Cells, viruses and oligonucleotides

GPCMV (strain 22122, ATCC VR682), first and second generation GPCMV BAC [20, 21] derived viruses were propagated on guinea pig fibroblast lung cells (GPL; ATCC CCL 158) in F-12 medium supplemented with 10% fetal calf serum (FCS, Life Technologies), 10,000 IU of penicillin/liter, 10 mg of streptomycin/liter (Gibco-BRL), and 7.5% NaHCO3 (Gibco-BRL). The second generation GPCMV BAC encodes a truncated version of GP129 (UL128 homolog) and as such is incapable of forming a complete homolog pentameric complex (gH/gL/GP129-GP133 homolog to HCMV gH/gL/UL128-131) and lacks tropism to epithelial cells (Coleman et al., paper in preparation). In order to restore epithelial tropism to the virus, a cDNA version of the full length GP129 was introduced into the GPCMV BAC at an intergenic locus (GP25/GP26 locus) of the GPCMV genome and placed under SV40 promoter control. Virus derived from this BAC was capable of forming a pentameric complex and had restored tropism to epithelial cells. A full description of this virus, pathogenicity, tropism, congenital infection rate and pentameric complex mutant studies is the subject of a pending paper from our lab (Coleman et al.). Virus titrations were carried out on six-well plates. Plaques were stained with 10% Giemsa stain or visualized by fluorescence microscopy. All oligonucleotides were synthesized by Sigma-Genosys (The Woodlands, TX) and are listed in S1 Table. Plasmids and viral PCR products were further verified by sequencing as necessary.

Protein structure analysis

The predicted protein sequences of the GPCMV glycoproteins were analyzed by various programs. (1) Signal peptide sequence was predicted by various on line programs: http://www.cbs.dtu.dk/services/SignalP/ [48]; http://sigpep.services.came.sbg.ac.at/signalblast.html; http://www.csbio.sjtu.edu.cn/bioinf/Signal-3L/ [49]. (2) Protein transmembrane domain predicted by http://www.cbs.dtu.dk/services/TMHMM/. (3) BLAST alignments performed on MacVector.

Ethics

Guinea pig (Hartley) animal studies were carried out under IACUC (Texas A&M University) permit 2013#013. All study procedures were carried out in strict accordance with the recommendations in the “Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.” Animals were observed daily by trained animal care staff, and animals requiring care were referred to the attending veterinarian for immediate care or euthanasia. Terminal euthanasia was carried out by lethal CO₂ overdose followed by cervical dislocation in accordance with IACUC protocol and NIH guidelines.

All animals were verified by anti-GPCMV ELISA to be seronegative prior to their inclusion in the study. Convalescent antisera to GPCMV was generated by subcutaneous inoculation of GPCMV negative guinea pigs (n = 6) with 1x10^5 pfu salivary gland (SG) stock GPCMV (strain 22122). Seroconverted animals were verified 1 month post infection by anti-GPCMV ELISA. A booster inoculation of virus was given and final bleeds were taken at 8 weeks post initial inoculation. Serum samples were subsequently pooled for studies. Control negative sera was obtained from GPCMV negative animals, verified by ELISA, prior to initial GPCMV challenge.

Cloning of GPCMV glycoprotein genes and generation of knockout shuttle vectors

The predicted GPCMV glycoprotein coding sequences were based on the complete viral genome sequence (Genbank accession #AB592928.1). The coding sequence co-ordinates are: GP55 (gB) 94,164–96,869; GP73 (gN) 117,644–118,045; GP74 (gO) 117,992–119,104; GP75 (gH) 119,553–121,724; GP100 (gM) 157,482–158,531; GP115 (gL) 180,216–180,992. All genes are encoded on the complementary strand with the exception of GP73. The predicted GPCMV encoded proteins were verified as homologs of HCMV glycoproteins based on the co-linear location of the GPCMV viral gene with the HCMV genome and the percentage identity of the predicted GPCMV protein with HCMV glycoproteins [9, 10, 19]. In order to carry out expression studies and a systematic knockout of the glycoprotein genes each individual GPCMV glycoprotein ORF was PCR cloned from the viral genome into plasmid vector pUC19 or pNEB193 (New England Biolabs) using specific oligonucleotide primer pairs (S1 Table) following the protocol described in McGregor et al. [24]. The following primer pair sets were used: for GP73 oligos FGP73 & RGP73; for GP74 oligos FGP74 & RGP74; for GP100 oligos GP100F & GP100R; for GP115 oligos FGPgL & RGPgL. The PCR primers also carried an additional 5’ EcoR I restriction enzyme site to introduce flanking EcoR I sites at the ends of the GP73, GP74, GP100 and GP115 coding sequences for ease of cloning of the PCR product. The various ORFs were cloned as EcoR I fragments into pNEB193 except for GP74 which was cloned as an EcoR I fragment into plasmid pNEB193dSalI, which had the Sal I site removed from the poly-cloning region. Individual glycoprotein gene plasmid clones were verified by sequencing. The GP75 ORF was cloned as a Hind III fragment using primers FGP75 and RGP75 tagged with Hind III sites (Table 1). The glycoprotein gene plasmids were designated: pNEBgNT for GP73; pNEBGP74 for GP74; pNEBGP100 for GP100; and pNEBgL for GP115 and pNEBgH for GP75. Next, all of the glycoprotein coding plasmids were individually modified by the insertion of a kanamycin (Km) drug resistance marker cassette [22] to disrupt each coding sequence and enable positive selection of mutant GPCMV BAC clones. The Km cassette was inserted into a unique restriction enzyme site as close as possible to the start of the ORF for each glycoprotein gene plasmid construct. The Km cassette was amplified by PCR from plasmid template pACYC177 (New England Biolabs) using oligonuceotide primers KmF and KmR (S1 Table). These primers also introduced unique restriction sites at the 5’ & 3’ ends of the cassette to enable cassette cloning into the different target glycoprotein genes. Individual PCR products were amplified, gel purified and digested with the appropriate restriction enzyme as previously described [24]. The KmF and KmR basic primer pairs were synthesized with additional 5’ sequence for the specific restriction enzyme site. The following restriction enzyme sites were added to individual Km PCR cassettes: Kpn I; EcoR V; Sal I; Nru I; or BamH I. Disruption of each glycoprotein gene on each shuttle vector was carried out as follows. Plasmid pNEBgNT was digested with Nru I and an EcoR V Km cassette was inserted to disrupt the GP73 (gN) ORF at codon 71 (pNEBgNTKm). Plasmid pNEBGP74 was digested with Sal I and a Sal I Km cassette inserted to disrupt the GP74 ORF at codon 110. Plasmid pNEBGP100 was transformed into a dam^- strain of E.coli K12 bacteria (ER2925, New England Biolabs) and cut with Bcl I and a BamH I Km cassette inserted to disrupt the GP100 ORF at codon 110 (pNEBGP100km). Plasmid pNEBgL was cut with EcoR V to insert an EcoR V Km cassette to disrupt GP115 ORF at codon 45 (pNEBgLKm). The GP75 gene in pNEBgH was digested with Nru I and EcoR V to delete 791 bp of the coding sequence. The linearized DNA was band isolated as previously described and a EcoR V Km cassette inserted to disrupt the GP75 (gH) ORF at codon 200 (pUCGP75Km). In the case of GP55, a shuttle vector was designed to encode the homolog gB AD-1 and trans-membrane domains of GP55 [45]. The GP55 coding sequence (codons 400–829, genome co-ordinates 95,359–96,649c) was PCR amplified using primers FgBEc and RgBPst (S1 Table) that also introduced unique EcoR I and Pst I restriction sites at the 5’ and 3’ ends of GP55 respectively. The GP55 PCR product was cloned into pUC19 cut with EcoR I and Pst I to generate pUCgB. An EcoR V Km cassette was next introduced into pUCgB at a unique EcoR V site that disrupts the GP55 coding sequence at codon 528. The modified shuttle vector was designated pUCgBKm. The various glycoprotein knockout shuttle vectors (carrying kanamycin insertion markers) were verified by sequencing and subsequently used to create individual glycoprotein gene mutants on the GPCMV BAC in bacteria. Modified plasmids encoding Km cassettes are described in S1 Fig.

