Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene

  • Cristina W. Cunha ,

    Contributed equally to this work with: Cristina W. Cunha, Kathryne E. Taylor

    Affiliation Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America

    ⨯
  • Kathryne E. Taylor ,

    Contributed equally to this work with: Cristina W. Cunha, Kathryne E. Taylor

    Affiliation Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

    ⨯
  • Suzanne M. Pritchard,

    Affiliation Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America

    ⨯
  • Mark G. Delboy,

    Affiliation Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, United States of America

    ⨯
  • Tri Komala Sari,

    Affiliation Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America

    ⨯
  • Hector C. Aguilar,

    Affiliations Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America, Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, United States of America

    ⨯
  • Karen L. Mossman ,

    mossk@mcmaster.ca (KLM); nicola@vetmed.wsu.edu (AVN)

    Affiliation Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

    ⨯
  • Anthony V. Nicola

    mossk@mcmaster.ca (KLM); nicola@vetmed.wsu.edu (AVN)

    Affiliations Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America, Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, United States of America

    ⨯

Abstract

Herpes simplex virus 1 (HSV-1) ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture.

Citation: Cunha CW, Taylor KE, Pritchard SM, Delboy MG, Komala Sari T, Aguilar HC, et al. (2015) Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene. PLoS ONE 10(7): e0131129. https://doi.org/10.1371/journal.pone.0131129

Editor: Luis M. Schang, University of Alberta, CANADA

Received: March 9, 2015; Accepted: May 27, 2015; Published: July 17, 2015

Copyright: © 2015 Cunha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are available via GenBank under accession numbers KR781517 and KR781518.

Funding: This work was supported by National Institutes of Health AI113619 to AVN and a grant from the Marvel Shields Autzen Foundation to AVN. This work was also supported by Canadian Institutes for Health Research MOP-57669 to KLM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Herpes simplex virus 1 (HSV-1) is a prototype virus of the Alphaherpesvirinae subfamily that causes lifelong latent infections in humans. Upon infection of the host cell, HSV-1, like all herpesviruses, executes a cascade of temporally regulated gene expression. Infected cell protein (ICP0) is an HSV-1 immediate early (IE) phosphoprotein that acts as a promiscuous transactivator of viral and cellular genes, and is required for low multiplicity infection [1,2,3,4,5]. ICP0 is important for progression to lytic infection and for reactivation from latency [6,7,8,9]. Although initially thought to function in the nucleus by targeting repressive cellular proteins for degradation using the E3 ubiquitin ligase activity of its RING finger domain (reviewed in [1], ICP0 has been more recently suggested to have additional functions in the cytoplasm [10,11,12], and many of its binding partners are not directed to the proteasome [13,14,15,16,17,18,19,20,21,22]. In addition to being expressed in the host cell, ICP0 is a minor structural component of the tegument layer of viral particles [23,24,25,26,27,28,29]. Tegument ICP0 has been proposed to regulate transport of entering viral capsids to the nuclear pore complex in a proteasome-dependent manner [30,31].

HSV-1 dl1403 is an ICP0 deletion mutant virus derived from wild type strain 17+. The ICP0 gene is present in two copies within the HSV-1 genome, one each in the TRL and IRL inverted repeat regions. Bearing a 2 kilobase deletion in both copies of the ICP0 gene, HSV-1 dl1403 was constructed via homologous recombination between the HSV-1 17+ genome and a plasmid specifying the ICP0 gene containing a 2 kb deletion [4]. The resultant virus has been used in many studies of ICP0 function [30,32,33,34,35,36,37,38,39]. Here, we demonstrate that HSV-1 dl1403 contains a previously unrecognized secondary mutation that renders it incapable of synthesizing the wild type gC gene product.

Materials and Methods

Cells and viruses

Vero, HEL and U2OS cells (American Type Culture Collection, Rockville, MD) were propagated in Dulbecco modified Eagle medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA). CHO-nectin-1 (M3A) cells (provided by Roselyn Eisenberg and Gary Cohen, University of Pennsylvania) are stably transformed with the nectin-1 gene and contain the E. coli lacZ gene under the control of the HSV-1 ICP4 promoter. The cells were propagated in Ham F-12 nutrient mixture (Invitrogen) supplemented with 10% fetal bovine serum, 150 ug of puromycin (Sigma, St. Louis, MO)/ml, and 250 ug of G418 sulfate (Fisher Scientific, Fair Lawn, NJ)/ml.

HSV-1 wild-type Glasgow strain 17 syn+ (17+) [40], its ICP0 mutant derivative dl1403, the rescuant dl1403R, and dl1403-derived mutants FXE and D8 were provided by Roger Everett, MRC Virology Unit, Glasgow, United Kingdom. HSV-1 dl1403 has a 2 kilobase lesion in both copies of the ICP0 gene [4]. FXE, D8 and the rescued virus dl1403R were obtained by co-transfection of mutant virion DNA and a plasmid containing a fragment specifying the ICP0 gene [41,42]. Wild-type HSV-1 strain KOS and its derivative 7134, which contains the lacZ gene in place of both inverted repeat copies of the ICP0 gene [43] and the KOS-derived ICP0-null virus n212 [44] were obtained from P. Schaffer (Harvard University). HSV-1 KOS-tk12 contains the lacZ gene under the control of the viral ICP4 promoter [45] and was obtained from P. Spear (Northwestern University). The ICP0-null virus 7910 derived from HSV-1 strain F was obtained from B. Roizman (University of Chicago). HSV-1 KOS-derived mutant gC∆2–3 (provided by Curtis Brandt, University of Wisconsin) lacks gC coding sequences [46]. 17+, dl1403, dl1403R, FXE, D8, n212, 7910 and 7134 virus stocks were grown and titered on U2OS cells. KOS and gC∆2–3 virus stocks were grown and titered on Vero cells.

