pittsburgh, Pa. Rabbit anti-ORF47 antibody was prepared as described elsewhere (4a). Rabbit anti-gE antibody was prepared by cloning ORF68 (gE) into pGEX-2T vector (Amersham Pharmacia Biotech, Inc.), expressing and purifying the glutathione S-transferase fusion protein (21), and using purified gE for rabbit immunization. Sera were collected before and after immunization and stored at −20°C. VZV proteins were detected with each primary rabbit antibody and secondary goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate (36).
Infection of SCID-hu skin xenografts.
Skin implants were made in homozygous C.B-17 scid/scid mice (35), using human fetal tissues obtained with informed consent according to federal and state regulations. Animal use was in accordance with the Animal Welfare Act and approved by the Stanford University Administrative Panel on Laboratory Animal Care. Eight weeks after implantation, mice were anesthetized and bilateral skin implants were exposed for inoculation with the test virus, which had been passed three times in primary human lung (HEL) cells. Control implants were inoculated with uninfected HEL cells. Implants were harvested at 12, 21, and 28 days postinoculation. Viral titers were measured by infectious focus assay (36). DNA from each implant was prepared by using the DNeasy tissue kit (Qiagen, Inc.). PCR for ORF71 and ORF62 was done with the primers described above. Primer sets for ORF4 were 5′-GACGTCCAAGTCCAATCA-3′ and 5′-CATGCGACGGGATGGTATGA-3′.
RESULTS
Effects of ORF62, ORF71, and dual ORF62/71 deletions.
Transfection of the three intact cosmids, pvFsp73, pvSpe14, and pvPme2, and intact pvSpe23 resulted in recovery of recombinant Oka (rOka), with 75% of melanoma cells exhibiting cytopathic effects (CPE) within 10 days (Table 2). Parallel transfections in which pvSpe23ΔORF62or pvSpe23ΔORF71 was substituted for pvSpe23 showed 75% CPE at 17 or 10 days, respectively. Two independently derived VZV recombinants were generated, using separately constructed pvSpe23Δ ORF62 or pvSpe23ΔORF71 mutant cosmids, and were designated rOkaΔORF62 and rOkaΔORF71. The plaque morphology of rOkaΔORF71 was indistinguishable from that of rOka; rOkaΔORF62 exhibited a smaller plaque phenotype after the initial transfection, but the plaque size became equivalent to that of rOka after passage. In contrast, transfection of pvSpe23ΔORF62/71 and the intact cosmids yielded no infectious virus in three separate experiments. The presence of the expected mutation in independently derived pvSpe23Δ ORF62/71 cosmids was confirmed by PCR, as described below (Fig. 2A and B, lanes 5). Cells were passed for 28 to 30 days to ensure that a virus with a slow-growth phenotype was not missed. These observations suggest that one copy of ORF62 or ORF71 is required for VZV replication in cell culture.
TABLE 2.
Recovery of infections virus after transfections of cosmids with deletions of ORF62, ORF71, or ORF62/71 and with insertion of ORF62 into a nonnative site in the US region
Cosmid cotransfected with pvFsp73, pvSpe14, and pvPme2 | CPEa | Days after transfection | Designation of recovered virus |
---|---|---|---|
pvSpe23 | +++ | 6-10 | rOka |
pvSpe23ΔORF62 | +++ | 13-17 | rOkaΔORF62 |
pvSpe23ΔORF71 | +++ | 6-10 | rOkaΔORF71 |
pvSpe23ΔORF62/71 | − | 28-30b | NAc |
pvSpe23ΔORF62/71(vORF62@Avr) | ++ | 9-13 | rOkaΔORF62/71(vORF62-R) |
pvSpe23ΔORF62/71(pORF62@Avr) | ++ | 9-13 | rOkaΔORF62/71(pORF62-R) |
Proportions of cells exhibiting altered morphology: +, 25 to 50%; ++, 50 to 75%; +++ 75 to 90%, −, no CPE.
Cells were split every 3 to 4 days to ensure that a slow-growth phenotype would not be missed.
NA, not applicable (no virus was recovered from multiple transfections).
FIG. 2.
PCR analysis of rOkaΔORF62, rOkaΔORF71, rOkaΔORF62/71(vORF62-R), and rOkaΔORF62/71(pORF62-R). PCR analysis was done with cosmid DNA (lanes 2 to 7) or DNA isolated from infected cells (lanes 8 to 12) as described in Materials and Methods. (A to C) PCR products obtained with the ORF71 primers (A), the ORF62 primers (B), or primers for insertion of the ORF62 gene at the AvrII site (C). The specimens tested are as follows: lanes 1 and 13, 1-kb DNA ladder; lane 2, pvSpe23; lane 3, pvSpe23 Δ ORF71; lane 4, pvSpe23ΔORF62; lane 5, pvSpe23ΔORF62/71; lane 6, pvSpe23ΔORF62/71(vORF62@Avr); lane 7, pvSpe23ΔORF62/71(pORF62@Avr); lane 8, rOka; lane 9, rOkaΔORF71; lane 10, rOkaΔORF62; lane 11, rOkaΔORF62/71(vORF62-R); lane 12, rOkaΔORF62/71(pORF62-R). (D) Expected PCR products. Lines 1 to 3, predicted primer annealing sites of primers 1, 2, 3, and 4 with the intact and deleted cosmids. The predicted fragment sizes for ORF71 PCR are also indicated. Lines 4 to 8, possible mechanism by which the 3,515-bp product in PCR with ORF71 primers and pvSpe23ΔORF62 was generated.
