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Journal of Virology, April 2005, p. 4819-4827, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4819-4827.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of the Varicella-Zoster Virus Gene Product Encoded by Open Reading Frame 35 in Viral Replication In Vitro and in Differentiated Human Skin and T Cells In Vivo

Hideki Ito,1,{dagger} Marvin H. Sommer,1 Leigh Zerboni,1 Armin Baiker,1,{ddagger} Bunji Sato,1,§ Ruibin Liang,2 John Hay,2 William Ruyechan,2 and Ann M. Arvin1*

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California,1 Department of Microbiology & Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, University of Buffalo, Buffalo, New York2

Received 21 September 2004/ Accepted 3 December 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOka{Delta}35(#1) and rOka{Delta}35(#2), and replaced ORF35 at a nonnative site, generating two rOka{Delta}35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOka{Delta}35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOka{Delta}35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOka{Delta}35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOka{Delta}35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOka{Delta}35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOka{Delta}35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Varicella-zoster virus (VZV) is an ubiquitous alphaherpesvirus that causes varicella (chicken pox) and herpes zoster (shingles) in the human host (2, 10). The VZV genome is a double-stranded DNA molecule that consists of the unique long (UL) and unique short segments, each of which is flanked by internal repeat and terminal repeat sequences. Complete sequencing of several VZV strains has demonstrated conservation of the linear organization of open reading frames (ORFs) and limited variation in the DNA sequence among isolates from different geographical regions (1, 13, 16, 17, 24). Although the VZV genome contains ORFs that are known or predicted to encode more than 70 distinct proteins, functions have been assigned to only about half of these VZV gene products (10). In many cases, the contributions of VZV genes to viral replication are presumed because of their partial sequence homologies with ORFs in herpes simplex virus type 1 (HSV-1), which is the prototype of the alphaherpesviruses (35). While such assumptions have proven to be useful, instances of function or requirements not corresponding to those inferred have also been documented, and HSV and VZV proteins with high apparent homology may not have complementing functions, as illustrated by DNA replication proteins (3, 7, 9, 10).

ORF35 is located in the UL region of the genome (13) and is predicted to encode a protein of about 285 amino acids. The development of VZV cosmid systems has allowed the direct analysis of VZV gene functions with VZV recombinants that have targeted changes in the viral genome (3, 5, 8, 23, 25, 31, 36). The aim of these experiments was to examine the contribution of VZV ORF35 to VZV replication by mutagenesis in the context of the viral genome using our five-cosmid system (5, 36). Genes related to ORF35 are conserved among all of the herpesviruses (4, 12, 14, 15, 18, 19, 21, 26, 39). The predicted ORF35 protein has substantial homology to HSV-1 UL24, with an identity of 33% at the amino acid level; comparisons with the corresponding gene product in pseudorabies virus and bovine herpesvirus 1 indicate 41 and 45% identity, respectively. The deletion of UL24 from the HSV genome was compatible with replication, but it was associated with decreased yields of infectious HSV, small plaques, and syncytium formation in cultured cells (21, 22). UL24 was important for HSV-1 infection of mouse sensory ganglia (22). UL24 exhibited a complex transcription pattern, generating four transcripts, although most UL24 protein function was associated with expression from the first start site and was regulated by ORF27 (33, 34). UL24 overlaps with UL23, the gene encoding the HSV thymidine kinase (TK), and its promoter on the opposite strand (21). Although VZV ORF35 and ORF36 also have a head-to-head orientation, ORF35 did not overlap with ORF36, the gene for VZV TK, by sequence analysis.