Antibodies

Mouse monoclonal antibody H1A027 (Virusys, North Berwick, ME) recognizes ICP0. R47 is a rabbit polyclonal antibody to gC [47], and DL6 is a mouse MAb to gD [48] (both provided by Gary Cohen and Roselyn Eisenberg). Mouse MAb H1817 (Virusys) recognizes gB, and mouse MAb AC-74 (Sigma) recognizes beta-actin.

SDS-PAGE and Western blot analysis

Samples in Laemmli buffer were separated by SDS polyacrylamide gel (4–20% gradient) electrophoresis. Gels were either fixed and stained with Coomassie blue (Sigma) or blotted onto nitrocellulose and probed with 1 μg of mouse monoclonal antibody (MAb)/ml specific for HSV gB, VP5 (MAbs H1359, H1A021, respectively, Santa Cruz), ICP0 (MAb 11060, Virusys, Sykesville, MD), or 0.01 μg MAb 1–21 to VP16 (Virusys). Nitrocellulose membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Pierce, Rockford, IL), developed with enhanced chemiluminescence detection reagents (Pierce), and exposed to X-ray film (Kodak) [49].

DNA sequencing

DNA sequence from HSV-1 17+, dl1403, and dl1403R viruses was amplified by PCR using the forward primer 5’ GAGGGGGAGGCGTCGG (this study) and reverse primer 5’ CGGACGACGTACACGATT [50]. PCR products were electrophoresed on a 1% agarose gel, and the 1520 bp band corresponding to the gC gene was cut from the gel. DNA was purified from gel using a MiniElute PCR purification kit (Qiagen) and sequenced with the PCR primers. Sequences were analyzed with the Vector NTI Advance (Life Technologies).

RT-PCR

Total RNA was extracted from Vero cells infected with HSV-1 17+ or dl1403 (MOI of 1) for 24 hours using the iPrep TRIzol Plus RNA kit per the manufacturer's instructions (Life Technologies), modified to include DNAse treatment. RNA was converted into cDNA using the iScript Advanced cDNA synthesis kit (Bio Rad). gC transcripts was detected using the CFX96 Real-Time PCR Detection System (Bio-Rad) and forward primer 5’GTCCACCCTGCCCATTTC (this work) and reverse primer 5’ CGGACGACGTACACGATT [50].

Effect of proteasome-inhibitor MG132 on HSV entry

Confluent CHO-nectin-1 cell monolayers grown in 96-well dishes were treated with culture medium containing MG132 for 15 min at 37°C. HSV-1 KOS, 7134, gC∆2–3, 17+ or dl1403 (multiplicity of infection [MOI] of 1) was added. Cells were incubated in the constant presence of agent for 7 h. 0.5% Nonidet P-40 (Sigma) cell lysates were prepared, chlorophenol red-beta-D-galactopyranoside (Roche Diagnostic, Indianapolis, IN) was added, and the beta-galactosidase activity was read at 595 nm with an ELx808 microtiter plate reader (BioTek Instruments, Winooski, VT). The MG132 treatments tested had no adverse effect on cell viability as measured by trypan blue exclusion [31]. Beta-galactosidase activity indicated successful entry [51]. Mean results and standard errors were calculated for four replicate samples.

Effect of heparin on HSV-1 infectivity

Confluent Vero cells were pre-chilled on ice. HSV-1 17+, dl1403, KOS, or gC∆2–3 (100 PFU per well) was mixed with indicated heparin concentrations in carbonate-free DMEM containing 5 mM HEPES and 0.2% BSA. Chilled inocula were added to cells and incubated at 4˚C on ice for 1 hr to allow virus binding to the cell surface. Cultures were washed thrice with PBS, and then incubated at 37˚C for 24 hr. Plaque formation was detected by immunoperoxidase staining with anti-HSV-1 polyclonal antibody HR50 (Fitzgerald Industries) [52]. Plaque formation in the untreated sample was set to 100%. The data are means of quadruplicate determinations with the standard error.

Viral growth assays

HEL cells were infected with the indicated viruses for 24 hr (MOI of 10). Cells and supernatant media were harvested, freeze-thawed three times, and then titered on U2OS cells in the presence of hexamethylene bisacetamide (HMBA) and 2% human serum. After three days, cells were fixed with methanol, stained with Giemsa (Sigma), and then plaques were counted.

Results and Discussion

HSV-1 dl1403 virions lack gC protein

ICP0-null virions have a protein composition similar to that of the wild type virions. Specifically, the HSV-1 proteins VP5, VP1/2, ICP4, VP16, VP22, VP13/14, gB, gD, gH, and gL are incorporated into extracellular dl1403 virions in the absence of ICP0 [24]. To continue this line of inquiry, equivalent VP5 units of wild type 17+ virions or dl1403 virions were analyzed by SDS-PAGE and Western blotting with polyclonal antibody to gC. Interestingly, gC was not detectable in dl1403 virions (Fig 1). As expected, tegument ICP0 was also not detected. Virion gB and gD did not appear to be reduced in the absence of ICP0 (Fig 1) in agreement with previous observations [24].

thumbnail
Download:
Fig 1. Virion gC is undetectable in the HSV-1 ICP0 deletion mutant, dl1403.