PCR analysis of mutant cosmids and rOkaΔORF62- and rOkaΔORF71-infected cell DNA.
As expected, PCR with primers 1 and 2 (Table 1) to amplify ORF71 generated a 7,468-bp product from pvSpe23 and 3,515-bp products from pvSpe23ΔORF71 and pvSpe23ΔORF62/71 (Fig. 2A). However, PCR of pvSpe23ΔORF62 resulted in a 3,515-bp product as well as the 7,468-bp product. A minor, nonspecific 4,200-bp band was observed in all lanes. The 3,515-bp band produced by PCR of pvSpe23ΔORF62 can be explained as illustrated in Fig. 2D. Primers 1 and 2 were designed to amplify regions containing ORF71, independent of the two possible orientations of the inverted repeats. Therefore, primer 1 anneals not only at nt 124947 to 124966 but also at nt 105137 to 105156. In the first few PCR cycles, annealing of primer 1 at nt 105137 to 105156 generates a short fragment containing ORF63 (and ORF64) with ORF62 deleted. After the denaturation step, this fragment could anneal to the fragment generated from primer 2. After these two fragments anneal, in the following elongation step, the short 3,515-bp product is generated. Once this short fragment is generated in the early PCR cycles, it becomes the predominant product. A control experiment using mixtures of pvSpe23 and pvSpe23ΔORF62 in different ratios showed that this 3,515-bp band was 100- to 1,000-fold more likely to be generated than the 7,468-bp band (data not shown). This result indicates that the 3,515-bp band in the PCR of pvSpe23Δ ORF62 (Fig. 2A, lane 4) was a result of the PCR conditions. A similar observation was made when primers 1 and 3 were used to amplify ORF62 (data not shown). PCR with primer 3 and primer 4, which anneal within the UL region, yielded the expected bands. Taken together, the PCR analyses indicated that the mutant cosmids, pvSpe23ΔORF62 and pvSpe23ΔORF71, were constructed as designed.
In contrast, PCR using DNA from cells infected with rOka ΔORF62, and rOkaΔORF71 yielded some unexpected results. PCR of rOka yielded single products of the expected sizes with the ORF71 and ORF62 primers (Fig. 2A and B, lanes 8). However, PCR of rOkaΔORF71 resulted in two products with each primer set (Fig. 2A and B, lanes 9). The 3,515-bp product generated with the ORF71 primers was expected for ORF71 deletion, but the 7,468-bp product was as expected for intact rOka (Fig. 2A, lane 9). With the ORF62 primers, the expected 8,510-bp band was observed, but a 4,557-bp band was also observed, suggesting recombination during transfection with repair of the deletion. PCR of rOkaΔORF62 with ORF71 primers also yielded both the faint 7,468-bp band and a 3,515-bp band, which is consistent with recombination; the 3,515-bp product could be due in part to PCR conditions, as was observed with pvSpe23ΔORF62 (Fig. 2A, lane 10). The data indicate that recombination occurred during the generation of rOkaΔORF71, and probably of rOkaΔORF62, from cosmids, as we reported with rOkaΔORF63 and rOkaΔORF64 mutants (54).
Southern blot analysis of rOkaΔORF71 and rOkaΔORF62.
Southern hybridization of cosmid DNA gave the expected single band (Fig. 3A, lanes 1 to 3 and 7 to 9), confirming the PCR data. Since PCR analyses suggested recombination, the same infected-cell DNA samples were digested with EcoRI and analyzed by Southern hybridization. Two bands were observed when rOka DNA was hybridized with the ORF71 and the ORF62 probes (Fig. 3A, lanes 4 and 10). This pattern is expected in VZV-infected cells because the VZV genome is a mixture of two isoforms with inversion of IRS -US -TRS (Fig. 3B, lines 1 and 2) (12, 13, 57) and shows that generating VZV genomes from cosmids results in creation of both isoforms, even though the pvSpe23 cosmid has the IRS -US -TRS sequence in a single orientation. The ORF71 probe should hybridize to a 3.8-kb band in rOkaΔORF71 DNA (Fig. 3B, lines 3 and 4), but hybridization results were the same as with rOka DNA. No 3.8-kb band was detected (Fig. 3A, lane 5), suggesting that intact rOka virus rather than rOkaΔORF71 was the primary recombinant virus generated. PCR showed that some DNA from which ORF71 had been deleted was also present in rOkaΔORF71-infected cells.
FIG. 3.
