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Journal of General Virology (2011), 92, 2586–2589 Short Communication DOI 10.1099/vir.0.031443-0 Glycoprotein J of infectious laryngotracheitis virus is required for efficient egress of infectious virions from cells Alice Mundt,1 Egbert Mundt,1 Robert J. Hogan2 and Maricarmen Garcı́a1 Correspondence Maricarmen Garcı́a [email protected] Received 16 February 2011 Accepted 11 July 2011 1 Poultry Diagnostic and Research Center, University of Georgia, Athens, GA, USA 2 Anatomy and Radiology, Department of Population Health, University of Georgia, Athens, GA, USA Glycoprotein J (gJ) of infectious laryngotracheitis virus (ILTV) represents a major viral antigen and is dispensable for replication in cell culture and chickens. We generated gJ deletion mutants derived from the United States Department of Agriculture standard challenge strain (USDA-ch), a GFP-expressing mutant GDgJ, a gJ deletion mutant void of any foreign DNA insertion (BDgJ) and a gJ rescue mutant gJR with US5 restored. GDgJ, BDgJ and gJR were characterized in cell culture and embryonated eggs. Entry kinetic assays showed that the gJ deletion mutants did not differ in their entry kinetics from gJR. Replication kinetics strongly indicated that gJ plays an important role during egress of the virus. Differences in the abilities of the mutants to replicate in chorioallantoic membranes of chicken embryos and to release infectious virus into the allantoic fluid supported a function of gJ during the egress of ILTV from infected cells. Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens that results in severe production losses to the poultry industry (Bagust et al., 2000; Dufour-Zavala, 2008). It is caused by infectious laryngotracheitis virus (ILTV) named Gallid herpesvirus 1. ILTV is closely related to Psittacid herpesvirus 1 (PsHV-1), both representing the genus Iltovirus within the subfamily Alphaherpesvirinae of the family Herpesviridae (Davison et al., 2009; Thureen & Keeler, 2006). Sequence analysis of the approximately 148 kbp genome revealed 76 ORFs, 63 of these ORFs have similar counterparts in the genome of the prototypic alphaherpesvirus herpes simplex virus 1 (HSV-1). Currently, 17 genes have been individually deleted from the ILTV genome (Fuchs et al., 2000, 2007; Devlin et al., 2006a, b; Pavlova et al., 2010), resulting in mutants with varied degrees of in vitro replication defects and levels of attenuation in chickens (Fuchs et al., 2007). ILTV glycoprotein J (gJ) is encoded by US5 in the unique short region of the viral genome and was named after its positional homologue in the HSV-1 genome. Fuchs et al. (2005) analysed transcription and protein expression and identified ILTV gJ as a true late glycoprotein, which is translated from a spliced and a non-spliced mRNA and appears as four high molecular mass proteins in SDSPAGE. A gJ deletion mutant constructed from the virulent ILTV A489 strain showed significant reduction in cell culture titres when compared with the wild-type virus and the corresponding gJ rescue virus. Here, we describe the generation of two gJ deletion mutants and their gJ rescue Supplementary material is available with the online version of this paper. 2586 mutant derived from United States Department of Agriculture standard challenge strain (USDA-ch) and their characterization in cell culture and embryonated chicken eggs. Our data indicate that gJ plays a role during release of infectious virus from cells. As a prerequisite for the generation and characterization of gJ deletion mutants and their rescue mutant, several tools were generated as described in the Supplementary Methods (available in JGV Online). A gJ-specific polyclonal rabbit antiserum was generated using baculovirus-expressed gJ as antigen. mAbs directed against gJ and gC were obtained after immunization of mice with purified ILT virions. A GFP-expressing gJ deletion mutant (GDgJ) was generated by marker rescue after co-transfection of viral DNA prepared from ILT (USDA-ch) virions with a recombination plasmid. In order to assign phenotypic differences of gJ deletion mutants to the absence of gJ and to exclude accidental alterations of the genome during the homologous recombination as responsible factors, a gJ rescue mutant gJR was generated using viral genomic DNA from the GFPexpressing gJ deletion mutant GDgJ. Viral DNA from GDgJ was also used to generate a gJ deletion mutant, BDgJ, which does not contain foreign DNA. Details on the generation of the mutants are presented in the Supplementary Methods and Supplementary Fig. S1 (available in JGV Online) confirmation of mutant phenotypes is presented in Supplementary Fig. S3 and S4 (available in JGV Online). Although gJ of ILTV is not essential for replication in cell culture, gJ deletion mutants were impaired with