Equivalent VP5 units of extracellular 17+ or dl1403 virions were analyzed by SDS-PAGE followed by Western blotting with antibodies against the indicated proteins. VP5 content of virion preparations was determined by SDS-PAGE and Coomassie staining.

https://doi.org/10.1371/journal.pone.0131129.g001

To address one possible reason for the absence of gC from dl1403 virions, we determined whether gC was present in the viral particles of a different ICP0-null HSV-1. We utilized the ICP0-null virus, HSV-1 7134, which was constructed in an HSV-1 wild type KOS background. gC was detected in 7134 virions (Fig 2B), suggesting that gC can be assembled into viral particles in the absence of ICP0.

thumbnail
Download:
Fig 2. Detection of gC in cells infected with HSV-1 dl1403.

A. U2OS cells were infected with the indicated virus (MOI of 1) for 18 h. Cell lysates were analyzed by SDS-PAGE followed by Western blotting with PAb R47 to gC, MAb H1A027 to ICP0, or MAb AC-74 to beta-actin (Sigma). B. Equivalent VP5 units of extracellular HSV-1 KOS or 7134 virions were analyzed by SDS-PAGE followed by Western blotting with antibodies against the indicated proteins. VP5 content of virion preparations was determined by SDS-PAGE and Coomassie staining.

https://doi.org/10.1371/journal.pone.0131129.g002

gC is not detectable in cells infected with HSV-1 dl1403

We next determined whether dl1403-infected cells expressed gC. HSV-1 dl1403-infected cell lysates were analyzed by SDS-PAGE and Western blot. There was no detectable gC (or ICP0) present in the dl1403-infected cells (Fig 2A). Lysates of the wild type 17+-infected cells contained detectable levels of gC and ICP0 (Fig 2A). gC was also readily detected in cells infected with ICP0-null viruses n212, 7134 (KOS strain) and 7910 (F strain) that were generated independently from dl1403 (data not shown). Thus, the results suggest that gC was undetectable in dl1403 virions (Fig 1) because gC protein was not expressed in the infected cells from whence they came. Similarly, cells infected with the dl1403-derived viruses, D8, in which the nuclear localization signal of ICP0 has been disrupted, and FXE, which lacks the RING finger domain of ICP0, also lacked detectable gC (data not shown). Therefore, the possibility that HSV-1 dl1403 contained a previously unrecognized mutation in its gC gene was investigated.

HSV-1 dl1403 contains a frameshift mutation in its gC gene

Sequencing the gC gene from HSV-1 dl1403 revealed a single nucleotide deletion of C186 relative to the wild type parent 17+, which results in a frameshift (Fig 3A). The new reading frame introduces a premature stop codon at nucleotide positions 356–358. Unlike the wild type gC polypeptide which is 511 amino acids, the predicted polypeptide encoded by the dl1403 gC gene consists of the first 62 native gC residues followed by 112 non-gC amino acids (Fig 3B and 3C). The sequence also revealed a G to A substitution at nucleotide 170 (Fig 3A), which corresponds to a predicted S56N amino acid mutation (Fig 3B). The dl1403 virus that was sequenced was the same passage that was analyzed in Figs 1 and 2. Identical sequencing results were obtained with the earliest passage virus to which our two laboratories had access (dl1403_ori). Thus, in addition to not expressing ICP0, HSV-1 dl1403 fails to express a detectable gC protein likely due to these mutations.

thumbnail
Download:
Fig 3. Sequence analysis of the HSV-1 gC gene from parental wild type 17+ and dl1403 derivatives.

A. Nucleotide alignment of a fragment of the gC gene from the current passage of HSV-1 dl1403 in our laboratory (dl1403), from the original dl1403 stock received by our lab (dl1403_ori), from the original HSV-1 dl1403R (ICP0 rescuant) received by our lab as well as from D8 and FXE, ICP0 mutant viruses derived from dl1403. Nucleotide differences are indicated in color between wild type 17+ (blue) and the virus derivatives (red). Deletion is indicated by "-". Numbering of nucleotides is indicated at the top. B. Alignment of gC proteins from the four virus preparations based on DNA sequence results. Sequences in gray have no homology to the gC protein expressed by the parental HSV-1 17+. Stop codons are indicated by “*”. Numbering of amino acids is indicated in the top. C. Schematic representation of the region of the genomes of HSV-1 17+ and dl1403 encoding the gC gene. The DNA sequence encoding gC is in black, and the frameshifted coding sequence that results in altered protein sequence is in gray. Forward and reverse primers used for sequencing are indicated.

https://doi.org/10.1371/journal.pone.0131129.g003

To explore further whether the mutations in the gC gene arose during recent passage in cell culture, we sequenced the gC gene from dl1403R, a dl1403 virus that was rescued with the wild type ICP0 gene, and from FXE and D8, ICP0 mutants derived from dl1403. The gC gene from each of these dl1403-derived viruses contained the same substitution and frameshift mutations that were detected in all preparations of dl1403 tested (Fig 3A). These results suggest that the gC mutations were introduced during the original construction of the dl1403 virus.