Because VZV is highly species specific in its host range, we developed SCIDhu mouse models to investigate the genetic mechanisms of VZV pathogenesis in vivo. In this model, human skin and T-cell xenografts are infected in vivo to define the effects of selected genetic mutations in the VZV genome on virulence (3, 5, 20, 27-31, 36, 37). Defining VZV genes required for infection of T cells and skin is important because VZV pathogenesis is characterized by tropism for these cells in the human host (2). ORF35 proved to be dispensable, providing the opportunity to evaluate the effects of deleting ORF35 on viral replication in cultured cells and to document a role for ORF35 gene expression in the pathogenesis of VZV infection of differentiated human skin and T cells in SCIDhu mice in vivo.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cosmids and plasmids. ORF35 is located in pvPme 19, nucleotides (nt) 63976 to 64753, in the VZV cosmids derived from vaccine Oka virus (23, 29). To facilitate manipulation of VZV gene sequences in the UL region, which includes ORF35, two new cosmids were made from pvPme19 (Fig. 1) (5, 36). The five-vaccine Oka virus cosmid set consisted of pvFsp4, pvSpe5, pvAfl30, pvAvr10, and pvSpe21. To delete ORF35, pvPme19 was digested with KpnI to obtain a 15.6-kb fragment, nt 57402 to 72955, that contained ORF35. This fragment was subcloned into pLitmus28 at the KpnI site, yielding pLitmus28/VZV3. Two sets of PCR primers were used to delete ORF35: primer 1, 5'-ACCACAAAAACACCCGACTC-3' (the 5' end anneals at nt 58261); primer 2, 5'-CCCATGCCTAGGAAAACAGC-3' (the 5' end anneals at nt 63987); primer 3, 5'-TAGCGGACCTAGGTGAAGTT-3' (the 5' end anneals at nt 64744); and primer 4, 5'-AACACTTCCGCAATACAAAC-3' (the 5' end anneals at nt 68263). Primers 1 and 4 anneal upstream and downstream of MscI restriction sites. Primers 2 and 3 anneal over the ORF35 stop and start codons and alter the nucleotides, indicated in boldface type in the sequences listed above, to create an AvrII site (CCTAGG). With pLitmus28/VZV3 as the template, PCRs were carried out with the Elongase Enzyme mix (GIBCO/BRL, Inc.), with primers 1 and 2 and primers 3 and 4. The 5,227- and 3,520-nt PCR products were isolated and digested with AvrII and MscI, resulting in 354- and 2,013-nt products, which were sequenced to verify that no additional mutations were introduced. The plasmid pLitmus28/VZV3 was digested with MscI, generating a 15.2-kb fragment. A triple ligation was done with the 354- and 2,013-nt PCR products and MscI-digested pLitmus28:VZV-3 to create plasmids from which ORF35 was deleted (pLitmus28/VZV3{Delta}ORF35). To transfer the sequence with the ORF35 deletion into pvAfl30, a shuttle vector was constructed by digestion of pvPme19 with AscI and AvrII and ligation of the 18.0-kb piece into pLitmus28 to generate pLitmus28/VZV4. pLitmus28/VZV4 was digested with MscI, producing an 18,064-nt fragment. Digestion of pLitmus28/VZV3{Delta}ORF35 with MscI yielded a 2,367-nt fragment that was then cloned into the 18.0-kb MscI-cut pLitmus28/VZV4, generating pLitmus28/VZV4{Delta}ORF35. pvAfl30 was digested with either AscI or AscI plus AvrII to isolate a 6.8-kb AscI vector and 7.6-kb AscI/AvrII DNA fragments, and pLitmus28/VZV4{Delta}ORF35 was partially digested with AvrII to isolate a 20.4-kb fragment. The 20.4-kb fragment was digested with AscI to generate a 17.6-kb AscI/AvrII{Delta}ORF35 fragment. A triple ligation was done using this 17.6-kb AscI/AvrII{Delta}ORF35 fragment, AscI-digested pvAfl30 (6.8 kb), and AscI/AvrII-digested pvAfl30 (7.6 kb). The resulting mutated pvAfl30 cosmid clones, lacking ORF35, were designated pvAfl30{Delta}ORF35.



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  		FIG. 1. Construction of cosmids with ORF35 mutations. Line 1 shows a schematic diagram of the VZV genome with the location of ORF35 in the UL region. Line 2 depicts the five overlapping VZV cosmids. Line 3 shows the subcloned KpnI fragment that includes ORF34, ORF35, and ORF36 with the nucleotide numbers of the ORF35 start and stop sites and adjacent ORFs. Line 4 depicts the deletion of ORF35 from rOka. To restore the gene, ORF35 was ligated into a unique AvrII site of pvSpe21 in each orientation, resulting in rOka{Delta}35/35@Avr#1 (right to left) and rOka{Delta}35/35@Avr#2 (left to right) (line 5). Line 6 shows ligation of Flag-tagged ORF35 into the AvrII site, used to make rOka{Delta}35/35Flag@Avr and rOka35Flag@Avr viruses.

 
ORF35 was inserted into the AvrII site in pvSpe21 to restore ORF35 at an alternative site in the VZV genome. Primer 1 (5'-CGGATAACACCTAGGTAATTTTAT-3') and primer 2 (5'-AATATCCCACCTAGGCAT TTATTC-3'), introduced the AvrII site (CCTAGG) at both ends of the PCR product, with nucleotide changes indicated in boldface type. The ORF35 sequence, including the putative promoter region and downstream elements (nt 63976 to 64753), was amplified, and the 1.3-kb PCR product was isolated, digested with AvrII, and reisolated. The pvSpe21 cosmid was digested at the unique AvrII site between ORF65 and ORF66 (Fig. 1). The linearized cosmid and the 1.3-kb PCR product were ligated to make pvSpe21ORF35@Avr.