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Table 1. GPCMV Glycoprotein Genes, predicted size, homology and knockout site.

https://doi.org/10.1371/journal.pone.0135567.t001

Generation of gene knockout GPCMV BACmids and analysis of GPCMV BAC mutants

An inducible ET recombination system (GeneBridges) was introduced into DH10B bacterial cells containing a first or second generation GPCMV BAC plasmid [20, 21] using a protocol previously described [24]. Individual GPCMV glycoprotein knockout targeting shuttle vectors were linearized with a unique restriction enzyme cutting outside the glycoprotein gene flanking sequence or alternatively the kanamycin cassette and glycoprotein gene flanking coding sequence were amplified by PCR using gene specific primers as described in S1 Table. Linearized DNA plasmids or PCR products were band isolated by agarose gel electrophoresis and recovered by gene clean kit (MP Biomedical). Concentrations of DNA were modified to introduce 1μg of linear DNA into each transformation reaction via electroporation [24]. Recombinant bacterial colonies of GPCMV BAC glycoprotein knockout mutants were isolated by chloramphenicol (12.5 μg/ml) and kanamycin (20 μg/ml) antibiotic selection in LB agar bacterial Petri dishes. Bacterial plates were initially incubated at 39°C to remove the ts ET recombination plasmid (Genebridges). Mutant GPCMV BAC DNA purified by maxiprep kit (Qiagen) were analyzed by separate EcoR I and Hind III restriction digestions to verify the accuracy of the predicted genome configuration after mutation [20, 21]. Insertion of the Km drug resistance cassette into the viral genome introduced a novel Hind III restriction enzyme site at the site of mutation to enable verification of locus modification. Specific glycoprotein gene modifications were confirmed by comparative PCR analysis between wild type and mutant GPCMV BACs using common flanking primers for each gene (S1 Table). PCR reactions were carried out using conditions described in McGregor et al. [24] except the extension time at 72°C was modified based on the size of each gene (based on 30 sec extension per 500 bases). The gene knockout for mutants was further verified by sequencing of the PCR product.

GPCMV glycoprotein knockout & BAC mutagenesis characterization results

GPCMV glycoproteins genes were individually knocked out by targeted mutagenesis of the GPCMV BAC in bacteria using shuttle vectors carrying a Km drug resistance marker to disrupt each ORF. The specific site of disruption for each glycoprotein is summarized in Table 1. Targeted recombination knockout of each glycoprotein gene on the GPCMV genome was performed in separate transformation reactions in both first generation [20] and second generation [21] GPCMV BACs and gene knockouts were selected by insertion of the Km cassette into the GPCMV BAC genome. S1 Fig shows the location of the genes in the viral genome as well as the restriction enzyme profile analysis of the various mutant GPCMV BAC clones. Analysis shown is for the mutagenesis of the second generation GPCMV BAC [21]. Insertion of the Km cassette also introduced a new Hind III site encoded in the Km ORF. Modified GPCMV genomes were analyzed separately by EcoR I and Hind III restriction enzyme profile analysis. In an effort to limit redundancy the profiles shown for each mutant are either Hind III or EcoR I analysis. Additionally, two clonal mutants were generated for each knockout but only one is described. Comparative restriction fragment profiles of wild type and mutant GPCMV BAC genomes correctly demonstrated specific sub-genomic fragment modification for all mutants. Original designated GPCMV restriction fragment band nomenclature described by Gao and Isom [50] is used to identify specific band shifts. EcoR I profiles are shown for GP73, GP74, GP75, GP100 and GP115 knockouts. Hind III profile is shown for GP55 knockout. The GP73 (117,644–118,045) ORF is encoded in the EcoR I ‘C’ fragment (18,026bp) and was modified by a 1.1 kb Km marker insertion. This resulted in the modified ‘C’ fragment shifting and partially overlapping with the EcoR I ‘B’ fragment (19,766bp), see S1(iii) Fig. Similarly, the GP74 (117,992–119,1104, complementary strand) ORF is encoded on the EcoR I ‘C’ fragment with a 1.1 kb shift in size because of the Km marker insertion, see S1(iv) Fig. The GP75 (119,553–121,724, complementary strand) ORF is encoded in both the EcoR I ‘C’ and ‘B’ fragments. An internal deletion within the ORF during mutagenesis (S3 Fig) resulted in the loss of the separate bands in the mutant and a fused modified ‘C+B’ bands which has a 1.1 kb Km cassette insertion and the shift with fused bands results in an overlap with the EcoRI ‘A+y’ band (38,915bp), see S1(v) Fig. The GP100 (157482–158531, complementary strand) ORF is in the EcoR I ‘E’ band (11,210bp) and a 1.1kb insertion results in a shift in the band to 12.3 kb which partially overlaps with the ‘D’ fragment (12,061bp), see S1 Fig. The GP115 (180,216–180,992, complementary strands) is encoded in the ‘F’ fragment (174,634–184,516) and 1.1 kb insertion modifies the subgenomic fragment from 9,882bp to 10,982bp, see S1(vii) Fig. The GP55 (94,164–96,869, complementary strand) ORF encoded in the Hind III ‘K’ fragment (8,056bp) was modified by Km cassette insertion which increased the size of the fragment by 700bp because of the presence of a Hind III site within the Km cassette, see S1(ii) Fig.