Despite the detected mutations in the gC gene, we addressed the formal possibility that HSV-1 dl1403 may lack the gC protein due to an inability to synthesize gC mRNA. gC transcripts containing a region downstream of the substitution and frameshift mutations were detected by RT-PCR in both dl1403-infected cells and wild type infected cells (data not shown). Together, the results suggest that dl1403 virions and infected cells lack detectable levels of gC, due to the identified mutations in the dl1403 gC gene.

Entry of ICP0-null mutants is resistant to inhibition by MG132, regardless of the presence of gC

ICP0 present in the virion tegument layer regulates the proteasome-dependent delivery of incoming viral capsids to the nuclear pore complex [30]. The entry of ICP0-null virions is less sensitive to inhibition by proteasome inhibitors. Specifically, we showed previously that the entry of wild type HSV-1 strain 17+ was inhibited by the proteasome inhibitor MG132, a peptide aldehyde, in a concentration dependent manner, but HSV-1 dl1403 was refractory to inhibition [30]. Since dl1403 lacks gC in addition to ICP0, we assessed directly whether gC contributes to the proteasome-dependence of HSV-1 entry. The effect of MG132 on the entry of a gC-null (ICP0+) virus, HSV-1 gC∆2–3, was determined. MG132 inhibited the entry of gC∆2–3 in a concentration-dependent manner as measured by beta-galactosidase reporter gene expression, similar to the wild type virus (KOS) from which it was derived (Fig 4A). The highest concentration of MG132 inhibited > 90% of entry of either virus. These results suggest that gC does not contribute to the reliance of HSV-1 entry on the degradative activity of the proteasome. In contrast, as demonstrated previously [29,30], the entry of viruses that lacked gC and ICP0 (dl1403; Fig 4B) or lacked ICP0 alone (7134; Fig 4C), were refractory to MG132 relative to matched viruses that contained ICP0.

thumbnail
Download:
Fig 4. Effect of proteasome inhibitor MG132 on the entry of HSV-1 that lacks gC.

CHO-nectin-1 cells (A, B) or Vero cells (C) were treated with MG132 for 15 min at 37°C. HSV-1 KOS, gC∆2–3, 17+, dl1403, KOS-tk12, or 7134 was added (MOI of ~1) for 6 h. The percent beta-galactosidase activity relative to that obtained in the absence of MG132 is indicated. The data are means of quadruplicate determinations with the standard error of the mean. Results are representative of three independent experiments.

https://doi.org/10.1371/journal.pone.0131129.g004

HSV-1 dl1403 is more resistant to inhibition by heparin than is its HSV-1 17+ parent

Envelope gC is the principal mediator of HSV-1 attachment to cell surface glycosaminoglycans, such as heparan sulfate [53,54,55]. In the absence of gC, gB mediates attachment of HSV-1 to heparan sulfate proteoglycans [46]. We assessed the ability of heparin to inhibit the infectivity of dl1403. HSV-1 dl1403 was inhibited by heparin to a lesser extent than the gC-containing parental virus 17+ (Fig 5). Likewise, the infectivity of gC-null HSV-1 gC∆2–3 was inhibited to a lesser extent than its parental wild type virus, KOS (Fig 5), consistent with previous reports [56]. The results from Fig 5 suggest that dl1403 behaves similarly to a gC-null virus in an assay of biological function.

thumbnail
Download:
Fig 5. Resistance of HSV-1 dl1403 to inhibition by heparin.

HSV-1 17+, dl1403, KOS, or gC∆2–3 (100 PFU per well) was added to Vero cells at 4˚C for 1 hr in the presence of 0 to 5 μg/ml heparin as indicated. Cultures were washed thrice with PBS, and then incubated at 37˚C for 24 hr. Plaque formation was detected by immunoperoxidase staining. Plaque formation in the untreated sample was set to 100%. The data are means of quadruplicate determinations with the standard error of the mean. Results are representative of three independent experiments.

https://doi.org/10.1371/journal.pone.0131129.g005

HSV-1 ICP0-null mutants grow to similar titers regardless of gC expression

Upon the initial generation of dl1403, marker rescue experiments showed that restoration of the ICP0 sequence returned viral replication to wild type levels in cultured cells [4]. If the loss of gC had affected viral growth in culture, a decrease in the growth of the rescued virus would have been expected, suggesting that in this background in cultured cells, the additional disruption of gC does not decrease the infectivity of dl1403. Similarly, when grown on HEL fibroblasts and titered on U2OS cells, as previously described [12], we found that dl1403, n212 and 7134 all reached similar titers (Fig 6). This suggests that despite the loss of gC, dl1403 is not more attenuated than other ICP0-null viruses with intact gC expression. Interestingly, all three of these viruses reach higher titers than what is observed for 7910, which may be a strain-specific difference.

thumbnail
Download:
Fig 6. Loss of gC does not render HSV-1 dl1403 more attenuated than other ICP0-null strains with intact gC expression.