respect to replication efficiency. The highest titres (TCID50) of virus Downloaded from www.microbiologyresearch.org by 031443 G 2011 SGM IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:18:03 Printed in Great Britain Infectious laryngotracheitis virus glycoprotein J stocks amplified in chicken kidney cells (CKC) were between 104 and 105 ml21 for BDgJ and 105 and 106 ml21 for GDgJ compared with the rescue mutant gJR and the parent virus USDA-ch, which had TCID50 of approximately 106 to 107 ml21. To determine whether gJ is involved in the process of entry of ILTV into cells, entry kinetic experiments were performed in the chicken liver tumour cell line (LMH) (Fig. 1). One hundred p.f.u. of gJ deletion mutants (GDgJ and BDgJ), the ILTV wild-type strain USDA-ch and the gJ rescue mutant gJR were adsorbed to LMH cells on ice. At different times after shifting the temperature to 39 uC, extracellular virus was inactivated and cells were overlaid with semi-solid medium and incubated for plaque assay. p.f.u. values obtained after 60 min incubation at 39 uC were defined as 100 % viral entry and p.f.u. values obtained at the indicated times are shown as percentages of the 60 min p.f.u. value. Per cent entry at all tested times did not differ significantly between the gJ deletion mutants and the rescue mutant, clearly showing that the gJ deletion mutants were not impaired in entry as compared with gJR (Fig. 1). In spite of greater SD, entry of the parental virus USDA-ch was significantly faster than the entry of gJR, GDgJ or BDgJ. This phenomenon may be due to the lack of homogeneity of the USDA-ch strain, which was not plaque-purified, contrary to the mutants, which are derivatives of one single virion. Still, since restoration of gJ expression in gJR did not alter entry kinetics as compared to GDgJ and BDgJ, gJ does not seem to play an important role for the entry of ILTV into LMH cells, although a penetration function of gJ cannot yet be ruled out. In order to assess whether virus egress was affected, a two-step replication kinetic experiment was performed. To this end primary CKC were infected with USDA-ch, GDgJ, BDgJ or gJR at an m.o.i. of 0.01. At 24, 48, 72 and 96 h post-infection (p.i.) cell-associated and extracellular viral titres were compared for each virus USDA-ch (a), gJ deletion mutant GDgJ (b), gJ rescue mutant gJR (c), gJ deletion mutant BDgJ (d) (Fig. 2). While differences between virus titres of cell-associated virus and virus titres of the corresponding supernatants declined markedly over time for USDA-ch (Fig. 2a) and the rescue mutant gJR Fig. 1. Entry kinetics of ILTV into LMH cells. Means of three independent experiments are shown, error bars represent SD, asterisks mark significant differences. http://vir.sgmjournals.org (Fig. 2c), they remained high for GDgJ (Fig. 2b) and BDgJ (Fig. 2d) over the duration of the experiment, indicating a defect in the release of infectious virus particles from the cells into the supernatant in both gJ deletion mutants. The observed impairment with respect to viral egress can be attributed to the absence of gJ expression, because the kinetics of cell-associated and supernatant viral titres of gJR were very similar to USDA-ch. The obtained data showed a severe impairment of release of infectious virus into the supernatant in the absence of gJ. The next experiments were performed to test if a similar phenotype for the gJ deletion mutants with significant deficits in egressed virus to the cell culture supernatant may be reflected in embryonated chicken eggs. To test whether release of virus into allantoic fluid (AF) was impaired in the gJ deletion mutants, viral titres in the AF of embryos infected with the different viruses by inoculation on the embryo chorioallantoic membranes (CAMs) were compared to viral titres of extracts of the inoculated portions of the CAMs. Egg shells of 9-days-old specific-pathogen-free (SPF) chicken embryos were sanitized by spraying with BioSentry 904 (Neogen). CAMs were inoculated with 104 TCID50 of gJR, GDgJ and BDgJ and the eggs were incubated for 6 days at 37 uC. The portion of CAM around the inoculation site was collected and stored in 400 ml Dulbecco’s minimal essential medium/ 2 % FBS at 280 uC prior to homogenization using the FastPrep system (MPbiomedical). After centrifugation at 12 000 g for 2 min, virus titres in the cleared supernatant were determined as TCID50 on CKC. AF was collected prior to extraction of CAM, frozen at 280 uC, quickly thawed and centrifuged at 14 000 g for 2 min. Supernatants were transferred to fresh tubes and titres were determined as TCID50 on CKC (Fig. 3). No infectious virus was detected in the AF of embryos infected with BDgJ and the mean CAM titre was 103.4 TCID50 ml21. The AF of embryos infected with GDgJ contained a mean titre of 101.8 TCID50 ml21, while