The modified loci for the GPCMV glycoprotein gene mutants were also confirmed by specific PCR analysis of wild type and disrupted glycoprotein genes using common flanking primers for each gene (S1 Table). The predicted sizes of PCR products for both wild type and mutated glycoprotein genes are shown in S2 Fig using common flanking primer pairs described in S1 Table. S3 Fig shows the actual PCR results for the GPCMV glycoprotein BAC wild type and mutant loci using the common flanking PCR primer pair for each gene which produced results as expected.

Recombinant Adenovirus vectors

Recombinant defective adenoviruses (serotype 5) encoding either GFP tagged gH or mCherry tagged gL or gB were generated as high titer stocks by Welgen Inc. on HEK293 cells. The C-terminal tagged ORFs from plasmids pAcGFPNgH and pmCherryNgL or the non-tagged complete ORF of gB from pACYCIEgB (described above) were each placed under HCMV MIE enhancer promoter control in the E1 locus of the defective Ad vectors using a IE1 shuttle vector (Welgen Inc.) to generate recombinant defective adenoviruses designated AdgHGFP, AdgLmCherry and AdgB respectively. A defective Ad vector encoding GFP (AdGFP) was also used in control expression studies.

Generation of mutant virus and rescue of knockout mutants

For generation of recombinant viruses, large-scale GPCMV BAC DNA was purified from E. coli DH10B strain using a maxi plasmid kit (Qiagen). BAC DNA was transfected onto GPL cells in six well dishes using Lipofectamine 2000 (Invitrogen) as previously described [51]. GPCMV BAC transfections were carried out with two independent clones for each gene knockout. Transfections were followed for at least 3–4 weeks for the production of viral plaques. GFP positive viral plaques were detected via microscopy [51]. Non-infectious mutants produced only single GFP positive cells that did not progress to viral plaques. GPCMV mutant BAC transfections were carried out multiple times (minimum of 6 times) for each clone.

For glycoprotein gene knockout mutants, each mutant was rescued back to wild type phenotype by co-transfection with the appropriate rescue plasmid. Rescue plasmids were generate as full length glycoprotein genes cloned individually into pUC19, pNEB193 (New England Biolabs) or pLITMUS28 (New England Biolabs). Rescue plasmids provided sufficient flanking homologous recombination sequence around the site of Km insertion on the mutated GPCMV BAC to enable efficient generation of rescue virus. All rescue viruses (GP55, GP73, GP74, GP75, GP100 and GP115) were generated as previously described [22, 24]. Additionally, gH knockout derived virus was supported on a complementing GPL cell line which expressed gH in trans. The GP75 ORF was placed under HCMV MIE promoter control in an existing sleeping beauty construct [52] and cell line generated by transposition of GPL cells and selection under neomycin (G418) resistance at a selection concentration of 400 μg/ml. Complemented or rescued virus was determined by the spread of GFP tagged virus on the various cell monolayers. Specific rescue was confirmed by DNA extraction from virus infected cells and PCR analysis using common flanking gene specific primers for appropriate loci (S1 Table) as previously described [24].

RT-PCR

Time point samples were taken from wild type GPCMV infected GPL cells in a six well dish (moi = 1 pfu/cell) at 0, 4, 8, 16, 24, 48 hr post infection. RT-PCR was performed essentially as described in McGregor et al. [51]. RT-PCR reactions were performed for GPCMV glycoprotein genes GP73, GP74, GP100, and control GAPDH using primers described in S1 Table at late stage infection. In another duplicate study, chemical inhibitors (cycloheximide at 100μg/ml or phosphonoacetic acid at 200μg/ml) were included to identify the different classes of transcripts (IE, E or L). Inhibitors were used as described by Yin et al. [53]. RT-PCR was also performed on the major immediate early transcripts for GPCMV IE2, GP122 unique exon, (data not shown) using primers previously described [51].

Immunodetection assays

ELISAs

Immunoprecipitation assay

Immunoprecipitation (IP) assays were carried out on plasmid transfected or recombinant Ad transduced fibroblast cells using commercial GFP-trap reagent (ChromoTek) following manufacturer’s protocol and inclusion of protease inhibitor cocktail (Pierce) in cell lysates. Samples were subsequently analyzed by SDS-PAGE (4–20% gradient gel) and western blot using specific anti-epitope tag antibodies: HA (Novus Biologicals); FLAG (Novus Biological); GFP (Santa Cruz); Myc-c (Novus Biologicals); and mCherry (Clontech). Appropriate secondary anti-mouse or anti-rabbit HRP conjugate (Cell Signaling Technology) were also used following standard western blot protocol.

GPCMV Neutralization assay

GPCMV neutralization assays were conducted using convalescent GPCMV positive sera. Serially diluted guinea pig serum was incubated with approximately 50 pfu of GFP positive GPCMV, vAM403 [20], in media containing 0.5% rabbit complement (Equitech Bio, Kerrville, TX) for 60 minutes at 37°C. Each dilution was done in duplicates and virus only was used as control. To show neutralization specificity, monoclonal antibody 29–29 was used at 1:500 dilution in place of anti-GPCMV sera [44]. The neutralization reactions were added to wells of a 12 well-plate containing confluent monolayers of GPL cells and incubated for 2 hours. The wells were washed twice with PBS before complete F12- media was added and incubated for 3–5 days at 37°C. The plates were fixed with 4% paraformaldehyde and GFP positive plaques counted using a fluorescent microscope. Control assays were also performed with GPCMV negative sera which did not inhibit virus infection at dilutions used.

GPCMV Glycoprotein gene expression

Based on the GPCMV genome sequence [10], the GPCMV glycoproteins associated with the homolog complexes in HCMV are predicted to be encoded by the GPCMV genes GP55 (gB), GP73 (gN), GP74 (gO), GP75 (gH), GP100 (gM), GP115 (gL). Analysis of the predicted proteins (http://www.cbs.dtu.dk/services/SignalP/) indicated potential N-terminal signal leader peptide sequence of 23 and 24 amino acids in gB and gH respectively (S4 Fig). N-terminal signal peptide sequences were also predicted (http://sigpep.services.came.sbg.ac.at/signalblast.html) for gM (26 amino acids), gL (31 amino acids) and gN (44 amino acids), see S4 Fig The gO homolog protein had insufficient conserved sequence (based on results from analysis programs used) to fully predict a signal peptide sequence. However, the inability to detect a N-terminal epitope/GFP tagged glycoprotein in transient expression assays (data not shown) in contrast to successful detection of C-terminal epitope tagged proteins in transient expression studies (see individual glycoprotein expression results) would suggest that all the GPCMV glycoproteins have N-terminal signal sequences. Previous transient expression studies with other N-terminal tagged, epitope (FLAG) or GFP, GPCMV proteins demonstrate the success of this approach for detection of proteins that lack an N-terminal signal peptide sequence. These previous GPCMV protein expression studies included: GP35 (UL35 homolog), GP44 (viral DNA polymerase subunit), GP82 (pp71 homolog), GP83 (pp65 homolog), GP84 (UL84 homolog), GP97 (viral kinase) [22–24, 55]. The size of the predicted GPCMV glycoproteins and the percentage identity to HCMV glycoproteins by BLAST analysis is indicated in Table 1. The gB (gCI homolog complex), gM and gN (gCII homolog complex) exhibit the highest identity to HCMV proteins. All of the viral glycoproteins have additional potential post-translational modification by glycosylation (O or N linked glycosylation) and the total number of potential sites are indicated in Table 1 and discussed in later sections. Most notably, the gO protein homolog encoded by GP74 is predicted to be heavily N-glycosylated as is the case for HCMV gO and the potential glycosylation sites are conserved between HCMV and GPCMV. This is discussed in more detail in a later section on the gH/gL/gO complex.

