Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex

Abstract

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.

Fig. 1

Construction of p-OKA cosmid vectors with VZV ORF25 deletion and substitution mutants. Line 1 shows a schematic diagram of the pOKA genome and the localization of orf25 in the unique long region. Line 2 depicts the overlapping segments of the pOKA genome subcloned into the respective pOKA cosmids: pvFspe73, pvSpe14, pvSpe23, and pvPme 2. Line 3 shows the splitting of the pvSpe14 segment into pNhe and pPvu. Line 4 shows the construction of the orf25 deletion mutant, where the second codon of orf25 was changed into a stop codon without affecting the integrity of the orf26 reading frame (black box). Line 5 depicts the position of the single AvrII site within pvSpe23 used for the insertion of orf25 rescue or substitution mutants. shows the construction of ORF25 mutants using a novel 5 cosmid system. Lane 1 shows the localization of ORF25 within the VZV genome and lane 2 the arrangement of the four cosmids including the whole VZV genome. Lane 3 shows how cosmid pvSpe14 was split into two overlapping cosmids. Lane 4 depicts the construction of the deletion mutant by introducing the stop codon TAA (black square) at aa position 2, which results in silent mutations for the overlapped ORF26. Lane 5 depicts the ectopic AvrII site of insertion of the rescue mutant.

Fig. 2

A) The upper panel shows MeWo cells that were infected with pOKA, fixed at 48 hpi and subjected to microscopic analysis. ORF25 was detected with a rabbit polyclonal anti- ORF25 and IE63 was detected with a mouse and nuclei were counterstained with DAPI. The lower panel shows MeWo cells transfected with the expression plasmid pCR3.1-N-Venus- ORF25 expressing full-length ORF25, fixed at 48 h post transfection, and subjected to microscopic analysis. B) Kinetics of ORF25 expression in VZV infected MeWo cells. The infected input cells were labeled with an orange cell tracker, which allows the selective analysis of newly infected cells. MeWo cells were infected with orange CellTracker labeled, pOKA-infected inoculum cells. The cells were fixed at 4, 6, 9 and 12 hpi and stained with specific antibodies for VZV proteins ORF25, ORF47 or ORF63. Cell nuclei were counterstained with DAPI. Pink arrows mark inoculum cells whereas green arrows indicate the corresponding VZV ORF.

Fig. 3

Self-interaction of ORF25. This figure shows the Y2H and LuMPIS results of the ORF25 self interaction assay as studied with the full-length protein as well as with its N- and C-terminal domains. The data were statistically analysed by ANOVA (*P<0.05) followed by Dunnett’s post hoc test.

Fig. 4

PPI of proteins putatively involved in encapsidation and nuclear egress as determined by three independent PPI screens. Panel A, B and C depict the results obtained by Y2H ^, , LuMPIS and BRET, a full box represents a positive interaction, under each matrix there is a scheme illustrating the different fusion proteins used in the respective assays. Panel D summarizes the number of in the intersections depict the overlap between the different screening systems.

Fig. 5

Model of the PPI network of proteins putatively involved in encapsidation and nuclear egress. Panel A shows the PPI considered as high fidelity (detected by all three screen systems) in black full lines, and those considered as medium fidelity (detected by two systems) in full grey line if detected by Y2H and LuMPIS and in dashed lines if detected by LuMPIS and BRET. Panel B depicts the interactions of the orthologues as already described in the literature ^{, , -, , , , , -}. Full dark gray lines represent interactions validated with other systems than Y2H and the dashed lines represent PPI found exclusively in Y2H screens. The numbers on the lines represent respective citations 1=^{, ,} , 2=, 3=, 4=^{, ,} , 5=, 6= ^{, , ,} , 7=^, , 8=^, , 9=, 10=^{, ,} , 11=, 12=, 13=, 14=^, , 15=, 16=^{, , , , ,} , 17=^{, ,} , 18=^{, , ,} , 19=, 20=.