HEL cells were infected with HSV-1 dl1403, n212, 7134, or 7910 for 24 hr (MOI of 10). Cells and supernatant were harvested, freeze-thawed three times, and then titered on U2OS cells in the presence of hexamethylene bisacetamide (HMBA). The data is the average of 3–4 independent replicates with the standard error of the mean. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison post-test. *** p <0.001, ns = not significant.

https://doi.org/10.1371/journal.pone.0131129.g006

In summary, HSV-1 dl1403 virions and strains derived from it, such as D8 and FXE, lack gC, and dl1403-infected cells fail to synthesize gC, likely due to detected mutations in the gC gene. Our results illustrate the potential for secondary mutations in the construction and evaluation of HSV-1 mutant strains. In general, it is desirable to construct and examine the same mutations in multiple wild type strains. Although our stocks of the parental 17+ strain lack the gC mutations, it is possible that the stock of 17+ from which dl1403 was originally derived was the original source of the mutations. Notably, laboratory strains of HSV-1 considered to be wild type also contain mutations in their genomes [57,58,59,60,61].

Several strains of HSV-1 that lack gC have been identified [62,63,64,65]. These strains are viable in cell culture, consistent with the non-essential role of gC in HSV-1 replication. While HSV-1 gC plays a well-defined role in virion attachment to cell surface glycosaminoglycans [66], it is not an absolute requirement for HSV-1 entry via endocytic and non-endocytic pathways [67,68]. Repair of the ICP0 deletion in HSV-1 dl1403 restores the virus to wild type phenotype in cell culture assays that test ICP0 functions [4,41]. For example, exogenous expression of ICP0 restores plaque formation efficiency of dl1403 to wild type in cell culture [4], suggesting that the observed phenotype of this mutant likely results from the loss of ICP0 and not gC. Studies using dl1403 in mouse models have also effectively used an ICP0 rescuant as a control [6]. HSV-1 gC also binds to complement component C3b, providing protection from antibody-independent neutralization [69,70]. Although this function of gC would not be necessary in cell culture, the loss of gC would be expected to have a more significant impact on experiments performed in vivo. Indeed, in some animal models, gC-null viruses are highly attenuated [69,71,72,73]. However, in others, the lack of gC does not affect disease outcome [71,74,75,76,77]. A previous study comparing the growth of 17 syn, dl1403, and the marker rescue virus R4 in mice indicated that R4 replicated similarly to 17 syn, although it did show some evidence of mild attenuation compared to the wild type virus [6]. This suggests that while the lack of gC in dl1403 may have produced minor additional defects in replication in vivo, the effect of the loss of ICP0 is much greater. Along with the present study, this also highlights the importance of using rescuants as controls in experiments using mutant HSV-1 strains. Phenotypes of HSV-1 dl1403 attributable to the absence of gC, but unrelated to ICP0 function, such as enhanced resistance to heparin (Fig 5), are also expected to manifest in other dl1403-derived viruses including ICP0 rescuants.

HSV-1 dl1403, dl1403R, D8 and FXE are widely used viruses. Thus, our finding that these strains contain a secondary mutation in gC impacts a large number of studies. We are currently in the process of creating repaired versions of dl1403, D8 and FXE with intact gC expression. Our findings suggest that caution is necessary in using the current viruses and also suggest that previous studies should be interpreted with the absence of both the ICP0 and gC genes in mind.

Acknowledgments

We thank Roger Everett for critical reading of the manuscript, and Curtis Brandt, Gary Cohen, Roselyn Eisenberg, Roger Everett, Priscilla Schaffer, Patricia Spear and Bernard Roizman for generous gifts of reagents.

Author Contributions

Conceived and designed the experiments: CWC KET SMP MGD KLM AVN. Performed the experiments: CWC KET SMP MGD. Analyzed the data: CWC KET SMP MGD TKS HCA KLM AVN. Wrote the paper: CWC KET HCA KLM AVN.