Expression kinetics of the GPCMV gB, gH and gL genes (GP55, GP75 and GP115 respectively) have been previously described.[45, 56, 57]. Transcription from genes GP73, GP74 and GP100 (encoding gN, gO and gM respectively) were analyzed by RT-PCR at various time points post infection (input virus moi = 1pfu/cell). The time course assay (Fig 1) indicated that the GP73, GP74 and GP100 genes were expressed under similar kinetics based on comparison with genes from different classes of expression: immediate early, IE, (GP122); early, E, (GP54); and late, L, (GP83) [51, 58, 59]. The glycoprotein genes (GP73, GP74 and GP100) are expressed as early/late genes. The use of transcription chemical inhibitors (cycloheximide and phosphonoacetic acid) more precisely place GP73 (gN), GP74 (gO) and GP100 (gM) as late transcripts (S5 Fig) as transcripts failed to be detected in the presence of inhibitors. As a positive control, the GPCMV IE2 transcript (GP122) could be detected in these samples (S5 Fig) which demonstrated the presence of viral transcripts and integrity of the samples.

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Fig 1. Electrophoresis of GPCMV glycoprotein gene expression by RT-PCR assay.

Agarose gel analysis of RT-PCR products. Expression of viral glycoprotein genes GP73 (gN), GP74 (gO) and GP100 (gM) was investigated at various time points post infection. Input virus was wild type GPCMV (strain 22122) on GPL cells (moi = 1 pfu/cell). RT-PCR was performed as described in materials and methods. RT-PCR analysis of time point samples 0, 4, 8, 16, 24, 48 hr post infection indicated. Controls: C1, no template; C2, infected cell lysate no reverse transcriptase stage. GAPDH control RT-PCR for all time samples.

https://doi.org/10.1371/journal.pone.0135567.g001

Transient expression studies of GPCMV homolog glycoproteins

In order to evaluate cellular location and interactions of the GPCMV glycoproteins, the various ORFs were cloned into commercial mammalian expression vectors and epitope tagged at the C-terminus. Construction of mammalian expression vectors encoding the GPCMV glycoprotein ORFs was as described in materials and methods. Transient expression studies were carried out on GPL cells and subsequently analyzed by: western blot, to verify protein size; immunofluorescence, for cellular co-localization of proteins; immunoprecipitation assay, to demonstrate specific protein:protein interactions.

GPCMV gM (GP100)/gN (GP73) studies

In HCMV, gM/gN proteins form a glycoprotein complex [60] and potentially GPCMV encodes a homolog complex. In GPCMV, gM is predicted to be a type III membrane protein with 8 possible membrane spanning domains and GPCMV gN is predicted to be a type I membrane protein with two potential transmembrane domains which is similar to HCMV. S6 Fig compares the predicted transmembrane domains of gM and gN in GPCMV and HCMV which show similarity in location of predicted transmembrane domains respectively. Expression plasmids for gN (GP73 mCherry or FLAG C-terminal tagged) and gM (GP100 C-terminal GFP tagged) were used to transiently express proteins individually (Fig 2). As with HCMV, GPCMV gN is predicted to be post-translationally modified by both O and N-linked glycosylation (Table 1) and two gN bands were detected with higher than expected molecular weight for both gNmCherry and gNFLAG. The predicted sizes for gN tagged proteins are shown in Fig 2, gNmCherry (42.8 kDa) and gN(f)FLAG (17.1 kDa). Two different molecular weight species were detected for each tagged gN protein: approximately 37 and 45 kDa (gNmCherry); and approximately 25 and 40 kDa (gN(f)FLAG). In order to demonstrate that the higher molecular weight protein was associated with post-translational modification of gN a N-terminal truncated gN (gN(s)FLAG) was generated. This ORF, which lacked the first 40 amino acids (as well as a complete predicted signal peptide sequence (S4 Fig), underlined, Fig 2A), initiated at the first internal methionine (codon 41 highlighted in red, Fig 2A). The full length gN (gN(f)FLAG) with an intact predicted signal peptide leader sequence (Fig 2A and S4 Fig) could undergo post translation modification. In contrast, the truncated gN(s) which lacked the leader N-terminal sequence would be impaired for post-translational modification. Western blot assays of plasmid transfected cell lysates detected a single species of protein for the truncated gN(s)FLAG of approximately 20 kDa in size, see Fig 2C(iii). This contrasted with the full length gN (gN(f)FLAG), where two species of gN protein could be detected (25 and 40 kDa), Fig 2C(ii). This indicated that the N-terminal end of the gN protein contained a peptide leader sequence (predicted size of 44 amino acids, S4 Fig) that was most likely necessary for post-translational modification of gN. Importantly, all of the potential glycosylation sites of full length gN were retained in the truncated gN(s) construct (Fig 2A). Tunicamycin (N-glycosylation inhibitor) was included in the transient expression of full length and truncated FLAG tagged gN proteins to further verify post translational modification. In the presence of tunicamycin, the full length gN (gN(f)FLAG) was detected as a single molecular weight species of approximately 20kDa (Fig 2D(i)), whereas the size of the truncated gN was unaffected by the presence of the inhibitor. We concluded that the higher molecular weight protein was a result of post-translation modification which was most likely N-glycosylation. However, even in the presence of tunicamycin, the truncated gN protein had a higher molecular weight than expected and potentially this might be due to predicted O-glycosylation of gN but this awaits further investigation.

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Fig 2. Transient expression of GPCMV gM and gN homologs and analysis of tagged proteins by western blot.

