The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment
© Melancon et al; licensee BioMed Central Ltd. 2007
Received: 19 October 2007
Accepted: 08 November 2007
Published: 08 November 2007
The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20-null virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.
Herpes simplex viruses (HSV) specify at least eleven virus-specified glycoproteins, as well as several non-glycosylated membrane associated proteins, most of which play important roles in multiple membrane fusion events during virus entry and intracellular virion morphogenesis and egress [1–8]. Spread of infectious virus occurs either by release of virions to extracellular spaces or through virus-induced cell-to-cell fusion. In vivo, the latter mechanism allows for virus spread without exposing virions to extracellular spaces containing neutralizing antibodies. Mutations that cause extensive virus-induced cell fusion predominantly arise in four genes of the HSV genome: the UL20 gene [9, 10], the UL24 gene [11, 12], the UL27 gene encoding glycoprotein B (gB) [13, 14], and the UL53 gene coding for glycoprotein K (gK) [15–19]. Of these four membrane associated proteins, only UL20 and gK are absolutely essential for the intracellular envelopment and transport of virions to extracellular spaces in all cell types [9, 20–23].
The most prevalent model for morphogenesis and egress of infectious herpes virions includes sequential de-envelopment and re-envelopment steps in transit to extracellular spaces: a) primary envelopment by budding of capsids assembled in the nuclei through the inner nuclear leaflet leading to the production of enveloped virions within perinuclear spaces; b) de-envelopment by fusion of viral envelopes with the outer nuclear leaflet leading to the accumulation of unenveloped capsids in the cytoplasm; c) assembly of sets of tegument proteins on the cytoplasmic capsids, as well as potentially on vesicle sites to be used for cytoplasmic envelopment; d) re-envelopment of cytoplasmic tegumented capsids into TGN-derived vesicles. This final event in cytoplasmic virion envelopment is thought to be largely mediated by interactions between tegument proteins and cytoplasmic portions of viral glycoproteins embedded within the TGN-derived membranes. Cytoplasmically enveloped viruses are thought to be transported to extracellular spaces within Golgi or TGN-derived vesicles (reviewed in: [7, 24, 25]).
The UL20 gene encodes a 222 amino acid non-glycosylated transmembrane protein that is conserved by all alphaherpesviruses. The UL20p is a structural component of extracellular enveloped virions and it is expressed in infected cells assuming a predominantly perinuclear and cytoplasmic distribution . An initial report indicated that partial deletion of the UL20 gene resulted in perinuclear accumulation of capsids indicating that the UL20 gene functioned, most likely, in the de-envelopment of enveloped virions found within perinuclear spaces . However, we showed previously that a precise deletion of the UL20 gene revealed that the UL20 gene strictly functioned in cytoplasmic envelopment of capsids . Importantly, syncytial mutations in either gB or gK failed to cause fusion in the absence of the UL20 gene, indicating that the UL20 protein was essential for virus-induced cell fusion . Furthermore, we showed that UL20 is required for cell-surface expression of gK and TGN localization, suggesting a functional interdependence between gK and UL20 for virus egress and cell-to-cell fusion [28, 29]. Recently, we delineated via site-directed mutagenesis the functional domains of UL20p involved in infectious virus production and virus-induced cell fusion. Importantly, we showed that both amino and carboxyl terminal portions of UL20p, which are predicted to lie within the cytoplasmic side of cellular membranes, function both in cytoplasmic virion envelopment and virus-induced cell fusion .
In this manuscript, we show that the amino and carboxyl termini of UL20p contain distinct domains that function in infectious virion production and intracellular gK/UL20 transport.
Mutagenesis of HSV-1 UL20
WT aa Sequence
Mut. aa Sequence
Complementation assay for infectious virus production
Complementation for virus-induced cell-to-cell fusion
Intracellular transport and TGN localization of UL20p mutants and gK
The effect of UL20 carboxyl terminal truncations on UL20p and gK TGN localization after endocytosis from cell surfaces
We showed previously that UL20 and gK are functionally interdependent for their intracellular transport, cell-surface expression and TGN localization  and that this interaction plays pivotal role in cytoplasmic virion envelopment and egress from infected cells . In this study, we investigated the ability of selected UL20 mutations reported previously, as well as a new set of UL20 mutants, on their ability to transport and colocalize with gK on cell-surfaces and in TGN-labeled intracellular compartments:
Previously, we characterized a series of carboxyl terminal truncations including the 204t and 211t encoding carboxyl terminal truncations of 18 and 11 aa respectively. These two UL20p truncations failed to complement for infectious virus production and virus-induced cell fusion, while the 216t coding for a 6 aa truncation enabled virus-induced cell fusion, but failed to complement for infectious virus production . We show here that the inability to complement for virus-induced cell fusion was not due to defects in intracellular transport and TGN localization, because 216t, as well as both 204t and 211t were efficiently transported to cell-surfaces and co-localized with gK in TGN-labeled membranes. Therefore, intracellular transport, cell-surface expression and TGN localization of UL20p and gK are not sufficient for infectious virus production. Based on these results, we can conclude that the carboxyl terminal six amino acids of UL20p function exclusively in intracellular virion envelopment and infectious virus production, while the UL20p domain spanning amino acids 204–211is important for both intracellular transport and virus-induced cell fusion.