References

  1. 1. Everett RD (2000) ICP0, a regulator of herpes simplex virus during lytic and latent infection. Bioessays 22: 761–770. pmid:10918307
  2. 2. Hagglund R, Roizman B (2004) Role of ICP0 in the strategy of conquest of the host cell by herpes simplex virus 1. J Virol 78: 2169–2178. pmid:14963113
  3. 3. Cai W, Schaffer PA (1992) Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells. J Virol 66: 2904–2915. pmid:1313909
  4. 4. Stow ND, Stow EC (1986) Isolation and characterization of a herpes simplex virus type 1 mutant containing a deletion within the gene encoding the immediate early polypeptide Vmw110. J Gen Virol 67 (Pt 12): 2571–2585.
  5. 5. Sacks WR, Schaffer PA (1987) Deletion mutants in the gene encoding the herpes simplex virus type 1 immediate-early protein ICP0 exhibit impaired growth in cell culture. J Virol 61: 829–839. pmid:3027408
  6. 6. Clements GB, Stow ND (1989) A herpes simplex virus type 1 mutant containing a deletion within immediate early gene 1 is latency-competent in mice. J Gen Virol 70 (Pt 9): 2501–2506.
  7. 7. Harris RA, Everett RD, Zhu XX, Silverstein S, Preston CM (1989) Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system. J Virol 63: 3513–3515. pmid:2545921
  8. 8. Leib DA, Coen DM, Bogard CL, Hicks KA, Yager DR, et al. (1989) Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J Virol 63: 759–768. pmid:2536101
  9. 9. Halford WP, Schaffer PA (2001) ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency. J Virol 75: 3240–3249. pmid:11238850
  10. 10. Liu M, Schmidt EE, Halford WP (2010) ICP0 dismantles microtubule networks in herpes simplex virus-infected cells. PloS one 5: e10975. pmid:20544015
  11. 11. Paladino P, Collins SE, Mossman KL (2010) Cellular localization of the herpes simplex virus ICP0 protein dictates its ability to block IRF3-mediated innate immune responses. PloS one 5: e10428. pmid:20454685
  12. 12. Taylor KE, Chew MV, Ashkar AA, Mossman KL (2014) Novel roles of cytoplasmic ICP0: proteasome-independent functions of the RING finger are required to block interferon-stimulated gene production but not to promote viral replication. Journal of Virology 88: 8091–8101. pmid:24807717
  13. 13. Van Sant C, Kawaguchi Y, Roizman B (1999) A single amino acid substitution in the cyclin D binding domain of the infected cell protein no. 0 abrogates the neuroinvasiveness of herpes simplex virus without affecting its ability to replicate. Proceedings of the National Academy of Sciences of the United States of America 96: 8184–8189. pmid:10393969
  14. 14. Kawaguchi Y, Van Sant C, Roizman B (1997) Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3. J Virol 71: 7328–7336. pmid:9311810
  15. 15. Kawaguchi Y, Bruni R, Roizman B (1997) Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1delta: ICP0 affects translational machinery. J Virol 71: 1019–1024. pmid:8995621
  16. 16. Kawaguchi Y, Tanaka M, Yokoymama A, Matsuda G, Kato K, et al. (2001) Herpes simplex virus 1 alpha regulatory protein ICP0 functionally interacts with cellular transcription factor BMAL1. Proc Natl Acad Sci USA 98: 1877–1882. pmid:11172044
  17. 17. Lomonte P, Thomas J, Texier P, Caron C, Khochbin S, et al. (2004) Functional interaction between class II histone deacetylases and ICP0 of herpes simplex virus type 1. J Virol 78: 6744–6757. pmid:15194749
  18. 18. Gu H, Liang Y, Mandel G, Roizman B (2005) Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells. Proc Natl Acad Sci USA 102: 7571–7576. pmid:15897453
  19. 19. Liang Y, Kurakin A, Roizman B (2005) Herpes simplex virus 1 infected cell protein 0 forms a complex with CIN85 and Cbl and mediates the degradation of EGF receptor from cell surfaces. Proc Natl Acad Sci USA 102: 5838–5843. pmid:15824310
  20. 20. Gu H, Roizman B (2009) The two functions of herpes simplex virus 1 ICP0, inhibition of silencing by the CoREST/REST/HDAC complex and degradation of PML, are executed in tandem. J Virol 83: 181–187. pmid:18945770
  21. 21. Liang Y, Roizman B (2006) State and role of SRC family kinases in replication of herpes simplex virus 1. J Virol 80: 3349–3359. pmid:16537602
  22. 22. Nagel CH, Albrecht N, Milovic-Holm K, Mariyanna L, Keyser B, et al. (2011) Herpes simplex virus immediate-early protein ICP0 is targeted by SIAH-1 for proteasomal degradation. J Virol 85: 7644–7657. pmid:21632771
  23. 23. Yao F, Courtney RJ (1992) Association of ICP0 but not ICP27 with purified virions of herpes simplex virus type 1. J Virol 66: 2709–2716. pmid:1313896
  24. 24. Delboy MG, Siekavizza-Robles CR, Nicola AV (2010) Herpes simplex virus tegument ICP0 is capsid associated, and its E3 ubiquitin ligase domain is important for incorporation into virions. J Virol 84: 1637–1640. pmid:19906912
  25. 25. Loret S, Guay G, Lippe R (2008) Comprehensive characterization of extracellular herpes simplex virus type 1 virions. J Virol 82: 8605–8618. pmid:18596102
  26. 26. Maringer K, Elliott G (2010) Recruitment of herpes simplex virus type 1 immediate-early protein ICP0 to the virus particle. J Virol 84: 4682–4696. pmid:20164220