A. Predicted amino acid sequence for GP73 (gN) with potential glycosylation sites shaded in green. A truncated version of gN was generated by deletion of the first 40 codons, which included the majority of the predicted signal peptide sequence (underlined, see also S4 Fig). The truncated gN(s) initiated from the first internal methionine (shaded in red). B. Figure of gN and gM expression constructs. Full length gN tagged with C-terminal mCherry was designated gNmCherry, B(i). Full length gN with C-terminal FLAG epitope tag was designated gN(f), B(ii), and truncated gN designated gN(s), B(iii). Full length gM was C-terminal tagged with GFP and designated gMGFP, B(iv). Size of predicted MW of tagged proteins indicated (kDa). C. Western blots were performed on transient plasmid expression of gN and gM tagged proteins in GPL cells. C(i) gNmCherry expression detected using anti-mCherry antibody. Lanes: (1) kDa ladder; (2) and (3) gNmcherry; (4) mock untransfected GPL cell lysate. C(ii) gN(f)FLAG expression detected using anti-FLAG antibody. Lanes: (1) kDa ladder; (2) and (3) gN(f)FLAG. C(iii) gN(s)FLAG expression detected using anti-FLAG antibody. Lanes: (1) kDa ladder; (2) and (3) gN(s)FLAG (4) mock untransfected GPL cell lysate. C(iv) gMGFP expression detected using anti-GFP antibody. Lanes: (1) kDa ladder; (2) and (3) gM; (4) mock untransfected GPL cell lysate. D. gN and gM expression in the presence or absence of glycosylation inhibitor. D(i) Western blot of gN(f)FLAG in the presence of tunicamycin (lane 2) or absence (lane 1). D(ii) Western blot of gN(s)FLAG in the presence of tunicamycin (lane 2) or absence (lane 1). D(iii) Western blot of gMGFP in the presence of tunicamycin (lane 2) or absence (lane 1). Control mock untransfected GPL cell lysate lane 3 D(i)-(iii).

https://doi.org/10.1371/journal.pone.0135567.g002

In contrast to gN, the gM (GP100) protein is not predicted to be as extensively glycosylated with two N-glycosylation sites predicted (see Table 1). Western blot analysis indicated that the gMGFP fusion protein was smaller than expected in size, approximately 55 kDa compared to the predicted size of 66.6 kDa (Fig 2B and 2C(iv)). HCMV gM [60] encodes a signal peptide leader sequence and a signal sequence is predicted for GPCMV gM (S4 Fig). Presumably, cleavage of the predicted signal peptide sequence (26 amino acids) accounted for the difference in size of the detected protein. Inclusion of tunicamycin in the transient expression assay of gMGFP did not appear to have any effect on the size of the detected protein (Fig 2D(iii)) which supported the assumption that gM is not extensively post-translationally modified by glycosylation. In gMGFP western blots, a fainter higher molecular weight species was detected at about 100 kDa which might represent a non-fully denatured form of the protein. It should be noted that gM samples in Laemmli denaturing loading reagent were heated at 37°C for 5 minutes prior to loading on a SDS-PAGE gel as opposed to the normal procedure of 95°C for 5 minutes as we found that samples treated at high temperature became partially insoluble and did not enter the gel and instead were retained in the wells of the gel. Presumably, the predicted 8 transmembrane structure of the protein (S6 Fig) accounted for the technical difficulty associated with analysis of this protein by SDS-PAGE. Future experiments with denaturing urea conditions might help further improve protein resolution.

Transient expression of gM and gN in plasmid transfected GPL fibroblast cells demonstrated a co-localization of both proteins in the cytoplasm (Fig 3). Co-localization of gN with gMGFP was similar regardless of tag used for full length gN. Additionally, the N-terminal truncated gN, gN(s), also co-localized with gM in a similar manner (Fig 3I–3L). Importantly, this results is also observed in HCMV [61]. Immunoprecipitation (IP) studies of transiently expressed proteins confirmed specific protein:protein interaction between gM (GFP tag) and gN (mCherry or FLAG tagged). Immunoprecipitation assays were carried out with a commercial IP system, GFP trap (ChromoTek), which consists of coupled recombinant single domain anti-GFP antibody fragments derived from alpaca as monovalent matrices (agarose beads). In addition to normal antibodies, Camelidae make a second antibody called heavy chain antibody that lack a light chain and bind their antigen via a single variable domain. This approach reduces potential background associated with the use of regular antibodies in IP assays. Manufacturer’s protocol (ChromoTek) was followed to immunoprecipitate any proteins that interacted with gMGFP (Fig 4) or control GP84GFP [51]. Proteins were detected at approximately 50 kDa and 100 kDa that corresponded to gMGFP (Fig 4i, 4iii and 4iv). Both mCherry and FLAG tagged full length gN proteins were precipitated by interaction with gMGFP and both high molecular weight and lower weight species of gN were equally precipitated (Fig 4i and 4iii). Immunoprecipitation of truncated gN(s) indicated both glycosylated and non-glycosylated versions of gN interact with gM (Fig 4iii and 4iv) and that the complex is not dependent upon the N-terminal domain of gN. The IP results confirmed the suggested co-localization/interaction data seen in Fig 3. Control IP of GFP tagged GP84 [51] with gN(f)FLAG failed to show any specific interaction, see Fig 4(ii), which demonstrated specificity of the assay. We concluded that in GPCMV a functional gM/gN homolog complex is formed. In HCMV, the gM/gN complex is considered an important antibody target. The immune response to GPCMV gM/gN homolog complex was evaluated by a newly established ELISA (see section on immune response to GPCMV glycoprotein complexes).

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Fig 3. Transient co-expression of GPCMV gM and gN.

Tagged versions of gN and gM were transiently co-expressed in GPL cells and cellular localization patterns investigated by immunofluorescence or autofluorescence assay. Panels A-D, gMGFP and gNmCherry co-localization studies. A and B show gM and gN separately within the same cell. C is the merged image for A and B. D is the overlay of C with DAPI stain to indicate location of the nucleus. E-H, gMGFP and gN(f)FLAG co-localization with G merged image for panels E and F. H the overlay for DAPI stain with merged image G. I-L, gMGFP and gN(s)FLAG co-localization with K merged image for I and J. L overlay with DAPI staining.

https://doi.org/10.1371/journal.pone.0135567.g003

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Fig 4. GPCMV gM /gN complex formation and immunoprecipitation (IP) assays.