To insert Flag-tagged ORF35 into pvSpe21, primers 1 (5'-CGCCGGATCCCGGTCGGATAACATAATTTT-3') and 2 (5'-CTTTGGTACCCC CATGGGAAAACATCCCGG-3') were designed to introduce a BamHI and a KpnI site at the ends of the PCR product (nucleotide changes are indicated in boldface type). ORF35 with the putative promoter region and downstream elements was amplified, and the 1.3-kb PCR product was isolated, digested with BamHI and KpnI, reisolated, and ligated into a Flag expression vector, pFlag-CMV-5a (Sigma, Inc., St. Louis, Mo.), that was linearized with BamHI and KpnI, yielding pFlag-CMV-5a-VZV-ORF35. Primers 3 (5'-GAATTCAAGCTTGCGGCCTAGGATCTATCGATCTGC-3') and 2 (5'-CATTCCACAGAAGCTGGCCTAGGCGTACCCAATTCAACA-3') introduced AvrII sites (bold) at both ends of the PCR product, consisting of the ORF35 putative promoter, the ORF35 coding region, the Flag tag, and hGH poly(A) from pFlag-CMV-5a-VZV-ORF35. The 2.0-kb fragment was digested with AvrII, reisolated, ligated to pvSpe21, and digested with AvrII, producing pvSpe21ORF35-Flag@Avr.

Cosmid transfection. VZV cosmid DNA was prepared as described previously (25). For transfections, pvAvr10 was digested with AscI and AvrII, and the other cosmids were digested with AscI and mixed in water to a final concentration of 100 ng/µl of pvFsp4, pvSpe5, pvAfl30, and pvAvr10; pvSpe21 or pvSpe21ORF35@Avr was used at a concentration of 50 ng/µl. Melanoma (Mewo) cells, grown in tissue culture medium (Dulbecco's modified Eagle's medium; Gibco) supplemented with heat-inactivated fetal calf serum, were transfected as described previously (6, 37); plaques appeared in 5 to 10 days.

PCR and sequencing. VZV cosmid DNA was purified with QIAGEN columns, and viral DNA was recovered from infected cells with DNazol (Gibco BRL, Inc., Grand Island, N.Y.). PCR was performed with Elongase enzyme mix (Gibco BRL, Inc.). The primers used to assess ORF35 deletions were primer 1 (5'-TCATACGCCCTCTTAACTCA-3') and primter 2 (5'-GGCCCGTTTGCTTACTCT-3'). To analyze inserts at AvrII, primer 3 (5'-CCACACAAACATCACCTG-3') was used with primer 4 (5'-TTACCACCGCTTCCATCA-3'). DNA was isolated with the QIAGEN (Chatsworth, Calif.) gel extraction kit, or PCR products were cloned into the pCR-TOPO cloning vector (Invitrogen, Carlsbad, Calif.). Sequencing reactions were primed with the T7 and T3 primers contained in the pCR-TOPO vector and pFlag-CMV-5a or with custom primers. To sequence the ORF35 region, the primer 5'-AATATCCCACATTTATTC-3' was used. This primer anneals within the end of ORF34 region. To sequence across the AvrII site, a primer (5'-CCACACAAACATCACCTG-3') was used.

Construction of ORF35-EGFP plasmids. ORF35-EGFP (for enhanced green fluroescent protein) plasmids were generated with two sets of primers: primer 1 (5'-CCGCTCGAGATGTCCGCTATGCGAATTCGGGC-3') and primer 2 (5'-CTCGGATCCCCATGGGAAAACATCCCGGTTAT-3') introduced an XhoI site and a start codon at the 5' end and a BamHI site at the 3' ends of the PCR products. Primer 3 (5'-CCGCTC GAGTGTCCGCTAGTCGAATTCGGGCC-3') and primer 4 (5'-CTCGGATCCTTACCCATGGGAAAACATCCC-3') introduced an XhoI site (bold, primer 3) at the 5' end and a BamHI site (bold, primer 4) and stop codon (underlined, primer 4) at the 3' ends. pvAfl30 was used as the template for PCR. PCR products were isolated and digested with XhoI and BamHI, and the resulting 774- and 777-nt products were reisolated, cloned into the GFP expression vectors pEGFP-N1 and pEFGP-C1 (BD Bioscience Clontech, Palo Alto, Calif.), and linearized with XhoI or BamHI. The pEGFP-N1-VZV-ORF35 and pEGFP-C1-VZV-ORF35 plasmids were sequenced.