  27. 27. Radtke K, Kieneke D, Wolfstein A, Michael K, Steffen W, et al. (2010) Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures. PLoS Pathogens 6: e1000991. pmid:20628567
  28. 28. Loret S, Lippe R (2012) Biochemical analysis of infected cell polypeptide (ICP)0, ICP4, UL7 and UL23 incorporated into extracellular herpes simplex virus type 1 virions. J Gen Virol 93: 624–634. pmid:22158881
  29. 29. Pritchard SM, Cunha CW, Nicola AV (2013) Analysis of herpes simplex virion tegument ICP4 derived from infected cells and ICP4-expressing cells. PLoS One 8: e70889. pmid:23940659
  30. 30. Delboy MG, Nicola AV (2011) A pre-immediate early role for tegument ICP0 in the proteasome-dependent entry of herpes simplex virus. J Virol 85: 5910–5918. pmid:21471243
  31. 31. Delboy MG, Roller DG, Nicola AV (2008) Cellular proteasome activity facilitates herpes simplex virus entry at a postpenetration step. J Virol 82: 3381–3390. pmid:18234803
  32. 32. Thompson RL, Sawtell NM (2006) Evidence that the herpes simplex virus type 1 ICP0 protein does not initiate reactivation from latency in vivo. J Virol 80: 10919–10930. pmid:16943285
  33. 33. Mossman KL, Saffran HA, Smiley JR (2000) Herpes simplex virus ICP0 mutants are hypersensitive to interferon. J Virol 74: 2052–2056. pmid:10644380
  34. 34. Preston CM (2007) Reactivation of expression from quiescent herpes simplex virus type 1 genomes in the absence of immediate-early protein ICP0. J Virol 81: 11781–11789. pmid:17715242
  35. 35. Kyratsous CA, Walters MS, Panagiotidis CA, Silverstein SJ (2009) Complementation of a herpes simplex virus ICP0 null mutant by varicella-zoster virus ORF61p. J Virol 83: 10637–10643. pmid:19656893
  36. 36. Morency E, Sabra M, Catez F, Texier P, Lomonte P (2007) A novel cell response triggered by interphase centromere structural instability. J Cell Biol 177: 757–768. pmid:17548509
  37. 37. Everett RD (2004) Herpes simplex virus type 1 regulatory protein ICP0 does not protect cyclins D1 and D3 from degradation during infection. J Virol 78: 9599–9604. pmid:15331692
  38. 38. Grant K, Grant L, Tong L, Boutell C (2012) Depletion of intracellular zinc inhibits the ubiquitin ligase activity of viral regulatory protein ICP0 and restricts herpes simplex virus 1 replication in cell culture. Journal of Virology 86: 4029–4033. pmid:22278229
  39. 39. Lilley CE, Chaurushiya MS, Boutell C, Landry S, Suh J, et al. (2010) A viral E3 ligase targets RNF8 and RNF168 to control histone ubiquitination and DNA damage responses. The EMBO journal 29: 943–955. pmid:20075863
  40. 40. Brown SM, Ritchie DA, Subak-Sharpe JH (1973) Genetic studies with herpes simplex virus type 1. The isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J Gen Virol 18: 329–346. pmid:4348796
  41. 41. Russell J, Stow ND, Stow EC, Preston CM (1987) Herpes simplex virus genes involved in latency in vitro. J Gen Virol 68 (Pt 12): 3009–3018.
  42. 42. Everett RD (1989) Construction and characterization of herpes simplex virus type 1 mutants with defined lesions in immediate early gene 1. J Gen Virol 70 (Pt 5): 1185–1202.
  43. 43. Cai W, Schaffer PA (1991) A cellular function can enhance gene expression and plating efficiency of a mutant defective in the gene for ICP0, a transactivating protein of herpes simplex virus type 1. J Virol 65: 4078–4090. pmid:1649316
  44. 44. Cai W, Astor TL, Liptak LM, Cho C, Coen DM, et al. (1993) The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reactivation from latency. Journal of Virology 67: 7501–7512. pmid:8230470
  45. 45. Warner MS, Geraghty RJ, Martinez WM, Montgomery RI, Whitbeck JC, et al. (1998) A cell surface protein with herpesvirus entry activity (HveB) confers susceptibility to infection by mutants of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus. Virology 246: 179–189. pmid:9657005
  46. 46. Herold BC, Visalli RJ, Susmarski N, Brandt CR, Spear PG (1994) Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B. J Gen Virol 75: 1211–1222. pmid:8207388
  47. 47. Eisenberg RJ, Ponce de Leon M, Friedman HM, Fries LF, Frank MM, et al. (1987) Complement component C3b binds directly to purified glycoprotein C of herpes simplex virus types 1 and 2. Microb Pathog 3: 423–435. pmid:2849025
  48. 48. Eisenberg RJ, Long D, Ponce de Leon M, Matthews JT, Spear PG, et al. (1985) Localization of epitopes of herpes simplex virus type 1 glycoprotein D. J Virol 53: 634–644. pmid:2578577
  49. 49. Dollery SJ, Delboy MG, Nicola AV (2010) Low pH-induced conformational change in herpes simplex virus glycoprotein B. J Virol 84: 3759–3766. pmid:20147407
  50. 50. Yu X, Liu L, Wu L, Wang L, Dong C, et al. (2010) Herpes simplex virus type 1 tegument protein VP22 is capable of modulating the transcription of viral TK and gC genes via interaction with viral ICP0. Biochimie 92: 1024–1030. pmid:20457214
  51. 51. Roller DG, Dollery SJ, Doyle JL, Nicola AV (2008) Structure-function analysis of herpes simplex virus glycoprotein B with fusion-from-without activity. Virology 382: 207–216. pmid:18950828