All IPs were performed with GFP-Trap (ChromoTek) as described in materials and methods to immunopreciptiate proteins that interacted with gMGFP or GFP control. (i) gMGFP and gNmCherry co-expression and IP. Lanes 1 and 4 are total cell lysate of gMGFP and gNmCherry, respectively. Lanes 3 and 6, IP reactions. Lanes 2 and 5, control mock (MI) cell lysate. For gM detection, anti-GFP antibody (lanes 1–3). For gN detection, anti-mCherry antibody (lanes 4–6). (ii) Control GFP IP. GP84 protein tagged with GFP [51] was co-expressed with gN(f)FLAG. Lanes 1 and 4 total cell lysate for GP84GFP and gN(f)FLAG respectively. Lanes 3 and 6, IP reactions for GP84GFP and gN(f)FLAG transfected cells. Lanes 2 and 5 mock control (MI). (iii) gMGFP and gN(f)FLAG co-expression and IP. Lanes, 1 and 4 are total cell lysates of gMGFP and gN(f)FLAG transfected cells respectively. Lanes 3 and 6, IP reactions for gMGFP and gN(f)FLAG transfected cells. Lanes 2 and 5 mock (MI) control cell lysate. 6. Detection for gN(f)FLAG by anti-FLAG antibody. (iv). gMGFP and gN(s)FLAG co-expression and IP. Lanes, 1 and 4 are total cell lysate of gMGFP and gN(s)FLAG transfected cells respectively. Lanes 3 and 6, IP reactions for gMGFP and gN(s)FLAG transfected cells. Lanes 2 and 5, mock control (MI). Detection for gN(s)FLAG by anti-FLAG antibody. Specific protein bands are indicated by an arrow. In gMGFP expressing cells a second higher MW protein (100 kDa) was detected and labelled x. All gels (4–20%) SDS-PAGE included a lane for a kDa ladder (MagicMark Protein Standard, Life Technologies). Ladder lanes not shown.

https://doi.org/10.1371/journal.pone.0135567.g004

GPCMV gH(GP75)/gL(GP115)/gO(GP74) studies

In HCMV, the gH glycoprotein interacts with gL and gO to form the gCIII triplex complex or with gL, UL128, UL130 and UL131 proteins to form the pentameric complex [32, 62, 63]. While both complexes are important immune targets, our focus in this paper is on the GPCMV homolog gH/gL/gO complex. Expression plasmids (Fig 5) or recombinant defective adenoviruses encoding GPCMV gH (AdgHGFP, GP75 GFP C-terminal tag) and gL (AdgLmCherry, GP115 mCherry C-terminal tag) were used in conjunction with wild type or defective mutant gO (GP74 3xFLAG C-terminal tag) expression plasmids to determine interactions between the homolog proteins in the presence and absence of virus.

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Fig 5. Transient expression constructs for GPCMV gH, gL, gO glycoproteins.

(i) Shows the structure of the C-terminal tagged ORFs and expression plasmids: gH (GP75) was tagged with GFP; gL (GP115) was tagged with mCherry; gO (GP74) both wild type and mutant ORFs were tagged with 3xFLAG tag. (ii) BLAST alignment of wild type and mutant gO ORFs. The 13 predicted N-glycosylation sites were knocked out in the mutant (gOdef) by substitution of an alanine (A) in place of threonine (T) to disrupt the N-glycosylation recognition sequence (N-X-T/S).

https://doi.org/10.1371/journal.pone.0135567.g005

As with HCMV, the GPCMV gO homolog protein, encoded by GP74, is predicted to be extensively modified post-translationally by N-linked glycosylation (codon recognition sequence NXT/S, 13 potential sites), see Table 1 and S2 Fig A defective gO ORF was generated synthetically which removed all 13 potential N-glycosylation sites by codon modification from T to A in the NXT motif. Fig 5(ii) shows BLAST alignment between wild type and mutant GP74 proteins. Both wild type and mutant gO were C-terminal FLAG tagged, underlined in Fig 5(ii). Separate transient expression studies of wild type and defective gO were performed under various conditions (Fig 6). Both versions of the gO protein have similar cellular localization (Fig 6). Western blot assays of transiently expressed gO proteins indicated that the wild type protein has a higher molecular weight (80kDa) than the mutant gO (45 kDa), but the predicted difference in size between the two proteins was 0.2 kDa. Additionally, both proteins should be approximately 45 kDa in size (Fig 6). Potentially, post-translation N-glycosylation associated with wild type, but not the mutant gO, (Fig 6E) accounted for the difference in size. When glycosylation inhibitor tunicamycin was included in experiments, the majority of the wild type gO protein was the lower molecular weight protein species (45 kDa). The mutant gO molecular weight size was unchanged (Fig 6J) which suggested that the difference in molecular weight was due to N-glycosylation. Transient expression of gO (wild type and defective) in the presence of virus resulted in the ability to detect both versions of wild type gO (N-glycosylated and non N-glycosylated), whereas the defective gO remained unchanged (Fig 6O). We concluded that gO is N-glycosylated and during GPCMV infection the level of gO N-glycosylation is modified possibly by disruption or modification of the secretory pathways but this awaits further investigation.

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Fig 6. Transient expression of wild type or mutant gO in the presence or absence of GPCMV.

The cellular location and molecular weight of gO protein was investigated by transient expression studies. Panels E, J and O are western blots for wild type or mutant gO using anti-FLAG antibody. Other panels are immunofluorescene images of wild type and mutant gO protein cellular localization by transient plasmid expression in GPL cells (A-D); GPL cells the presence of tunicamycin (glycosylation inhibitor, 2.5 ug/ml) (F-I); GPL cells plus GPCMV (K-N). Matched paired panels for gO (FITC) or gO (FITC) and DAPI (merged): A and B; C and D; F and G; H and I; K and L; M and N. Western blots: for wild type or gO mutant (E); wild type or gO mutant in the presence of tunicamycin (J); wild type or gO mutant in the presence of GPCMV (O).

https://doi.org/10.1371/journal.pone.0135567.g006

Fluorescence and immunofluorescence assays of transiently expressed GPCMV gH, gL and gO homolog proteins demonstrated that gH and gL co-localized in the cytoplasm of GPL fibroblast cells (Fig 7A–7D). Consequently, initial interactions between gH/gL would not appear dependent upon gO. However, in the absence of gO, gH/gL appeared to form aggregates in the cytoplasm. In the presence of gO, the gH/gL proteins are more uniformly distributed in the cytoplasm (Fig 7E–7H). Co-localization of gO with gH/gL was independent of the N-linked glycosylation state of gO as both wild type and mutant gO co-localized with gH/gL in a similar manner (Fig 7I–7L). In order to further evaluate specific protein:protein interactions, immunoprecipitation studies of transiently expressed proteins (gH, gL and gO) were carried in GPL cells using GFP trap (ChromoTek) to pulldown all proteins that interacted with GFP tagged gH or GFP control. Initial studies confirmed specific protein:protein interaction between gH (GFP tag) and gL (mCherry tag) (Fig 8A), whereas control IP of GFP and gL failed to pulldown gL (Fig 8B). Pulldown studies also demonstrated a specific interaction of gO with gH and gL (Fig 8D and 8E) which was not dependent upon the presence of virus (Fig 8E). As expected, based on co-localization studies, the gO mutant protein (GP74def) was also immunoprecipitated which demonstrated complex formation was independent of N-glycosylation status (Fig 8F). An additional control IP assay with FLAG tagged GP44, viral polymerase subunit protein [51], and gHGFP demonstrated that there was no non-specific interactions with gH (Fig 8).