Southern analysis of rOkaΔORF62 and rOkaΔORF71 deletion viruses. (A) Southern hybridization of cosmids and viruses with the ORF71probe (lanes 1 to 6) and the ORF62 probe (lanes 7 to 12). The specimens tested are as follows: lanes 1 and 7, pvSpe23; lanes 2 and 8. pvSpe23ΔORF71; lanes 3 and 9, pvSpe23ΔORF62; lanes 4 and 10, rOka; lanes 5 and 11, rOkaΔORF71; lanes 6 and 12, rOkaΔORF62. (B) EcoRI mapping of the inverted region of the VZV genome. Underlined EcoRI indicates the introduced EcoRI site created by deletion of the ORF62 or ORF71 gene. Boxes indicate IRs and TRs. Arrows indicate ORF62 or ORF71 and the orientation of the gene. The ORF62 probe (closed box) was designed to anneal just before the unique AvrII site in the US region, including part of the ORF65 coding region. The ORF71 probe (open box) was designed to anneal in the ORF68 coding region, just after the unique EcoRI site in the US region. The two predicted isoforms of the intact rOka and mutant genomes are illustrated. Predicted band sizes for hybridization of each probe are shown on the right.
The analysis of rOkaΔORF62 DNA also showed that intact rOka was present (Fig. 3A, lane 6). The ORF71 probe should hybridize to 13.0- and 8.0-kb bands in rOkaΔORF62, as in rOka DNA (Fig. 3B, lines 5 and 6). However, a third 3.8-kb band was detected which should only appear in hybridizations with rOkaΔORF71. The ORF62 probe should hybridize only to the 8.0-kb band in rOkaΔORF62, because of the EcoRI site introduced in deleting ORF62 (Fig. 3B, lines 5 and 6). However, hybridization detected not only the 8.0-kb band but also 16.8- and 13.0-kb bands, as with rOka (Fig. 3A, lane 12). The analysis of rOkaΔORF62 suggested recombination, accounting for the 3.8-kb band, yielding a mixture of intact rOka and rOkaΔORF62 (Fig. 3B, lines 7 and 8). Southern blot analysis of recombinant viruses that had been generated in two independent transfections with separately derived cosmids gave the same patterns (data not shown), indicating that the result was not due to contamination during transfection or cosmid preparation.
Replication of rOkaΔORF71 and rOkaΔORF62 in vitro.
When growth kinetics were evaluated over 6 days, no significant difference in viral titers was observed for rOka, rOkaΔ ORF71, or rOkaΔORF62 (see Fig. 5A). However, the kinetic analysis must be interpreted in the context of the PCR and Southern blot evidence of recombination in vitro, resulting in mixtures of intact rOka and single-deletion ORF62 and ORF71 mutants.
FIG. 5.
Replication kinetics of rOka and mutant viruses. Virus-infected melanoma cells were seeded onto fresh monolayers of melanoma cells. At days 1 through 6 after infection, the infected monolayer was harvested, and the infected cells were serially diluted and used to infect monolayers of melanoma cells in triplicate. At 6 days after infection, the melanoma cell monolayers were stained with crystal violet and the number of plaques was counted. The error bars indicate standard deviations. (A) Comparison of rOka with single-copy viruses and repaired viruses. (B) Comparison of rOkaΔORF62/71(pORF62-R) with point mutant viruses.
Construction and characterization of rOkaΔORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R).
To confirm that failure to recover VZV from transfections done with pvSpe23ΔORF62/71 was due to absence of the ORF62/71 diploid gene, the pvSpe23ΔORF62/71 cosmid was further modified to insert the pOka ORF62 or the vOka ORF62 sequence into the AvrII site between ORF65 and ORF66 (Fig. 1). The inserted sequence contained ORF62 along with the full intragenic region between ORF62 and ORF63 and 70 bp of ORF63, which was predicted to contain the complete or essential components of the ORF62 promoter. The requirement for at least one copy of the ORF62 or ORF71 gene and its promoter for VZV replication was demonstrated by recovery of infectious viruses, designated rOkaΔORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R), in all transfections done with pvSpe23ΔORF62/71(pORF62@Avr) or pvSpe23ΔORF62/71(vORF62@Avr) and the three intact cosmids in two separate experiments (Table 2). However, initial plaque sizes were smaller than those of rOka, and repaired viruses produced only 50% CPE in melanoma cells at 13 days after transfection, suggesting attenuated growth.
Sequencing of ORF62 and ORF71 in the respective pOka and vOka cosmids revealed that these two duplicated ORFs were identical in pOka and also in vOka. When the ORF62/71 sequences from the pOka and vOka cosmids were compared, only two differences were identified, both of which resulted in an amino acid change. An A-to-G transition at position 2872 (A2872G) resulted in an arginine-to-glycine substitution at position 958 (R958G), and a T-to-C substitution at position 3172 (T3172C) produced a tyrosine-to-histidine substitution at position 1058 (Y1058H). Sequencing of ORF62 in plasmid clones made from cells infected with vaccine Oka virus has shown considerable heterogeneity, as confirmed in two independent reports (3, 17). The A2872G substitution present in the vOka cosmid was one of the numerous mutations that have been identified in ORF62 from vaccine Oka virus stocks, but the T3172C mutation has not been reported previously.
PCR analysis of mutant cosmids and rOkaΔORF62/71(pORF62-R)- and rOkaΔORF62/71(vORF62-R)-infected cell DNA.