the mean titre in CAM extracts was 105.8 TCID50 ml21, which is 10 000 times higher. In contrast, the mean viral titre in the AF of embryos infected with the rescue mutant gJR was 107.2 TCID50 ml21. This was eightfold higher compared with the corresponding mean CAM titre of 106.3 TCID50 ml21. For comparison, 9-day-old chicken embryos were inoculated with 104 TCID50 of USDA-ch and viral titres of AF and CAM were determined after 6 days of incubation. Means of seven embryos were TCID50 of 106.9 ml21 for AF and 106.7 ml21 for CAM. As shown for gJR, the viral titre was 1.6-fold higher in AF than in CAM (Fig. 3). These findings were in line with the results of the two-step replication kinetic in cell culture and indicate that the lack of gJ likely hinders egress of infectious virus into the AF of infected chicken embryos. In this study, two gJ-negative recombinant ILTV were derived from the USDA-ch strain of ILTV. During characterization of the mutants in cell cultures and embryonated chicken eggs data were obtained supporting a possible role for gJ in the release of infectious virus from infected cells. ILTV gJ was previously identified and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:18:03 2587 A. Mundt and others Fig. 2. Replication kinetics of different ILTV in CKC. Means of three independent experiments are shown, error bars represent SD. The titres are shown as negative log10 of the TCID50. characterized by Fuchs et al. (2005). They showed that gJ deletion mutants replicated to lower titres in cell cultures than the corresponding rescue mutant and wild-type virus and that the lack of gJ did not influence cell-to-cell spread. In this study, the gC expression from the gJ deletion mutants exhibited a slightly reduced mobility in SDS-PAGE, however, in infected cells using two different gC mAbs this shift was not observed in Western blots of purified virions (Supplementary Data). The underlying mechanism responsible for this phenotype was not addressed experimentally and remains speculative. One possible explanation would be that post-translational processing of glycoproteins in the gJ deletion mutants is less efficient, resulting in accumulation of immature forms of gC in the infected cells, while virions contain mostly fully processed gC. Lack of reactivity of the anti-gJ mAb in gJ deletion mutant virions or infected cells demonstrated absence of the epitope, but did not entirely rule out expression of truncated forms of gJ from the 668 bp remnant of US5 that was retained in the mutants. Existence of such truncated gJ forms was shown to be very unlikely with the lack of binding of the polyclonal rabbit serum directed against gJ. Complete absence of gJ expression in the gJ deletion mutants is of importance if these mutants become candidates for marker vaccines, which should be serologically distinguishable from wt virus and live attenuated vaccine virus. While the polyclonal anti-gJ rabbit serum reacted with the four different forms of gJ, the anti-gJ mAb only bound to the two largest gJ forms of 220 and 160 kDa (Supplementary Data). As shown by Fuchs et al. (2005), the large forms of gJ are translated from the unspliced mRNA containing 986 codons, whereas the short forms of gJ are translated from the spliced mRNA containing 612 codons. Lack of binding of mAb#3 to the short forms of gJ indicates that the epitope recognized by the mAb might be encoded by the partially spliced exon. Fig. 3. Comparison of viral titres in CAM extracts and AF. Means of viral titres in CAM extracts (dark-grey bars) and AF (light-grey bars) are shown. Error bars indicate SD. Entry kinetics showed no impairment of the gJ deletion mutants compared with the gJ rescue mutant, indicating that gJ does not play an important role during viral entry. 2588 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:18:03 Journal of General Virology 92 Infectious laryngotracheitis virus glycoprotein J The fact that absence of gJ in the virion does not delay entry upon infection increases the value of the gJ deletion mutant for use as a live vaccine. In concordance with the earlier description of gJ deletion mutants of ILTV (Fuchs et al., 2005), gJ deletion mutants GDgJ and BDgJ did not appear impaired in their ability to spread from cell to cell, since plaque sizes were greater rather than smaller compared with plaque sizes induced by USDA-ch or the gJ rescue virus gJR on LMH cells (data not shown), indicating that gJ is not required for cell-to-cell spread of ILTV in cell culture. Replication kinetics of gJ deletion mutants, the rescuant and the parental virus titres in the supernatants and cells separately showed that gJ-expressing viruses were efficiently released, while for the gJ deletion mutants the titre differences between supernatants and cells remained high, indicating an important function