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Fig 7. Transient expression and cellular co-localization of GPCMV gH, gL and gO.

Expression plasmids described in Fig 5 were used to transiently express the viral glycoproteins in GPL cells. Panels A-D, gHGFP and gLmCherry co-expression. A and B cellular location of gH and gL within the same cell respectively. C, overlay of A and B. D, merge of overlay and DAPI stain to indicate location of nucleus. Panels E-H, gHGFP, gLmCherry and gOFLAG. Individual cellular localization panels E, F and G. Merged panel H, shows co-localization of all three protein and DAPI stained nucleus. Panels I-L are for gHGFP, gLmCherry and gO(def)FLAG. Individual panels I, J and K. Merged image (L) for all three panels plus DAPI stained nucleus.

https://doi.org/10.1371/journal.pone.0135567.g007

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Fig 8. GPCMV gH/gL/gO complex formation and immunoprecipitation (IP) assays.

All immunoprecipitations were performed with GFP-Trap (ChromoTek) as described in materials and methods to demonstrate protein:protein interaction with GFP tagged gH. Proteins were detected by specific antibodies to tags (GFP, mCherry or FLAG). Transient expression assays on GPL cells were carried out in different plasmid combinations as indicated A-F. Samples shown for each combination are total cell lysate (Lys) and IP from total cell lysate. Control proteins were GFP or FLAG tagged GP44 (polymerase subunit protein). A. gHGFP and gLmCherry IP: A(i) gHGFP western; A(ii) gL(mCherry) western. B. GFP control and gLmCherry control: B(i) GFPwestern; B(ii) gL (mCherry) western. C. GP44 and gHGFP: (i) gHGFPwestern; (ii) GP44(FLAG) western. D. gHGFP and gO (wt) in the presence of GPCMV: (i) gH western; (ii) gO(FLAG) western. E. gHGFP, gLmCherry and gO (wt): (i) gH western; (ii) gO (FLAG) western IP. F. gHGFP, gLmCherry and gO(def): (i) gH western; (ii) gO(def) western.

https://doi.org/10.1371/journal.pone.0135567.g008

A series of further transient co-expression studies were carried out in GPL cells with gHGFP and gO (both wild type and mutant versions) to evaluate the ability of gH to complex with gO in the absence of gL. Fig 9 demonstrated that the gO mutant formed a dimer complex with gH. However, in order for wild type gO to form a detectable complex with gH, the glycosylation inhibitor tunicamycin needed to be present during transient expression. If wild type gO formed a dimer complex with gH in the absence of tunicamycin then it was below the sensitivity of detection for our IP assay (data not shown). We concluded that a homolog gH/gL complex can form in GPCMV and that a triplex with gO is possible but is independent of the N-glycosylation state of gO. Additionally, gH can form a dimer with gO but only with non-N-glycosylated gO but the significance of this novel dimer awaits further investigation, as does the influence of gH glycosylation status upon protein interaction. A specific gH/gO dimer complex has not been identified in HCMV and potentially this might be due to the lack of studies with a mutant gO protein deficient in predicted N-glycosylation sites. This awaits studies in HCMV with specific gO mutants. The immune response to GPCMV gH/gL homolog complex is potentially an important component of the protective immune response in convalescent animals. The gH/gL immune response was evaluated by a newly established ELISA (see section below on immune response to GPCMV glycoprotein complexes).

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Fig 9. GPCMV gH/gO complex formation and immunoprecipitation (IP) assays.

All immunoprecipitations were performed with GFP-Trap (ChomoTek) as described in materials and methods. Proteins detected by specific antibodies to tags (GFP or FLAG). Transient expression assays were carried out in different plasmid combinations as indicated A-E. Samples shown for each combination are total cell lysate (Lys) and IP from total cell lysate. Control protein was GFP. gHGFP and gOdefFLAG IP in presence (A) or absence (B) of gLmCherry: A(i) gH western and A(ii) gO (def) western; B(i) gHwestern; (ii) gO (def) western. C. GFP and gO(def) FLAG control: C(i) GFPwestern; C(ii) gO(def) western. D. gHGFP and gO(wt) FLAG in the presence of gLmCherry and tunicamycin: D(i) gH western; D(ii) gOgO western. E. gHGFP and gO(wt) FLAG in presence of tunicamycin: E(i) gH western; E(ii) gOwestern.

https://doi.org/10.1371/journal.pone.0135567.g009

Immune response to GPCMV glycoprotein complexes

The immune response to GPCMV and viral glycoprotein homolog complexes were investigated for pooled sera from animals convalescent for GPCMV. An in-house anti-GPCMV ELISA (as described in materials and methods) showed high sensitivity for viral antigens when compared to a commercial anti-GPCMV ELISA kit (Xpress Bio), see Fig 10A. The in-house anti-GPCMV ELISA sensitivity level (end point >1:2560) was twice as great as the commercial ELISA kit (end point 1:1280), based on net OD values and the negative cut-off of 0.2. The OD values of negative serum were similar for both in-house and commercial assays. Neutralizing antibody assay of GPCMV convalescent serum on GPL cells demonstrated a neutralizing titer of 1:640 (Fig 10). This was similar to values previously reported [46]. The immune response to GPCMV gB, gH/gL and gM/gN homolog complexes was evaluated by newly established ELISAs as described in materials and methods (Fig 10B). Convalescent sera from GPCMV infected animals demonstrated a specific immune response to GPCMV viral glycoprotein complexes. The GPCMV gB specific ELISA had an endpoint of 1:1280 dilution. The gB protein is the immunodominant glycoprotein complex in both HCMV and GPCMV [64, 65] and our assays confirmed this observation. Depletion of anti-gB antibodies from convalescent sera was confirmed by gB specific ELISA (Fig 10D). Additionally, S7 Fig demonstrated by Western blot the successful depletion of the gB specific immune response. However, the gB antibody depleted sera in anti-GPCMV ELISA only showed a slight decrease in endpoint titer (Fig 10C). Furthermore, depleted sera retained neutralizing capability (1:320) which emphasized the importance of other glycoprotein target antigens in protection against GPCMV infection. In HCMV, the immune response is directed to the gH/gL complex rather than the gH protein alone [56,57]. A GPCMV gH/gL ELISA demonstrated a specific immune response with an endpoint of 1:640 serum dilution (Fig 10B). A relatively robust immune response is similar to that seen in HCMV convalescent sera. Although gH/gL can complex with gO to form a triplex, we found that gO is not required as a component in the ELISA assay (data not shown). It remains to be determined if the triplex is required in GPCMV to enhance the immune response. In preliminary immunogenicity studies it would appear that gH/gL is immunogenic in the absence of gO (data not shown; Choi and McGregor unpublished observation). An ELISA to gH only could not give an accurate reading above background at the dilutions used. However, specific immune response to gH could be demonstrated by western blot analysis (data not shown). The immune response to the gM/gN complex was lower than the response to gB or gH/gL with an end titer of 1:320 (Fig 10B). Anti-gB and anti-GPCMV antibodies have been demonstrated to neutralize virus infection on fibroblast cells. Here we demonstrate specific immune response to the various glycoprotein complexes as well as neutralizing antibodies that target viral glycoproteins in addition to gB. The importance of these specific neutralizing antibodies in the context of virus entry into various cell types awaits further study.