PCR with the AvrII site primers 5 and 6 (Table 1) showed that the ORF62 gene was inserted into cosmids at the AvrII site in pvSpe23ΔORF62/71(vORF62@Avr) and pvSpe23ΔORF62/71(pORF62@Avr), oriented towards ORF66 (Fig. 2C). PCR with primers 1 and 2 to amplify ORF71 generated a 3,515-bp product from pvSpe23ΔORF62/71(vORF62@Avr) and pvSpe23ΔORF62/71(pORF62@Avr) (Fig. 2A, lanes 6 and 7). PCR of rOka and repaired viruses rOkaΔORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R) yielded a single product of the expected size with either the ORF62 or ORF71 primers (Fig. 2A and B, lanes 8, 11, and 12). PCR with the AvrII site primers further confirmed that rOkaΔORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R) had a single copy of ORF62 at the AvrII site, as expected (Fig. 2C, lanes 11 and 12). In contrast to the case for single deletions, no evidence of recombination in vitro was observed when the pvSpe23ΔORF62/71(vORF62@Avr) or pvSpe23ΔORF62/71(pORF62@Avr) cosmids were used to generate these repaired viruses.
Effects of targeted mutations in ORF62.
The IE62 protein has been shown previously to bind to the IE4 protein (55) and to the whole ORF9 protein (56). The Ruyechan and Hay laboratories have mapped the region important for IE4 binding to aa 161 to 299 in the IE62 sequence by deletion analysis in in vitro expression experiments. Mutations in ORF62 were designed from their evidence that IE4 binding to IE62 protein was eliminated by phosphorylation of IE62 at or near threonine 250, which contains a common phosphorylation site for protein kinases C and A, and a second protein kinase C site at serine 245 (55, 56). To further explore these interactions, the point mutations S245A and T250A were introduced into ORF62 in the repaired single-copy pOka ORF62 gene. In addition, A28P, which was designed to disrupt the predicted alpha-helical structure of the putative ORF9 binding site (Ruyechan and Hay, unpublished observations), was transferred into ORF62. Infectious virus was recovered from all transfections done with the cosmids with these point mutations in the single-copy ORF62, indicating that these mutations were not lethal. These viruses were designated rOkaΔORF62/71(pORF62-R(S245A)), rOkaΔORF62/71(pORF62-R(T250A)), and rOkaΔ ORF62/71(pORF62-R(A28P)), respectively.
Replication of repaired rOkaΔORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R) and recombinants with targeted ORF62 mutations in vitro.
The repaired viruses, rOkaΔ ORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R), exhibited a consistent small-plaque phenotype in initial transfections and after passage in melanoma cells (Fig. 4A). The mean plaque size (± standard deviation) for rOkaΔORF62/71(pORF62-R) was smaller than that for rOka (0.53 ± 0.10 mm versus 1.04 ± 0.10 mm; P < 0.0001), and rOkaΔORF62/71(vORF62-R) also exhibited smaller plaque size than rOka (0.64 ± 0.13 mm versus 1.04 ± 0.10 mm; P < 0.0001). Analysis of the ORF62 point mutants showed smaller plaques with rOkaΔORF62/71(pORF62-R(A28P)), in which the putative ORF9 binding site was disrupted, compared to rOkaΔORF62/71(pORF62-R) (0.52 ± 0.09 mm versus 1.14 ± 0.12 mm; P < 0.0001). However, plaque sizes of rOkaΔORF62/71(pORF62-R(S245A)) and rOkaΔORF62/71(pORF62-R(T250A)), altering IE4 protein binding sites, were not further diminished (data not shown).
FIG. 4.
Plaque sizes of rOkaΔORF62, rOkaΔORF71, rOkaΔ ORF62/71(vORF62-R), and rOkaΔORF62/71(pORF62-R) and restoration of rOkaΔORF62/71(vORF62-R) and rOkaΔORF62/71(pORF62-R) plaque size by gE complementation. (A) The mean plaque size (± standard deviation) was determined for rOka and each mutant virus by measuring ≥ 40 plaques in melanoma cells. The asterisks indicate a significant difference from rOka plaque size (P < 0.001). (B) A doxycycline-inducible gE-expressing melanoma cell line, the Met-gE cell line, was used to examine the effect of gE complementation on plaque size (33). The mean plaque size (± standard deviation) was determined for ≥ 40 plaques for rOka, rOkaΔORF62/71(vORF62-R), and rOkaΔORF62/71(pORF62-R) in the presence (+) and absence (−) of doxycycline.