Domain I is the largest domain (63 aa) and it includes stretches of acidic amino acid (D, E) clusters, which could form electrostatic interactions with other proteins . Furthermore, the amino terminus of UL20p contains acidic clusters, as well as the amino acid motif YXXΦ (YSRL), which have been shown to function in endocytosis of alphaherpesvirus envelope proteins from plasma membranes to the TGN [32–36]. The acidic cluster motifs appear to direct TGN localization by binding to a cellular connector protein, PACS-1, which connects the glycoproteins to the AP-1 complex , while the YXXΦ motif binds adaptor proteins directly [2, 3, 40]. The YXXΦ (YSRL) amino acid sequence overlapping the CL49 mutated sequence, is conserved in HSV-1, HSV-2, and cercopithecine herpesvirus 1 and 2, but not in varicella zoster (VZV) or pseurodabies virus (PRV) (not shown). Mutagenesis of the Y residue of a YXXΦ(YTKI) motif within gK domain IV, shown to lie in the cytoplasmic side of membranes, produced a gK-null phenotype . Similarly, mutagenesis of either Y38 or Y49, or both residues resulted in loss of infectious virus production, while the UL20p mutants carrying either mutation or a combination of both mutations allowed efficient intracellular transport and TGN localization. This result is similar to the results obtained with the UL20p carboxyl terminal domains and suggests that amino terminal domains of UL20p that function in cytoplasmic virion envelopment can be functionally separated from those that function in UL20p and gK intracellular transport and TGN localization. Interestingly, the Y49A mutant allowed some virus-induced cell fusion caused by either the gBsyn3 or gKsyn1 mutation suggesting that the requirement of this residue for infectious virus production is more stringent that the requirement for virus-induced cell fusion.
We reported previously that the Y49A, CL49 and 216t mutant viruses produced syncytial plaques, although their ultrastructural phenotypes seemed to be similar to that of the UL20-null virus . We show here these phenotypes are consistent with the findings that these UL20p mutations allowed efficient intracellular transport, cell-surface expression and TGN localization. However, mutagenesis of both Y38 and Y49 amino acid residues in the amino terminus of UL20p, inhibited virus-induced cell fusion, while allowing efficient intracellular transport and TGN localization. This result suggests that the Y38 and Y49 residues together play important roles in cytoplasmic virus envelopment, but they are not required for proper UL20p/gK intracellular transport. The Y38A mutation seemed to affect both virion production and virus-induced cell fusion, although the Y49A mutation appeared to inhibit virion production, but allowed some cell fusion to occur. As is the case with the carboxyl terminus of UL20p discussed earlier, these results suggest that the amino terminus of UL20p contains functionally separable domains involved in cytoplasmic virion envelopment and intracellular glycoprotein transport. Furthermore, the Y49A mutation allowed some virus-induced cell fusion, but not infectious virus production to occur suggesting that domains within the UL20p amino-terminus involved in cytoplasmic virion envelopment may be functionally separated from domains functioning in UL20p/gK intracellular transport and virus-induced cell fusion.
These results show that UL20p domains required for UL20p and gK intracellular transport and TGN localization can be functionally segregated from domains involved in infectious virus production and virus-induced cell fusion. The results suggest that virus-induced cell fusion mechanisms are not required for cytoplasmic virion envelopment.
Materials and methods
Cells and viruses
African green monkey kidney (Vero) cells were obtained from ATCC (Rockville, MD). The Vero-based UL20 complementing cell line, G5, was a gift of Dr. P. Desai, (John Hopkins Medical Center) . Cells were maintained as previously described [20, 29, 38]. The parental wild-type strain used in this study HSV-1 (KOS) was originally obtained from P. A. Schaffer (Harvard Medical School). Δ20DIV5, Δ20gBsyn3 and Δ20gKsyn1DIV5 viruses were as described previously . Virus stocks were grown on the UL20 complementing cell line Fd20-1, the construction of which was described previously . In this paper, for simplification purposes, the Δ20DIV5 virus is referred to as Δ20 virus and the Δ20syngK1DIV5 virus is referred to as Δ20gKsyn1 virus .
pCR2.1-UL20, which was used as the parental vector for UL20 mutagenesis, was generated by cloning a 773 bp DNA fragment containing the UL20 gene, obtained by PCR amplification of HSV-1(KOS) viral DNA, into pCR2.1/TOPO (Invitrogen) as described in detail previously . The generation of UL20 cluster to alanine mutants CL38, CL49, CL153, and CL209, the single point mutant Y49A, and truncation mutants, 204t, 211t, 216t were reported previously . A set of new UL20 mutants generated for this study included a UL20 mutant containing both the CL38 and CL49 mutations (CL38 – CL49), the alanine cluster UL20 mutant CL61, the single point mutant Y38A, and the UL20 mutant Y-Y containing both the Y38A and Y49A mutations. The cluster mutations, the additional single point UL20 mutants, as well as the double mutants were generated by splice-overlap extension (SOE) PCR  as described previously . All mutations were verified by sequencing of the final plasmid construct.