Nested reverse transcription-PCR (RT-PCR). Melanoma cells infected with rOka or rOka{Delta}35 were harvested after 3 days, and RNA was extracted with an RNeasy Minikit (QIAGEN, Inc.). First-strand cDNA synthesis was performed using a SuperScript III for RT-PCR kit (Invitrogen) with gene-specific primers. For the initial primers, 34fs (5'-CCCTGGAGAGTTATTGCCCCTTGCC-3'), 35fs (5'-GCCATGGTATCCCTCAGC-3'), and 36fs (5'-GAACAGGCTCTGAAAATG-3'). Nested PCRs were then performed using the following sets of primers: 34F (5'-ATGACGGCGAGATATGGGTTCGG-3'), 34R (5'-CCCTGGAGAGTTATTGCCCCTTGCC-3'), 35F (5'-ATGTCCGCTAGTCGAATTCGGGCC-3'), 35R (5'-GCCATGGTATCCCTCAGC-3'), 36F (5'-ATGGGCGTTTTGCGTAT-3'), and 36R (5'-GAACAGGCTCTGAAAATG-3') were used. For the nested primers, 34nF (5'-GATCTATCTCGTTTCC-3'), 34nR (5'-CAAGTACACCAGGGTG-3'), 35nF (5'-CAAGTGTTTTCGTTTG-3'), 35nR (5'-GGCGCATACCCTCGCAAAACTGGTG-3'), 36nF (5'-GGACGGGGCGTATGGAATTGG-3'), and 36nR (5'-GCCGTGAGGCGTTGTGCGTG-3')were used. Initial and secondary reactions were performed for 36 cycles at 94, 72, and 60°C for 30 s each. Products were visualized by agarose gel electrophoresis. Predicted product sizes were as follows: ORF34, 445 bp; ORF35, 364 bp; and ORF36, 200 bp.

Infectious focus, plaque size, and immunofluorescence assays. Six-day growth curves were determined by infectious focus assay with melanoma, Vero, or human embryonic lung (HEL) cells inoculated with approximately 1.0 x 103 PFU of the test virus, as described previously (20). A doxycycline-inducible glycoprotein E (gE)-expressing melanoma cell line, the Met-gE cell line, was also used for plaque assays (27). The mean size of 40 plaques was measured for each VZV mutant and control at 6 days after inoculation and compared by Student's t test.

Melanoma cells in chamber slides (Lab-Tek, Inc., Naperville, Ill.) were inoculated with test viruses, fixed, and stained as described previously (20). Primary antibodies were anti-VZV gE rabbit polyclonal antibody (1:200 dilution) (34) and monoclonal anti-adaptin (Ap1) antibody, specific for the Golgi adaptor complex (1:250 dilution) (Sigma, Inc.). ORF35-Flag expression was evaluated with anti-Flag polyclonal antibody (1:200 dilution) (Sigma, Inc.) and anti-gE monoclonal antibody (1:100 dilution) (Chemicon International, Inc., Temecula, Calif.). Secondary antibodies were goat anti-mouse immunoglobulin G (IgG), conjugated with Texas red, or goat anti-rabbit IgG, conjugated with fluorescein isothiocyanate (Jackson ImmunoResearch, West Grove, Pa.). Expression from ORF35-EGFP and the control (pEGFP-N1 or pEGFP-C1) was evaluated in melanoma cells transfected with Lipofectamine (Invitrogen, Inc).

Infection of human xenografts in SCIDhu mice. Skin or T-cell xenografts were made in homozygous CB-17scid/scid mice, using human fetal tissues obtained with informed consent according to federal and state regulations, as described previously (3, 5, 20, 28-32, 36, 37). Animal use was in accordance with the Animal Welfare Act and approved by the Stanford University Administrative Panel on Laboratory Animal Care. Xenografts were inoculated with VZV recombinants and passed three times in HEL cells; titers were determined for each inoculum at the time of injection. Skin xenografts were harvested at 14 and 21days and T-cell xenografts were harvested at 10 and 18 days after inoculation and analyzed by infectious focus assay, PCR, and sequencing.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ORF35 is dispensable for VZV replication in vitro. Recombinant viruses were recovered consistently from melanoma cells transfected with the intact cosmids pvFsp4, pvSpe5, pvAvr10 and pvSpe21 in combination with two independently derived pvAfl30{Delta}ORF35 cosmids, yielding rOka{Delta}35(#1) and rOka{Delta}35(#2) (Fig. 1). PCR and sequencing confirmed the ORF35 deletion (data not shown). Nested RT-PCR demonstrated that ORF35 was transcribed during VZV infection and that the ORF35 deletion did not disrupt transcription of ORF34 or ORF36 (data not shown). No ORF35 transcript was detected in RNA extracted from cells infected with rOka{Delta}35, whereas the ORF34 and ORF36 transcripts were present at levels that appeared equivalent to those seen in rOka-infected cells.