of gJ during virus egress. The function of gJ during egress was also confirmed, albeit indirectly, by investigating replication in embryonated chicken eggs. Both ILTV encoding gJ (USDA-ch and gJR) replicated very efficiently in CAM, while the gJ deletion mutants showed significantly lower titres. Release of virus into AF after inoculation on the CAM was severely impaired p.i. with gJ deletion mutants, while the reconstitution of gJ expression restored the ability of the virus to exit into the cell culture supernatant, respectively AF. The data further suggest the possible role of gJ in the process of egress of infectious virus from infected cells. Comparing the phenotypes of the two gJ deletion mutants GDgJ and BDgJ, one observation was unexpected. Although both mutant viruses were unable to express gJ they showed slight differences in replication efficacies in vitro as well as in vivo. The deleted section in the viral genome was almost the same for both gJ deletion viruses and the analysis of the genome insertion of the constructs revealed no obvious hint at the cause of the differences. Speculative possible reasons could be that (i) BDgJ contains an additional accidental mutation elsewhere in the genome impairing its fitness with the consequence that the missing gJ function cannot be sufficiently compensated for or crippling a function unrelated to the function of gJ, (ii) insertion of the GFP-expression cassette exerts a beneficial effect for GDgJ, (iii) shortening of the genome by 2185 bp in the insert-free mutant BDgJ exerts a detrimental effect on virus replication that is greater than after deletion of 2300 bp as in the GDgJ mutant. Since we did not investigate biosynthesis and functionality of the other gene products of ILTV besides gJ it cannot be excluded that the deletion of a section of US5 resulted in an indirect effect by affecting http://vir.sgmjournals.org expression of other genes. Since US5, US6 and US7 form a 39 co-terminally transcribed gene cluster (Fuchs et al., 2005) an effect on the expression of these adjacent genes is conceivable. For the betaherpesvirus, human cytomegalovirus, it was shown that deletion of an upstream cistron in a bicistronic transcript affected expression of the downstream ORF (Isomura et al., 2010). We have generated and are currently characterizing a novel gJ-deleted ILTV in which the deleted section of US5 is replaced by a spacer of the same length and GC content, but it does not code for any protein. The phenotype of this novel gJ deletion mutant will be compared to GDgJ, BDgJ and gJR. References Bagust, T. J., Jones, R. C. & Guy, J. S. (2000). Avian infectious laryngotracheitis. Rev Sci Tech 19, 483–492. Davison, A. J., Eberle, R., Ehlers, B., Hayward, G. S., McGeoch, D. J., Minson, A. C., Pellett, P. E., Roizman, B., Studdert, M. J. & Thiry, E. (2009). The order Herpesvirales. Arch Virol 154, 171–177. Devlin, J. M., Browning, G. F. & Gilkerson, J. R. (2006a). A glycoprotein I- and glycoprotein E-deficient mutant of infectious laryngotracheitis virus exhibits impaired cell-to-cell spread in cultured cells. Arch Virol 151, 1281–1289. Devlin, J. M., Browning, G. F., Hartley, C. A., Kirkpatrick, N. C., Mahmoudian, A., Noormohammadi, A. H. & Gilkerson, J. R. (2006b). Glycoprotein G is a virulence factor in infectious laryngotracheitis virus. J Gen Virol 87, 2839–2847. Dufour-Zavala, L. (2008). Epizootiology of infectious laryngotracheitis and presentation of an industry control program. Avian Dis 52, 1–7. Fuchs, W., Ziemann, K., Teifke, J. P., Werner, O. & Mettenleiter, T. C. (2000). The non-essential UL50 gene of avian infectious laryngo- tracheitis virus encodes a functional dUTPase which is not a virulence factor. J Gen Virol 81, 627–638. Fuchs, W., Wiesner, D., Veits, J., Teifke, J. P. & Mettenleiter, T. C. (2005). In vitro and in vivo relevance of infectious laryngotracheitis virus gJ proteins that are expressed from spliced and nonspliced mRNAs. J Virol 79, 705–716. Fuchs, W., Veits, J., Helferich, D., Granzow, H., Teifke, J. P. & Mettenleiter, T. C. (2007). Molecular biology of avian infectious laryngotracheitis virus. Vet Res 38, 261–279. Isomura, H., Stinski, M. F., Murata, T., Nakayama, S., Chiba, S., Akatsuka, Y., Kanda, T. & Tsurumi, T. (2010). The human cytomegalovirus UL76 gene regulates the level of expression of the UL77 gene. PLoS ONE 5, e11901. Pavlova, S. P., Veits, J., Blohm, U., Maresch, C., Mettenleiter, T. C. & Fuchs, W. (2010). In vitro and in vivo characterization of glycoprotein C-deleted infectious laryngotracheitis virus. J Gen Virol 91, 847–857. Thureen, D. R. & Keeler, C. L., Jr (2006). Psittacid herpesvirus 1 and infectious laryngotracheitis virus: comparative genome sequence analysis of two avian alphaherpesviruses. J Virol 80, 7863–7872. 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