A significant difference in viral titers was observed between rOka and the repaired viruses, rOka Δ ORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R), at days 1 through 6 (P < 0.05) (Fig. 5A). This experiment suggested that growth of rOka ΔORF62/71(pORF62-R) was slower than that of rOkaΔ ORF62/71(vORF62-R) (P < 0.01), but a second experiment showed no difference between the two repaired viruses (data not shown), whereas the slower growth and lower peak titers of rOkaΔORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R) compared to rOka was confirmed. This viral titration data were consistent with less efficient cell-cell spread, as suggested by the small-plaque phenotype of the repaired viruses in vitro, and was further evidence for attenuation associated with insertion of the single copy of ORF62 into a nonnative site in the VZV genome. Viral titers of ORF62 point mutants were also compared with those of rOkaΔORF62/71(pORF62-R) and rOka (Fig. 5B). Only the mutant rOkaΔ ORF62/71(pORF62-R(A28P)) exhibited slower growth kinetics than rOkaΔORF62/71(pORF62-R), which was consistent with the plaque size analysis. This observation suggested that IE62 protein interactions with ORF9 protein, predicted to involve this region of the IE62 protein (Ruyechan and Hay, unpublished observations), were important for replication and warrants further investigation. However, the N-terminal region of the IE62 protein also has an acidic activation domain (49); the possibility that the A28P mutation could affect the transactivating activity of the IE62 protein, as well as interfering with ORF9 binding, must be examined (29, 56).
Expression of VZV proteins by rOkaΔORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R).
To investigate the attenuated growth of the repaired viruses in vitro, VZV protein synthesis was examined in melanoma cells infected with rOka, rOkaΔORF62/71(pORF62-R), and rOkaΔ ORF62/71(vORF62-R) and evaluated by Western blotting at days 1 and 4 (Fig. 6). In addition to IE62 protein, expression of IE4 protein, ORF47 protein kinase (an early gene product), and gE (a late gene product) were analyzed. As shown in Fig. 6, a reduced amount of IE62 protein was detected in cells infected with repaired viruses on days 1 and 4. The limited amount of IE62 protein produced by the repaired viruses was associated with expression of IE4 and ORF47 proteins at levels equivalent to those observed in rOka-infected cells on both days 1 and 4. Little or no gE was detected on day 1 in any specimens, which is consistent with its classification as a late gene product. However, gE remained markedly reduced in cells infected with rOkaΔORF62/71(pORF62-R) or rOkaΔ ORF62/71(vORF62-R) on day 4, which is consistent with a requirement for IE62 protein in inducing gE expression. Since the VZV gE-gI protein complex is required for cell-cell spread (1, 45, 46), reduced gE expression, secondary to limited IE62 protein expression, can account for the smaller plaque sizes and slower growth kinetics observed in melanoma cells infected with rOkaΔORF62/71(pORF62-R) or rOkaΔORF62/71(vORF62-R).
FIG. 6.
Viral protein expression analysis of mutant viruses. Melanoma cells (107) were infected with 5 × 104 PFU of each virus and harvested at 1 and 4 days postinfection. Cell lysates were subject to Western blotting. Expression of IE proteins (ORF62 and ORF4), the early viral kinase (ORF47), and the late glycoprotein E (ORF68) was analyzed with rabbit polyclonal antiserum specific for each protein. The specimens tested were as follows: lane 1, rOka; lane 2, rOkaΔORF71; lane 3, rOkaΔORF62; lane 4, rOkaΔORF62/71(vORF62-R); lane 5, rOkaΔORF62/71(pORF62-R); lane 6, mock infected.
Restoration of plaque size by infection of gE-expressing melanoma cells with rOkaΔORF62/71(pORF62-R) and rOka ΔORF62/71(vORF62-R).
To further examine the relationship between reduced gE expression and the attenuated phenotypes of the repaired viruses, Met-gE cells, which are a melanoma cell line with tetracycline-inducible gE expression (33), were inoculated with rOkaΔORF62/71(pORF62-R) or rOkaΔ ORF62/71(vORF62-R). In the absence of doxycycline, the mean plaque size for rOkaΔ62/71(pORF62-R) in Met-gE cells was less than that for rOka (0.75 ± 0.14 mm versus 1.26 ± 0.07 mm; P < 0.0001); the rOkaΔORF62/71(vORF62-R) plaque size was 0.75 ±0.10 mm, which was also reduced compared to that of rOka (P < 0.0001) (Fig. 4B). However, in the presence of doxycycline (1.0 μ g/ml), the mean plaque sizes of rOkaΔORF62/71(pORF62-R) and rOkaΔORF62/71(vORF62-R) were 1.21 ± 0.10 mm and 1.23 ± 0.09 mm, respectively, and were not significantly different from that of rOka, which was 1.25 ± 0.09 mm (Fig. 4B). The rOka plaque size was not affected by doxycycline.
Infectivity of rOkaΔORF71, rOkaΔORF62, rOkaΔORF62/71(vORF62-R), and rOkaΔORF62/71(pORF62-R) in SCID-hu skin xenografts.