Effects of the deletion of ORF35 on plaque formation and growth kinetics in vitro. When compared to rOka, the rOka{Delta}35 recombinants exhibited a small plaque phenotype in initial transfections, which persisted upon passage in melanoma cells. The mean plaque size (± standard deviation) was 0.63 ± 0.07 mm for rOka{Delta}ORF35(#1) and 0.62 ± 0.07 mm for rOka{Delta}35(#2), compared to 1.1 ± 0.13 mm for rOka plaques (P < 0.001). Plaque sizes did not differ between cells infected with the rOka{Delta}35/35Avr mutants and rOka. In previous experiments, rOka mutants, designated rOka{Delta}ORF62/71(pORF62-R), which have only a single copy of ORF62, exhibited a small plaque phenotype associated with diminished IE62 and gE expression (36). The small plaque phenotype was corrected when Met-gE cells with tet-regulated gE expression were infected with the single-copy ORF62 mutants and induced to express gE. To investigate whether the small plaque phenotype of the rOka{Delta}35 mutants was compensated by cellular gE expression, Met-gE cells were inoculated with rOka or rOka{Delta}35 in the presence and absence of doxycyline. Without doxycycline, the mean rOka{Delta}35 plaque size in Met-gE cells was less than that of rOka (0.77 ± 0.1 mm versus 1.26 ± 0.07 mm; P < 0.01). Adding doxycycline (1.0 µg/ml) had no effect; rOka{Delta}35 plaque sizes were 0.78 ± 010 mm compared to 1.25 ± 0.09 mm in rOka-infected, induced Met-gE cells. The expected increase in plaque size, based upon previous observations (35), was demonstrated in Met-gE cells infected with the single-copy ORF62 mutant (data not shown).

Effects of the deletion of ORF35 on VZV growth kinetics in vitro. Melanoma cells, Vero cells, and HEL cells were infected with rOka, rOka{Delta}35(#1), or rOka{Delta}35(#2), and virus yields were determined over 6 days. The titers of rOka in melanoma cells were significantly higher than those of rOka{Delta}35 mutants at all time points (P < 0.05) (Fig. 2A). In Vero cells, the titers were equivalent at days 1 to 3, but rOka replicated to higher titers than the rOka{Delta}35 mutants at days 4 to 6 (P < 0.05 for all comparisons) (Fig. 2B). rOka, rOka{Delta}35(#1) and rOka{Delta}35(#2) replication was indistinguishable in HEL cells by infectious focus assay (Fig. 2C).



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  		FIG. 2. Growth kinetics of rOKA{Delta}35 and repaired viruses. Melanoma (A), Vero (B), and HEL (C) cells were inoculated with rOka{Delta}35(#1), rOka{Delta}35(#2), or rOka at 103 to 104 PFU/ml. Infectious virus yields were determined for 6 days after inoculation. (D) The growth of the repair mutants rOka{Delta}35/35@Avr(#1) and rOka{Delta}35/35@Avr(#2) was compared to that of rOka in melanoma cells. Titers are expressed as means for quadruplicate wells at each time point. The x axes indicate the days after inoculation when infected cell monolayers were harvested, and the y axes indicate the number of PFU determined by infectious focus assay.

 
Growth characteristics of rOka{Delta}35/35@Avr mutants in vitro. To document that the decreased plaque sizes and growth kinetics of the rOka{Delta}35 mutants were attributable to the ORF35 deletion, the gene was restored at the Avr site in, pvSpe21ORF35@Avr#1 (right-left orientation) and pvSpe21ORF35@Avr#2 (left-right orientation) (Fig. 1, line 5). Infectious virus was recovered regardless of the ORF35 orientation, yielding rOka{Delta}35/35@Avr(#1) and rOka{Delta}35/35@Avr(#2). PCR and sequencing of products from cosmids and infected cell DNA verified the deletions and insertions of ORF35 (data not shown).

The rOka{Delta}35/35@Avr(#1) and rOka{Delta}35/35@Avr(#2) viruses exhibited mean plaque sizes of 0.98 ± 0.1 mm and 0.97 ± 0.1mm, respectively, which was not significantly different from the mean rOka plaque size of 1.05 ± 0.13 mm. Plaque sizes of rOka{Delta}35/35@Avr(#1) and rOka{Delta}35/35@Avr(#2) were significantly larger than that of rOka{Delta}35 (P < 0.01) (data not shown). The growth kinetics of rOka{Delta}35/35@Avr(#1) and rOka{Delta}35/35@Avr(#2) did not differ from rOka, whereas virus yields were significantly higher than rOka{Delta}35 at all time points (Fig. 2D).