UL20 complementation assay for infectious virion production
Confluent Vero monolayers in six well plates were transfected with 2 μg of wild-type or mutant UL20 plasmid with Lipofectamine 2000 as described by the manufacturer (Invitrogen). Six hours post-transfection, the monolayers were infected with a UL20-null virus at an MOI of 1. Infections were placed on a rocker for 1 hour at 4°C, and then transferred to 37°C for 2 hours. Residual virus was inactivated using an acid wash (PBS containing .5 M glycine, pH3) for 2 min, and monolayers were subsequently washed 3 times with DMEM to restore the pH to a normal level. Infections were incubated at 37°C for 24 hours. After repeated freeze/thaw cycles, virus stocks were titered in triplicate on Fd20-1 cells, which effectively complement the UL20-null defect . The complementation ratio for each mutant was calculated with the formula (virus titer of mutant/virus titer of negative control).
UL20 complementation assay for virus-induced cell-to-cell fusion and virus spread
The complementation assay was performed essentially as we described previously for addressing the role of the HSV-1 UL11 protein in virion morphogenesis . Briefly, confluent Vero monolayers in six-well plates were transfected with 2 μg of wild-type or mutant UL20 plasmid with Lipofectamine 2000 as described by the manufacturer (Invitrogen). 18 hours post transfection, the monolayers were infected at an MOI of 0.1 with either Δ20gKsyn1 or Δ20gBsyn3 viruses. Infections were placed on a rocker at room temperature for 1 hour, then transferred to 37°C for 30 minutes. Cells were overlaid with DMEM containing 1% methylcellulose. 24 hours post-infection, cell fusion was determined by visualization of syncytia formation by light microscopy. Cells were stained with a polyclonal HRP conjugated HSV-1 antibody as directed by the manufacturer (DakoCytomation). Briefly, cells were washed with PBS to remove methylcellulose media, and fixed with 4°C methanol for 15 minutes. TBS containing a 1:750 dilution of the polyclonal HSV-1 antibody was added to the cells and placed on a rocker at 4°C for 1 h. Cells were washed with TBS and developed using the Vector NovaRED peroxidase substrate kit as directed by the manufacturer (Vector, Inc). In this assay,
Complementation of the UL20-null virus by transient expression of the wild-type UL20 gene caused the production of up to 40% of total viral plaques appearing to have similar morphology and size to the HSV-1(F) parental virus.
Cell monolayers were grown on coverslips in six-well plates. Cell monolayers were transfected with the indicated UL20 and/or gK plasmid combinations by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and prepared for confocal microscopy approximately 30 h posttransfection. Cells were washed with TBS and fixed with electron microscopy-grade 3% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, Pa.) for 15 min, washed twice with phosphate-buffered saline-50 mMglycine, and permeabilized with 1.0% Triton X-100. Monolayers were subsequently blocked for 1 h with 7% normal goat serum and 7% bovine serum albumin in TBS (TBS blocking buffer) before incubation for 2 h with either anti-V5 (Invitrogen, Inc.), for recognition of gK, or anti-FLAG (Sigma Chemical, Inc.), for recognition of UL20p, diluted 1:500 in TBS blocking buffer. Alternatively, simultaneous detection of gK and UL20p in cotransfected cells was accomplished by concurrent incubation with murine anti-V5 and rabbit anti-FLAG (Sigma Chemical, Inc.) diluted 1:500 in TBS blocking buffer. Cells were then washed extensively and incubated for 30 min with Alexa Fluor 594 and/or Alexa Fluor 647-conjugated anti-immunoglobulin G diluted 1:500 in TBS blocking buffer. After incubation, excess antibody was removed by washing five times with TBS. TGN were identified with a donkey anti-TGN46 primary antibody and an Alexa Fluor 488-conjugated sheep anti-donkey secondary antibody . Specific immunofluorescence was examined using a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems, Exton, Pa.) fitted with a CS APO 63× Leica objective (1.4 numerical aperture). Individual optical sections in the z axis, averaged six times, were collected at the indicated zoom in series in the different channels at 1,024- by 1,024-pixel resolution as described previously [27, 29, 42]. Images were compiled and rendered with Adobe Photoshop. Image analyses were generated and analyzed using the Leica confocal microscopy software package and were modified from protocols described previously .
UL20p/gK cell surface internalization assay
Internalization assays were modified from similar assays performed previously [35, 44, 45]. Briefly, Vero cells were transfected with pgKDIV5 and either pUL20-3 × FLAG or a variant containing the indicated UL20 mutation . Twenty hours posttransfection, cells were incubated under live conditions for 6 h at 37°C with mouse anti-V5. Cells were extensively washed, fixed with paraformaldehyde, and processed for confocal microscopy as described above, with the exception that the internalized antibodies served as the primary antibody for gK (mouse anti-V5).
This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (AI43000) to K.G.K. J. M. M. and P. A. F. were supported by Louisiana Economic Development Graduate Assistant Fellowships. We acknowledge financial support by the LSU School of Veterinary Medicine to BIOMMED.
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