Skin xenografts were inoculated with equivalent titers of rOka, rOkaΔORF71, rOkaΔORF62, rOkaΔORF62/71(vORF62-R), and rOkaΔORF62/71(pORF62-R), and implants were harvested at days 12, 21, and 28 (Fig. 7). No difference in infectivity was observed between rOka, rOkaΔ71, and rOkaΔ62. This observation was not unexpected, since intact rOka was generated in transfections of the ORF62 and ORF71 single-deletion cosmids, as shown by PCR and Southern hybridization (Fig. 2 and 3). When skin xenograft tissue was analyzed by PCR using primers for amplifying ORF71, ORF62, and ORF4, 2 of 10 implants inoculated with rOkaΔ71 yielded the single-copy recombinant (Fig. 8A, lanes 6 and 7), 5 had mixed single-copy rOkaΔ71 and rOka viruses, and intact rOka virus was predominant in 3 implants (Fig. 8A, lanes 12, 14, and 15). Seven of nine implants inoculated with rOkaΔ62 had both rOkaΔ62 and rOka viruses, and two implants yielded predominantly intact rOka (Fig. 8A, lanes 17 and 21).
FIG. 7.
Infectivities of mutant viruses in SCID-hu skin implants. Skin implants in SCID-hu mice were inoculated with rOka (▪), rOka Δ ORF71 (▩), rOkaΔORF62rev (□), rOkaΔORF62/71(vORF62-R) (▨), or rOkaΔORF62/71(pORF62-R) (▤). Viral titers were measured by infectious focus assay (36). Each bar represents the mean titer of infectious virus recovered from infection of three animals at 12, 21, or 28 days after inoculation. ○̶, no viral growth in any implants; N.D., not done. Error bars represent standard deviations. An asterisk indicates that titers were significantly lower than those for rOKA (P < 0.05) at the same time point. The titers of the inocula are shown at the left.
FIG. 8.
PCR analysis of viruses recovered from SCID-hu skin xenografts. (A) The bars in the upper section depict the titers of infectious virus that was recovered from each implant. The specimens tested are as follows: bars 1 to 5, rOka; bars 6 to 15, rOkaΔORF71; bars 16 to 24, rOka ΔORF62. The lower section shows the PCR analysis of each of the same implants, using primers for ORF71, ORF62, and ORF4, as described in Materials and Methods. (B) PCR analysis of skin implants inoculated with the repaired viruses and evaluation for IE4 protein by Western blotting. The specimens tested are as follows: lanes 1 to 5, rOkaΔORF62/71(vORF62-R); lanes 6 to 10, rOkaΔORF62/71(pORF62-R); lane 11, uninfected control. Lane M, molecular size marker. As shown in the Western blot, no IE4 protein was detected from any implants inoculated with repaired viruses.
Infectious virus was recovered at low titers from only a single skin xenograft that was tested at day 12 after inoculation with rOkaΔORF62/71(vORF62-R) and from none of those inoculated with rOkaΔORF62/71(pORF62-R). Further, no rOka ΔORF62/71(vORF62-R) or rOkaΔORF62/71(pORF62-R) was recovered from 10 SCID-hu skin implants tested at 21 days after infection. By PCR, two of five xenografts inoculated with rOkaΔORF62/71(vORF62-R) and tested at day 12 had detectable ORF71 DNA. At day 21, all xenografts inoculated with rOkaΔORF62/71(pORF62-R) had detectable ORF71 DNA (Fig. 8B). Western blot analysis for IE4 protein expression was negative in rOkaΔORF62/71(vORF62-R)- and rOkaΔORF62/71(pORF62-R)-infected implants evaluated at day 21 (Fig. 8B). Despite failure to recover infectious virus, detection of VZV DNA in some implants by PCR suggested that rOkaΔORF62/71(pORF62-R) might replicate at a very low rate in vivo. Therefore, a second cohort of mice with skin xenografts were inoculated with rOkaΔORF62/71(vORF62-R) or rOkaΔORF62/71(pORF62-R) and evaluated after 28 days. PCR, virus titrations, and Western blot analysis for VZV protein expression of all of these skin implants were negative for both of the repaired viruses (data not shown). Thus, rOkaΔ ORF62/71(vORF62-R) and rOkaΔORF62/71(pORF62-R) were not infectious in vivo or replicated to a very limited extent in only one implant of all of the skin xenografts that were inoculated with these viruses. This defect in replication in differentiated cells in vivo was observed even though insertion of a single copy of vORF62 or pORF62 into the nonnative AvrII site was associated with restoration of the capacity to replicate in vitro, albeit with an attenuated phenotype.
DISCUSSION
The mutational analysis of ORF62 and ORF71, encoding the IE62 protein, demonstrated that one copy of the duplicated gene is required for VZV replication in vitro, either at one of the native locations in the repeat regions flanking the US region of the genome or in the nonnative insertion site in the US region. The requirement for IE62 protein fits the model in which IE62 protein must localize to the nucleus of the infected cell, along with uncoated viral DNA, as an essential first step in VZV replication. These experiments using VZV recombinant viruses support experiments with transient-expression systems showing that no other VZV regulatory protein can substitute for the transactivating effects of IE62 protein on viral gene promoters (19, 40). IE62 transactivation of promoters representative of IE, early, and late kinetic classes of VZV genes has been demonstrated in expression systems (25). The IE62 protein binds to the IE4 and IE63 proteins and is required to recruit other viral regulatory proteins to sites of transcription initiation, such as IE63 to the gI promoter (29, 55, 56). In addition, IE62 protein interacts with cellular proteins involved in viral gene transcription (32). Introducing pOka or vOka ORF62 into the nonnative US site was associated with the recovery of infectious virus. These experiments define ORF62 as an essential gene by showing that failure to generate infectious virus from pvSpe23ΔORF62/71 was due to the simultaneous deletion of ORF62 and ORF71 and not to disruption of promoter sequences or other regulatory regions affecting adjacent genes in the repeat segment or to random, unidentified mutations elsewhere in the VZV genome. The conclusion that the IE62 protein is an essential gene product parallels observations that deleting the duplicate ORFs encoding HSV ICP4 from the repeat regions flanking the US segment is lethal, except in complementing cell lines (47), and that mutating the ICP4 DNA binding domain is also incompatible with HSV replication (2).