Deletion of ORF35 disrupts formation of syncytia in vitro. VZV replication in cultured cells is characterized by the appearance of polykaryocytes and extensive cell fusion, generating large syncytia in vitro (2, 10). Infection of melanoma cells with the ORF35 deletion mutants was associated with disrupted formation of syncytia (Fig. 3). Infected cells had multiple nuclei, but their arrangement was disorganized compared to the usual appearance of a centralized Golgi structure, as detected by Ap-1 staining of melanoma cells infected with rOka. The typical viral highways extending between cells were not detected in rOka{Delta}35-infected cells. The localization of gE was altered to a diffuse pattern, in contrast to its usual distinct expression on plasma membranes, as well as in the cytoplasm of cells infected with rOka. These changes were confirmed in two separate experiments testing both rOka{Delta}35(#1) and rOka{Delta}35(#2). Polykaryocyte formation was indistinguishable between melanoma cells infected with rOka{Delta}35/35@Avr mutants, in which ORF35 expression was restored, and rOka (Fig. 3). In these multinucleated cells, the nuclei were organized in a regular, circular pattern around centralized Golgi bodies, shown by Ap-1 staining. Viral highways, which are visible as plasma membrane projections expressing gE extending between cells, were readily apparent whether ORF35 was expressed from its native location or at the AvrII site (Fig. 3).



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  		FIG. 3. Effects of ORF35 deletion on syncytium formation and gE localization in infected cells. Immunofluorescence microscopy analysis was performed on permeabilized melanoma cells infected with rOka, rOka{Delta}ORF35, or rOka{Delta}35/35@Avr or mock-infected cells at 96 h. Monolayers were stained with polyclonal anti-VZV gE (green) antibody, monoclonal anti-Ap1 antibody, a Golgi complex marker (red), and the nuclear marker DAPI (4',6'-diamidino-2-phenylindole) (blue). Magnification, x32.

 
Localization of ORF35 protein in VZV-infected cells. Initial experiments to assess the intracellular localization of ORF35 were done with pEGFP-N1-VZV-ORF35 and pEGFP-C1-VZV-ORF35, which have EGFP at either terminus of the ORF35 product. The ORF35 product was detected predominantly in the nuclei of transfected cells, regardless of the position of the EGFP tag (data not shown). To investigate the localization of the ORF35 product in VZV-infected cells, we used a Flag tag to label ORF35 in the VZV genome. This Flag tag was 8 amino acids and was expressed along with ORF35 from the native promoter, whereas EGFP was approximately 250 amino acids and used the human cytomegalovirus immediate early promoter, which might lead to overexpression of EGFP-ORF35. Two independently derived VZV recombinants were generated with pvSpe21ORF35-Flag@Avr cosmids and designated rOka{Delta}35/35Flag@Avr (Fig. 1). Two rOka35Flag@Avr mutants were also made with pvSpe21ORF35-Flag@Avr, yielding viruses with ORF35 at the native site as well as Flag-tagged ORF35 at the AvrII site. The plaque sizes and 6-day growth kinetics of the rOka{Delta}35/35Flag@Avr and rOka35Flag@Avr mutants were indistinguishable from those of rOka (data not shown). The expected deletions of ORF35 and insertions of Flag-tagged ORF35 into the AvrII site were confirmed by PCR and sequencing. When the intracellular expression of ORF35 was examined in melanoma cells infected with rOka{Delta}35/35Flag@Avr and rOka35Flag@Avr viruses, the ORF35 product was detected predominantly in the nuclei of infected cells at 24 and 96 h (Fig. 4), as was observed under transient expression conditions with EGFP-tagged ORF35. The pattern of ORF35 localization was the same when the only copy of ORF35 was the ORF35-Flag@Avr and when the mutant had intact ORF35 at the native site along with the ORF35-Flag insertion. No nonspecific staining was observed in mock-infected cells incubated with the Flag-tag antibody or in cells infected with the Flag-tagged viruses and tested with secondary antibodies only.



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  		FIG. 4. Localization of Flag-tagged ORF35 protein in infected melanoma cells. Melanoma cells infected with rOka{Delta}35/35Flag@Avr were fixed and permeabilized at 24 and 96 h after inoculation. Infected cells were incubated with primary polyclonal rabbit antibody to the Flag tag, fluorescein isothiocyanate-conjugated anti-rabbit IgG (green), monoclonal anti-VZV gE antibody, Texas red-conjugated anti-mouse IgG (red) antibody, and the nuclear marker, DAPI (blue).

 
Infectivity of rOka{Delta}35 and rOka{Delta}35/35@Avr mutants in SCIDhu skin and T-cell xenografts in vivo. When skin xenografts were inoculated with VZV mutants lacking ORF35, replication was decreased significantly compared to rOka, as shown by delayed growth and reduced peak titers (Fig. 5A). At day 14, the mean titers of rOka{Delta}35(#1) and rOka{Delta}35(#2) in skin xenografts were 51 and 40 PFU, respectively, compared to the mean rOka titer of 7.3 x 103 PFU (P < 0.01). The mean titers of rOka{Delta}35(#1) and rOka{Delta}35(#2) were also significantly lower than rOka at day 21 (P < 0.05), although the difference was less striking than at day 14. Growth in skin xenografts was restored when ORF35 was inserted at the AvrII site, as shown in the comparison of rOka{Delta}35/35@Avr(#1) and rOka (Fig. 5A). These experiments indicated that the altered virulence of the rOka{Delta}35 mutants was due to the deletion of ORF35 and not to another unidentified mutation. PCR and sequencing of rOka, rOka{Delta}35(#1), rOKA{Delta}35(#2), and rOKA{Delta}35/35@Avr(#1) from preparations used to inoculate the skin xenografts confirmed that input viruses were as designed. The persistence of the expected mutations was confirmed in viruses recovered from six skin xenografts harvested at day 21 after inoculation with rOka, rOka{Delta}35(#1), rOka{Delta}35(#2), and rOka{Delta}35/35@Avr(#1) (data not shown).



