Development of recombinant varicella-zoster viruses expressing luciferase fusion proteins for live in vivo imaging in human skin and dorsal root ganglia xenografts

Abstract

Varicella-zoster virus (VZV) is a host specific human pathogen that has been studied using human xenografts in SCID mice. Live whole-animal imaging is an emerging technique to measure protein expression in vivo using luminescence. Currently, it has only been possible to determine VZV protein expression in xenografts postmortem. Therefore, to measure immediate early (IE63) and late (glycoprotein E [gE]) protein expression in vivo viruses expressing IE63 or gE as luciferase fusion proteins were generated. Viable recombinant viruses pOka-63-luciferase and pOka-63/70-luciferase, which had luciferase genes fused to ORF63 and its duplicate ORF70, or pOka-gE-CBR were recovered that expressed IE63 or gE as fusion proteins and generated luminescent plaques. In contrast to pOka-63/70-luciferase viruses, the luciferase gene was rapidly lost in vitro when fused to a single copy of ORF63 or ORF68. IE63 expression was successfully measured in human skin and dorsal root ganglia xenografts infected with the genomically stable pOka-63/70-luciferase viruses. The progress of VZV infection in dorsal root ganglia xenografts was delayed in valacyclovir treated mice but followed a similar trend in untreated mice when the antiviral was withdrawn 28 days post-inoculation. Thus, IE63-luciferase fusion proteins were effective for investigating VZV infection and antiviral activity in human xenografts.

Fig. 1

Construction of VZV recombinants expressing IE63 and gE as luciferase fusion proteins. (A) Schematic of the VZV genome. UL—unique long; US—unique short; TRL—terminal repeat long; IRL—internal repeat long; IRS—internal repeat short; TRS—terminal repeat short. (B) The four cosmids (genome fragments in nucleotides (nt)), pvFsp73 (nt 1–33,128), pvSpe14 (nt 21,796-61,868), pvPme2 (nt 53,756-96,035) and pvSpe23 (nt 94,055-125,123), used to generate recombinant pOka and the luciferase fusion viruses. (C) The location of ORF63 and ORF70 (IE63) and 68 (gE) in the pvSpe23 cosmid. (D–F) The luciferase gene was inserted at the 3′-termini of ORFs 63 (D), 63/70 (E) and 68 (F). Shaded boxes show ORFs. Transfection of these cosmids into melanoma cells yielded recombinant viruses designated pOka-63-CBG68, pOka-63-CBR, pOka-63/70-CBG68, pOka-63/70-CBR and pOka-gE-CBR.

Fig. 2

Western blot of IE63- and gE-luciferase fusion proteins from infected melanoma cells. Cell lysates were prepared from elanoma cells infected with pOka (A), pOka-gE-CBR (B) and pOka-63-CBR (C). IE63 and IE63-CBR were detected with rabbit polyclonal anti-IE63. gE and gE-CBRwere detected with monoclonal antibody 3B3. α-Tubulin was detected with a monoclonal antibody. UI—uninfected. Molecular masses (M_r) for the proteinswere calculated from a standard curve derived from a protein marker.

Fig. 3

Replication in vitro of VZV recombinants expressing IE63 and gE as luciferase fusion proteins. Melanoma cells were infected with pOka or VZV recombinants and tested for replication kinetics over 6 days. (A) Replication of pOka, pOka-63-CBG68, pOka-63-CBR and pOka-gE-CBR. (B)Replication of pOka, pOka-63/70-CBR and pOka-63/70-CBG68. Standard error of the mean is shown on both graphs.

Fig. 4

Cellular localization of IE63 and gE expressed as luciferase fusion proteins. Melanoma cells were infected with VZV recombinants, expressing gE (pOka-gE-CBR) or IE63 (pOka-63-CBR, pOka-63/70-CBR) as luciferase fusion proteins and examined by confocal microscopy at 24 h post-inoculation. IE63—rabbit polyclonal antibody to IE63, gE—monoclonal antibody MAB8612 and Nuclei—Hoescht 33342. The white bar on the merge panels indicates 50 µm. Similar results were seen in melanoma cells infected with pOka-63-CBG68 and pOka-63/70-CBG68.