When ORF62 or ORF71 was removed from the IRS or TRS region, respectively, transfections of the mutated cosmid with the single copy of ORF62 or ORF71, along with the other three VZV cosmids, often resulted in the recovery of intact rOka as well as rOkaΔ62 or rOkaΔ71 mutants. While experimental information is quite limited, VZV is presumed to undergo rolling-circle DNA replication, with theta intermediates, as described for HSV (8, 51, 52). Segment inversion, occurring by intramolecular recombination between the inverted repeats during replication, generates the variant isoforms (7, 8, 52). When the four fragments of VZV DNA are introduced by cosmid transfection, assembly of initial full-length genomes results from recombination due to the overlap between fragments. As shown in our Southern hybridization experiments, segment inversion occurred even though the pvSpe23 cosmid contains the IRS -US -TRS in one orientation only, presumably during early replication of the initial genomes. In transfections with the single deletion cosmids, pvSpe23ΔORF62 and pvSpe23ΔORF71, replication was associated with repair of the ORF62 or ORF71 deletion, creating VZV DNA with full-length IRs and TRs. It is of interest that experiments with HSV-1 IRS deletion mutants did not identify similar recombination events (50). In VZV, recombination is likely to be facilitated by the presence of the two R4 repeat element in the IRS and TRS sequence, which are located in an intragenic region adjacent to each of the two VZV origins of replication (OriS); the R4 repeats are between ORF62 and ORF63 or between ORF71 and ORF70, respectively. R4 is one of the five such elements in the VZV genome, and this R4 repeat is made up of variable numbers of a 27-bp sequence (5, 8). As noted in our previous report about single-copy ORF63 and ORF70 deletions, this sequence is predicted to be single stranded during early replication and could provide the site for a specific crossover event (54). Thus, the intact IRS, or the intact TRS, could be introduced in place of the sequence from which ORF62 or ORF71 had been deleted. Our experiments with the single ORF62 and ORF71 deletion mutations in cell culture and in skin xenografts in vivo indicate that conditions of VZV DNA replication, and presumably cleavage and packaging, favor the restoration of full-length IRS and TRS sequences in the context of the viral genome.
Introducing a single copy of ORF62 into the nonnative US site permitted recovery of infectious viruses, rOkaΔORF62/71(vORF62-R) and rOkaΔORF62/71(pORF62-R), in cell culture, but changing the genomic location of ORF62 had an attenuating effect on VZV replication in vitro and was essentially lethal for infection of skin xenografts in vivo. These experiments, which were done repeatedly with independently derived cosmids, pvSpe23ΔORF62/71(pORF62@Avr) or pvSpe23ΔORF62/71(vORF62@Avr), indicate that the ORF62/71 sequence cannot be restored from this foreign location to its native sites during replication. The attenuation phenotypes of rOkaΔORF62/71(vORF62-R) and rOkaΔ ORF62/71(pORF62-R) were characterized by small plaques and delayed and diminished production of infectious virus compared to rOka. Introducing point mutations, S245A and T250A, to disrupt the IE4 binding site of the IE62 protein, as mapped by in vitro expression experiments, was not lethal and did not further diminish replication in vitro. However, altering the putative ORF9 binding site with the substitution of A28P was associated with a log decrease in virus yields at each time point in the kinetic analysis; this mutation may also affect the IE62 N-terminal acidic activation domain and warrant further analysis because of the attenuating effect. Mutations interfering with IE62 binding to IE4 and potentially with ORF9 protein are predicted to affect VZV infectivity in vivo, but failure of rOkaΔORF62/71(pORF62-R) to infect skin xenografts prevented an assessment of the consequences of these mutations in vivo. Experiments introducing these targeted mutations into both copies of ORF62/71 in their native locations will be necessary to address this question.