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  		FIG. 5. Replication of rOka{Delta}35 mutants in skin and T-cell xenografts in SCIDhu mice. (A) Skin xenografts were inoculated with (viewing bars from from left to right) rOka, rOka{Delta}35/35@Avr, rOka{Delta}35#1, or rOka{Delta}35#2. Each bar represents the mean titer of infectious virus ± standard deviation, recovered from at least four xenografts harvested at 14 or 21 days after inoculation. (B) T-cell xenografts were inoculated with rOka (bars on left) and rOka{Delta}35#1 (bars on right). Each bar represents the mean titer of infectious virus ± standard deviation recovered from three to four implants harvested at 10 or 18 days. Inoculum titers are shown on the vertical axes. An asterisk indicates that titers were significantly lower than that of rOka (P ≤ 0.05).

 
The replication of rOka{Delta}35#1 and rOka in T-cell xenografts inoculated with equivalent titers of infectious virus was assessed at days 10 and 18 (Fig. 5B). At day 10, the growth of rOka{Delta}ORF35 was significantly lower than that of rOka in T-cell xenografts, although the relative difference in titers was much less than in skin xenografts at the early time point. The mean peak titer of rOka{Delta}35 was 1.5 x 103 PFU at day 10, compared to 1.9 x 104 PFU in rOka-infected T-cell xenografts (P < 0.01). The rOka{Delta}35 and rOka titers declined with similar kinetics and were not significantly different at day 18. PCR analysis of all isolates recovered at days 10 and 18 showed no change from the viruses used for inoculation (data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These experiments represent the first analysis of VZV ORF35 and its contributions to viral replication in vitro and to virulence in differentiated human skin and T cells in vivo. Although genes homologous to VZV ORF35 are considered to be among the core genes of the herpesvirus family, information about the role of these gene products in viral replication and their significance for the pathogenesis of infection is limited. Deleting ORF35 was compatible with VZV replication in vitro, although yields of infectious VZV were reduced compared to intact rOka in melanoma and Vero cells but not in fibroblasts. Removal of the ORF35 coding sequence did not alter transcription of ORF34, which encodes a capsid protein, or ORF36, which is the VZV TK gene. The effects of deleting ORF35 on infectious virus yields and plaque formation were reversed when ORF35 was inserted into a nonnative site of the viral genome, as shown with two independently generated rOka{Delta}35/35@Avr viruses and with rOka{Delta}35/35Flag@Avr recombinants. Removing ORF35 had effects on VZV replication that are similar to those observed when HSV-1 UL24 was deleted. Among the herpesvirus genes that are related to ORF35, the HSV-1 UL24 homologue has been studied most extensively (12, 21, 22, 33, 34). UL24 mutants have been generated in which the mutation was within one of the five regions that are most highly conserved among the homologous genes (21). Fourteen of 15 such mutants exhibited reduced yields of infectious virus and small plaques in cultured cells. These changes in HSV replication were also observed when UL24 mutagenesis was targeted to eliminate UL24 expression without disrupting the overlapping UL23 gene encoding HSV-1 TK. Experiments with the rOkaD35/35Flag@Avr and rOka35Flag@Avr viruses demonstrated that the intracellular localization of ORF35 was predominantly nuclear by 24 h in VZV-infected cells. Nuclear localization was also observed with transient expression of EGFP-labeled ORF35. Although anti-UL24 antibody reagents were not effective for staining infected cells, the HSV-1 UL24 protein was nuclear by 12 h when it was analyzed in subcellular fractionation experiments (33).