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Fig 10. Antibody immune response of convalescent guinea pigs to GPCMV and GPCMV glycoprotein complexes determined by ELISAs.

Pooled sera from GPCMV infected convalescent animals was used to evaluate immune response to GPCMV (A) or to specific glycoprotein complexes (B). (A) Anti-GPCMV ELISA. The immune response to GPCMV antigens was analyzed by an in-house ELISA (blue line square) compared to commercially available GPCMV ELISA kit from Bioexpress (green line circle). GPCMV sera negative for both assays are shown in blue and green dotted lines. (B) Immune response of convalescent pooled guinea pig serum to individual glycoprotein complexes. Anti-gB, light blue line (triangle); Anti-gH/gL, orange line (closed circle); Anti-gM/gN, purple line (star). Convalescent sera depleted of anti-gB antibody (-gB) (as described in materials and methods) was retested by ELISA to (C) anti-GPCMV ELISA (solid green line) to demonstrate retention of antibody response to other viral antigens, and (D) anti-gB specific ELISA (solid light green line) to demonstrate gB depletion compared to the original undepleted sera. All sera were diluted from 1:80 to 1:2560 in doubling dilutions. Control sera from animals negative for GPCMV were used for base line in all assays. ELISAs performed as described in materials and methods. Base level for background indicated by horizontal dotted red line.

https://doi.org/10.1371/journal.pone.0135567.g010

Characterization of the essential nature of GPCMV glycoproteins

In order to determine the essential nature of the GPCMV glycoproteins, each gene was individually knocked out by targeted mutagenesis of the GPCMV BAC in bacteria using shuttle vectors carrying a kanamycin (Km) drug resistance marker to disrupt each ORF as described in materials and methods. The specific site of disruption for each glycoprotein is summarized in Table 1. Targeted recombination knockout of each glycoprotein gene on the GPCMV genome was performed in separate transformation reactions in both first generation and second generation GPCMV BACs and gene knockouts were selected by insertion of the Km cassette into the GPCMV BAC genome. S1 Fig shows the location of the genes in the viral genome as well as the restriction enzyme profile analysis of the various mutant GPCMV BAC clones compared to wild type genomes. Analysis shown is for the mutagenesis of the second generation GPCMV BAC [21]. Modifications to the BAC genomes for each mutant were as expected. More in depth analysis of the characterization of the GPCMV BAC profiles is provided in the Materials and Methods section. The modified loci for the GPCMV glycoprotein gene mutants were also confirmed by specific PCR analysis of wild type and disrupted glycoprotein genes using common flanking primers for each gene. S3 Fig shows the predicted sizes of PCR products for both wild type and mutated glycoprotein genes using common flanking primer pairs described in S1 Table. S4 Fig shows the actual PCR results for the GPCMV glycoprotein BAC wild type and mutant loci using the common flanking PCR primer pair for each gene. The actual size modification to the PCR product for each knockout glycoprotein locus (GP55, GP73, GP74, GP75, GP100 and GP115) in comparison to the wild type locus was as predicted. The GPCMV mutant PCR products were additionally sequenced to further verify knockout mutations (data not shown).

DNA from each individual knockout mutant GPCMV BAC was transfected separately onto GPL cells to determine if the specific glycoprotein gene knockout was lethal for the virus. Duplicate clones were generated for each mutant (data not shown). Additionally, each mutant BAC was co-transfected with the appropriate rescue plasmid or rescue PCR product encoding the respective wild type glycoprotein gene to generate rescue virus from lethal knockout mutants (see materials and methods). The results shown in Fig 11 demonstrated that all of the encoded GPCMV glycoproteins (gB, gH, gL, gM, gN, gO) are essential for virus replication in tissue culture. The presence of a GFP reporter gene encoded in the viral genome enabled real time tracking of virus development and spread across the cell monolayer. Non-infectious GPCMV BAC mutants remained as a single GFP positive transfected cell during the course of the assay. Rescue of the mutant locus enabled development of rescue virus which was detected by GFP virus spread (Fig 11). Overall, the results for GPCMV are similar to that obtained for HCMV [27, 66] with the exception of gO which is essential in GPCMV lab adapted virus (virus lacking epithelial tropism with defective UL128-131 homolog locus) but non-essential/ semi-essential in virus with epithelial tropism (intact homolog UL128-131 genes). All knockout GPCMV mutants could be successfully rescued by co-transfection of the appropriate rescue plasmid to generate wild type virus (Fig 11).

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Fig 11. Transfection of glycoprotein mutant KO GPCMV BACs onto GPL cells.

Individual mutant GPCMV BACs were separately transfected onto GPL fibroblast cells to regenerate virus. GFP reporter gene encoded in the viral genome enabled real time tracking of the development of virus from individual transfected cells. Glycoprotein mutant GPCMV BACs were either transfected individually (panels A, C, E, G, I, K and L) or in combination with a rescue plasmid encoding a wild type locus to restore the mutant back to wild type phenotype where GFP virus could be detected spreading across the cell monolayer (panels B, D, F and H). The gH mutant was also transfected onto a cell line expressing gH in trans to support virus growth (panel J). A gH rescue virus was also generated by co-transfection with a rescue locus plasmid (data not shown). Panels K and L show the outcome for a gO knockout mutant based on the back drop of a virus carrying (L) or lacking (K) epithelial cell tropism. Only gO mutant GPCMV with epithelial tropism could grow on GPL cells. A rescue virus of panel K was generated by co-transfection of the gO GPCMV mutant with a wild type locus plasmid to restore wild type virus phenotype (data not shown). Images taken between day 16–18 post transfection.

https://doi.org/10.1371/journal.pone.0135567.g011

The viable gO mutant virus was highly cell associated and produced virtually no detectable cell release virus. Viral spread on GPL cells (and primary guinea pig embryo fibroblasts, data not shown) was relatively restricted to primary plaques and was incapable of spreading across the full monolayer of cells. This was presumably due to the limited ability of the fibroblast cells to enable virus entry via endocytosis pathway. In epithelial cells, the gO mutant virus could successfully spread across the complete monolayer but had delayed growth kinetics compared to wild type virus (data not shown, Coleman et al., paper in preparation). Given the lack of cell release virus produced by the gO mutant virus, it is likely that the gO protein has a role in viral maturation but confirmation awaits further study.