  52. 52. Dollery SJ, Wright CC, Johnson DC, Nicola AV (2011) Low-pH-dependent changes in the conformation and oligomeric state of the prefusion form of herpes simplex virus glycoprotein B are separable from fusion activity. J Virol 85: 9964–9973. pmid:21813610
  53. 53. Shieh MT, WuDunn D, Montgomery RI, Esko JD, Spear PG (1992) Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans. J Cell Biol 116: 1273–1281. pmid:1310996
  54. 54. WuDunn D, Spear PG (1989) Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J Virol 63: 52–58. pmid:2535752
  55. 55. Herold BC, WuDunn D, Soltys N, Spear PG (1991) Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. J Virol 65: 1090–1098. pmid:1847438
  56. 56. Herold BC, Gerber SI, Belval BJ, Siston AM, Shulman N (1996) Differences in the susceptibility of herpes simplex virus types 1 and 2 to modified heparin compounds suggest serotype differences in viral entry. J Virol 70: 3461–3469. pmid:8648678
  57. 57. Haugo AC, Szpara ML, Parsons L, Enquist LW, Roller RJ (2011) Herpes simplex virus 1 pUL34 plays a critical role in cell-to-cell spread of virus in addition to its role in virus replication. J Virol 85: 7203–7215. pmid:21561917
  58. 58. Macdonald SJ, Mostafa HH, Morrison LA, Davido DJ (2012) Genome sequence of herpes simplex virus 1 strain McKrae. J Virol 86: 9540–9541. pmid:22879612
  59. 59. Macdonald SJ, Mostafa HH, Morrison LA, Davido DJ (2012) Genome sequence of herpes simplex virus 1 strain KOS. J Virol 86: 6371–6372. pmid:22570244
  60. 60. Szpara ML, Tafuri YR, Parsons L, Shreve JT, Engel EA, et al. (2014) Genome Sequence of the Anterograde-Spread-Defective Herpes Simplex Virus 1 Strain MacIntyre. Genome Announcements 2.
  61. 61. Negatsch A, Mettenleiter TC, Fuchs W (2011) Herpes simplex virus type 1 strain KOS carries a defective US9 and a mutated US8A gene. J Gen Virol 92: 167–172. pmid:20861322
  62. 62. Heine JW, Honess RW, Cassai E, Roizman B (1974) Proteins specified by herpes simplex virus. XII. The virion polypeptides of type 1 strains. J Virol 14: 640–651. pmid:4369085
  63. 63. DeLuca N, Bzik DJ, Bond VC, Person S, Snipes W (1982) Nucleotide sequences of herpes simplex virus type 1 (HSV-1) affecting virus entry, cell fusion, and production of glycoprotein gb (VP7). Virology 122: 411–423. pmid:6293179
  64. 64. Ruyechan WT, Morse LS, Knipe DM, Roizman B (1979) Molecular genetics of herpes simplex virus. II. Mapping of the major viral glycoproteins and of the genetic loci specifying the social behavior of infected cells. J Virol 29: 677–697. pmid:219254
  65. 65. Zezulak KM, Spear PG (1984) Mapping of the structural gene for the herpes simplex virus type 2 counterpart of herpes simplex virus type 1 glycoprotein C and identification of a type 2 mutant which does not express this glycoprotein. J Virol 49: 741–747. pmid:6321760
  66. 66. Shukla D, Spear PG (2001) Herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry. J Clin Invest 108: 503–510. pmid:11518721
  67. 67. Barrow E, Nicola AV, Liu J (2013) Multiscale perspectives of virus entry via endocytosis. Virol J 10: 177. pmid:23734580
  68. 68. Nicola AV, Straus SE (2004) Cellular and viral requirements for rapid endocytic entry of herpes simplex virus. J Virol 78: 7508–7517. pmid:15220424
  69. 69. Friedman HM, Cohen GH, Eisenberg RJ, Seidel CA, Cines DB (1984) Glycoprotein C of herpes simplex virus 1 acts as a receptor for the C3b complement component on infected cells. Nature 309: 633–635. pmid:6328323
  70. 70. Friedman HM (2003) Immune evasion by herpes simplex virus type 1, strategies for virus survival. Transactions of the American Clinical and Climatological Association 114: 103–112. pmid:12813914
  71. 71. Drolet BS, Mott KR, Lippa AM, Wechsler SL, Perng GC (2004) Glycoprotein C of herpes simplex virus type 1 is required to cause keratitis at low infectious doses in intact rabbit corneas. Curr Eye Res 29: 181–189. pmid:15512965
  72. 72. Lubinski JM, Jiang M, Hook L, Chang Y, Sarver C, et al. (2002) Herpes simplex virus type 1 evades the effects of antibody and complement in vivo. J Virol 76: 9232–9241. pmid:12186907
  73. 73. Lubinski JM, Wang L, Soulika AM, Burger R, Wetsel RA, et al. (1998) Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo. J Virol 72: 8257–8263. pmid:9733869
  74. 74. Minagawa H, Liu Y, Yoshida T, Hidaka Y, Toh Y, et al. (1997) Pathogenicity of glycoprotein C-deficient herpes simplex virus 1 strain TN-1 which encodes truncated glycoprotein C. Microbiol Immunol 41: 545–551. pmid:9272700
  75. 75. Sakai Y, Minagawa H, Ishibashi T, Inomata H, Mori R (1994) Stromal keratitis induced by a unique clinical isolate of herpes simplex virus type 1. Int J Ophthalmol 208: 157–160.
  76. 76. Sunstrum JC, Chrisp CE, Levine M, Glorioso JC (1988) Pathogenicity of glycoprotein C negative mutants of herpes simplex virus type 1 for the mouse central nervous system. Virus Res 11: 17–32. pmid:2845681
  77. 77. Liu Y, Sakai Y, Minagawa H, Toh Y, Ishibashi T, et al. (1993) Induction of bilateral retinal necrosis in mice by unilateral intracameral inoculation of a glycoprotein-C deficient clinical isolate of herpes simplex virus type 1. Arch Virol 129: 105–118. pmid:8385909