Analysis of the kinetics of viral protein synthesis by rOkaΔ ORF62/71(vORF62-R) and rOkaΔORF62/71(pORF62-R) in vitro showed that IE62 protein was reduced at day 1, while IE4 and ORF47 protein were not affected, and that delayed IE62 production was associated with reduced synthesis of the essential late VZV gE protein at day 4. Thus, the primary effect of relocating ORF62 and providing only one copy of ORF62 in the VZV genome was to decrease and delay its transcription, which is mediated by the transactivating function of its own gene product, IE62 protein, and ORF10 protein (39), and to reduce late ORF68 (gE) transcription. These observations are of interest because of the evidence that transcription of ORF4 and ORF47 did not require the usual concentrations of IE62 protein present at day 1 after VZV infection of cultured cells. Of note is that eliminating HSV ICP4, the IE62 homolog, resulted in high levels of expression of all IE genes, attributed to diminished repressor activity (9). The promoters of ORF4 and ORF47 have not been mapped, but both contain predicted binding sites for cellular transactivators based on computer sequence analysis; cellular proteins may function in combination with low levels of IE62 protein to mediate their transcription. With regard to glycoproteins, recent analyses have demonstrated that HSV ICP4 contributed to TFIID binding to a minimal gC promoter, consisting of the TATA box and a gC start site sequence that resembled the eukaryotic initiator (Inr) element, thereby facilitating formation of preinitiation complexes (18). Subsequent experiments showed that ICP4 was required for high levels of late HSV glycoprotein gene expression and that this function was mediated by ICP4 interaction with Inr start site regions (24). Since the ORF68 sequence, encoding VZV gE, has this conserved Inr element, IE62 protein may play a similar role in enhancing late gE expression. Characterizing the effects of the ecotopic insertion of ORF62 on the kinetics of VZV protein expression in vivo was not possible because of the failure of these attenuated viruses to replicate in differentiated skin xenografts.
In contrast to the case for IE62, we have not seen any effects on expression in vitro when other essential VZV genes and their promoters, including ORF63, gE (ORF68), and gK (ORF5), were introduced at the unique AvrII site in the US (33, 34, 54). When ORF62 was transferred to the AvrII site, 1,658 bp of upstream sequence was included, containing the complete ORF62-ORF63 intragenic region and 70 bp of ORF63. ORF62 and ORF63 are transcribed in opposite directions, and this region is presumed to contain promoter sequences for both genes. The IE62 promoter has three TAATGARAT motifs and other sites that are known to bind cellular and viral protein complexes, which were preserved in the inserted sequence (11, 31, 41, 42). We cannot exclude the possibility that reduced early IE62 protein synthesis was due to absence of promoter elements located farther upstream within the ORF63 coding sequence. In addition, the lack of recombination events, which occurred with the single ORF62 or ORF71 deletions, meant that only one copy of ORF62 was present in all progeny viruses generated by using the pvSpe23 ΔORF62/71(pORF62@Avr) cosmid, which could affect total IE62 protein synthesis. It is also possible that the insertion of the additional OriS altered IE62 expression from the ORF62 gene at its nonnative site.
The rOkaΔORF62/71(vORF62-R) and rOkaΔORF62/71(pORF62-R) mutants had a small-plaque phenotype in vitro, and infectious virus was recovered from only one skin xenograft inoculated with these mutants in vivo. In addition to its association with limited gE production, the small-plaque phenotype was corrected when melanoma cells with inducible gE expression (Met-gE cells) were induced and infected with rOka ΔORF62/71(vORF62-R) or rOkaΔORF62/71(pORF62-R). These experiments suggest that the attenuation phenotype was related directly to gE concentrations in infected cells. While dispensable in other alphaherpesviruses, VZV gE is essential for viral replication in vitro, probably because it is required for cell-cell fusion (23, 33). VZV infection of cultured cells is known to be highly cell associated and is characterized by extensive formation of multinucleated syncytia (4). VZV gE and gI, encoded by ORF67, form heterodimer complexes that are required for normal gE trafficking within infected cells and to the plasma membrane (1, 20, 30, 45, 46, 61). In our experience, mutations that eliminated gI expression by ORF67 deletion or significantly reduced gI expression by altering binding sites for the cellular transactivating proteins, Sp1 and USF, in the gI promoter have also resulted in recombinants with a small-plaque phenotype; in these experiments, plaque morphology was corrected by insertion of the intact gene and its promoter into the nonnative Avr II site (20, 30). Like for the rOka ΔORF62/71(vORF62-R) and rOkaΔORF62/71(pORF62-R) recombinants, the small-plaque phenotype of rOkaΔgI and the dual Sp1/USF gI promoter mutants in vitro was associated with no infectivity or much decreased replication in differentiated human skin cells in SCID-hu xenografts (20, 38). Other mutations that have no effects on plaque size or infectious virus yields in vitro can also be lethal for skin infection in vivo, notably deletion of the ORF47 protein kinase or elimination of its kinase function, by other mechanisms (4a, 37). However, our interpretation of experiments with the small-plaque rOkaΔORF62/71(vORF62-R) and rOkaΔ ORF62/71(pORF62-R) recombinants, in which gE expression was down-regulated, as well as with the small-plaque gI deletion and gI promoter mutants, is that the pathogenic effects of VZV in skin in vivo are critically dependent upon intact mechanisms of cell fusion mediated by gE-gI complex formation.
In summary, these experiments constitute the first analysis of the ORF62/71 gene pair in the context of the VZV genome and demonstrate the essential role of the IE62 protein in VZV replication in vitro and for the pathogenesis of VZV infection in skin in vivo.
Acknowledgments
This work was supported by grants AI36884 and AI053846 from the National Institute of Allergy and Infectious Diseases to A.A. and by grants from Fujisawa Pharmaceutical Co., Ltd., to B.S.
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