Fig. 5

Western blot of nuclear and cytoplasmic expression of VZV proteins in cells infected with recombinants expressing IE63-luciferase fusion proteins.Nuclear (N) and cytoplasmic (C) fractions were recovered from melanoma cell lysates collected at 48 h post-inoculation with pOka, pOka-63/70-CBR or pOka-63/70-CBG68. IE4—rabbit polyclonal antibody to IE4, IE62—rabbit polyclonal antibody to IE62, IE63—rabbit polyclonal antibody to IE63, gE—monoclonal antibody 3B3 and α-tubulin—monoclonal antibody to α-tubulin. The anti α-tubulin antibody was used to control for the purity of the cytoplasmic and nuclear fractions. UI—uninfected. Molecular masses (M_r) for the proteins were calculated from a standard curve derived from a protein marker.

Fig. 6

Southern blot detection of ORF63 and ORF70 plus the luciferase genes, CBR and CBG68 in cosmids and recombinant virus-infected cells. Southern blots were performed with cosmid or DNA extracted from HELFs infected with pOka or recombinants and digested with restriction endonuclease EcoRI. DNA probes to ORF63/70 or the CBR and CBG68 click beetle luciferases were used to detect the genes. Cosmid DNA: (A) pvSpe23, (B) pvSpe23-63-CBR, (C) pvSpe23-63/70-CBR, (D) pvSpe23-63-CBG68, (E) pvSpe23-63/70-CB68. Viral DNA: (A) pOka, (B) pOka-63-CBR, (C) pOka-63/70-CBR, (D) pOka-63-CBG68, (E) pOka-63/70-CBG68. UI—uninfected HELFs. The white numbers mark the DNA fragments (kbp) that were expected (1–16.8; 2–8.0; 3–18.5; 4–9.6) or generated from viral genomic reorganization (5–12.7/12.2; 6–14.3/13.8).

Fig. 7

Luminescence generated in vivo by IE63 expression in human tissue xenografts infected with pOka and pOka-63/70-luciferase viruses. Images of the infected mice in the figure were taken at 15 days post-inoculation. The intensity of the luminescence (photons/s/cm²) is indicated by the false colour scale. (A) (number of xenografts); 1—pOka (n = 10), 2—pOka-63/70-CBR (n = 10) and 3—pOka-63/70-CBG68 (n = 10). The graph shows the levels of luminescence generated by IE63-luciferase in skin xenografts for 21 days. Implants infected with pOka-63/70-CBR or pOka-63/70-CBG68 that did not generate luminescence were excluded fromthe graph with the exception of the pOka controls, which were used to determine the background levels of luminescence. (B) (number of xenografts); 1—pOka (n = 4) and 2—pOka-63/70-CBR (n = 4). The graph shows the levels of luminescence generated by IE63-luciferase in dorsal root ganglia xenografts for 56 days. (C) Luminescence generated in vivo by IE63 expression in dorsal root ganglia xenografts infected with pOka-63/70-CBR with (n = 8) or without (n = 10) valacyclovir treatment. Valacyclovir was added to the drinking water (1 mg/ml) and taken ad libitum. Valacyclovir was removed at day 28 (black arrow head) and two mice from each group were euthanized for analysis. The open arrow head indicates that three mice from the treated group and three mice from the untreated group were removed from the data set as they received additional treatments. The grey arrow head indicates that three mice in the untreated group were euthanized. Corrected luminescence—luminescence values were normalized by subtracting the luminescence values emitted from dorsal root ganglia infected with wild type pOka from those infected with pOka-63/70-CBR. Standard error of the mean is shown on all graphs.