Extensive cell-cell fusion, resulting in the formation of large polykaryocytes, is a hallmark of VZV replication, not only in vitro but also in vivo in VZV-infected skin (2, 6). In cells infected with the rOka{Delta}35 mutants, formation of the characteristic VZV syncytia was aberrant, and plaque sizes were reduced. Polykaryocytes in rOka{Delta}35-infected cells had a disrupted arrangement of nuclei, which typically form a uniform ring around centralized Golgi within fused cells. VZV gE, with its heterodimer partner, gI, appears to be critical for cell fusion and cell-cell spread of the virus, as well as being a major envelope glycoprotein (11). Expression of gE is usually prominent on plasma membranes of VZV-infected cells, including polykaryocytes in vitro and in VZV-infected skin (6). Trafficking of gE to plasma membranes was disrupted in the absence of the ORF35 gene product. We have identified other mutations of the VZV genome that yield viruses with a small plaque phenotype, associated with gE mislocalization. For example, VZV mutants as different as those in which binding sites for cellular transactivators within the gI promoter are disrupted, and the single-copy ORF62 mutants have a small plaque phenotype (20, 36). Although localization of gE to plasma membranes was reduced in rOka{Delta}35-infected cells, the small plaque phenotype persisted in melanoma cells expressing gE. These observations suggest that deleting ORF35 may have effects on VZV replication other than disrupting gE trafficking. Interestingly, mutations of HSV-1 UL24 enhanced syncytium formation, which is unusual in HSV-infected cells except when HSV gK, gB, or UL20 are disrupted (reviewed in reference 35). The Syn phenotype of HSV-1 UL24 mutants suggested a direct or indirect role for UL24 in membrane fusion events and a requirement for UL24 protein at early times to regulate steps in virion assembly and to inhibit cell fusion until the appropriate time in the replication cycle (34). VZV replication in cultured cells depends on cell-cell spread because cell-free virus is not produced (2). As noted, these experiments with the rOka{Delta}35 mutants demonstrated that syncytium formation occurred but that the organization of nuclei surrounding the Golgi within fused cells was aberrant. Our previous investigations of VZV gene functions by viral mutagenesis have revealed that VZV genes may be important as determinants of polykaryocyte morphology independently of their effects on cell fusion. For example, VZV mutants from which glycoprotein gI has been deleted have a small plaque phenotype and aberrant polykaryocytes (25), whereas VZV mutants with disrupted ORF47 protein kinase function have normal plaque sizes but form abnormal polykaryocytes (6). For both the VZV gI null mutants and the ORF47 protein kinase-deficient mutants, the aberrant polykaryoctes were associated with marked impairment of virion assembly (6, 38). The presence of ORF35 in HSV-2 virions has been described previously (19), but experiments to document its association with HSV-1 virions have not been reported. We were unable to determine whether ORF35 protein is part of the VZ virion because we were unable to generate antibodies to ORF35 protein. Repeated attempts to use the Flag-tagged viruses and anti-Flag antibodies were unsuccessful, despite various detergents and other experimental conditions. It is possible that VZV ORF35 and HSV UL24 proteins are required to optimize virion assembly, which is consistent with the reduced yields of infectious virus observed with both VZV and HSV mutants that do not express this conserved gene product.

The comparison of rOka{Delta}35 with rOka and rOka{Delta}35/35@Avr viruses demonstrated that VZV infection was impaired in skin and T cells in the absence of ORF35. However, ORF35 appeared to be more critical as a determinant of VZV virulence in skin than in T cells, based on comparative growth kinetics and infectious virus yields of rOka{Delta}35 and rOka in vivo. HSV-1 UL24 has been shown to be important for virulence as well. The corneal inoculation of mice with HSV-1 UL24 mutants was associated with a marked reduction in the recovery of infectious virus from sensory ganglia harvested after 3 days, whereas replication in corneal epithelium was comparable to intact HSV at the same time point (22). Reactivation of UL24-deficient viruses from ganglia was decreased by 12 fold. The VZV ORF35 gene product was particularly important as a determinant of VZV virulence in skin, which is consistent with our observations that efficient polykaryocyte formation and cell-cell spread are necessary for optimal replication in differentiated human epidermal cells in vivo (6, 28). rOka{Delta}35 replication was delayed but not eliminated in T-cell xenografts. Based on our evidence that virion assembly and release of infectious particles are required for VZV T-cell tropism, these findings suggest that while ORF35 may facilitate virion assembly, its contribution to these processes is not equally important in all cell types required for the life cycle of VZV in the human host.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI053846 and AI20459). H.I. received fellowship support from the Jikei University School of Medicine and Grant-in Aid for Young Scientists (B: 15790603).

We thank Cheryl Stoddart, Gladstone Institute, University of California, San Francisco, for assistance with the thymus/liver xenografts.


   FOOTNOTES
 
* Corresponding author. Mailing address: Stanford University School of Medicine, G-311, 300 Pasteur Dr., Stanford, CA 94305. Phone: (650) 498-6227. Fax: (650) 725-8040. E-mail: aarvin{at}stanford.edu. Back

{dagger} Present address: Department of Dermatology, Jikei University School of Medicine, 3-19-18 Nishishinbashi Minato, Tokyo, Japan. Back

{ddagger} Present address: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Munich, Germany. Back

§ Present address: Fermentation Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Tokodai, Tsukuba, Ibaraki 300-2698, Japan. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, April 2005, p. 4819-4827, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4819-4827.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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