Levels of selected transcripts differ between virus producers and viral particles

random integration (hCMV, Terhune et al., 2004), or are enriched in virus particles as in the case of HSVI (Sciortino et al., 2002). To analyze the situation with EBV, I compared the transcript levels in B95.8 cells and viral particles that these cells release.

EBV producing cells express the viral glycoprotein gp350 on their surface (Flamand et al., 1993; Yuan et al., 2006). The fraction of gp350+ cells in the B95.8 cell line depends on culture conditions and ranges between 1-10%. I enriched the gp350+ fraction of B95.8 cells by FACS-sorting and obtained an almost pure population (figure 3.5A) that was subjected to RNA preparation and reverse transcription for subsequent qPCR analysis.

Transcript levels in two different samples can be compared by normalization to a housekeeping transcript that is expressed at equal levels in both samples. This requirement could not be assured in my approach addressing producer cells and viral particles. I chose the BMRF1 transcript as reference, which showed high abundance in both B95.8 cells and B95.8 virus particles. Relative levels of several transcripts in sorted gp350+ cells and viral particles were calculated and plotted as shown in figure 3.5B.

Based on this calculation, none of the transcripts showed equal levels in lytic cells and particles. The ratio between gp350+ B95.8 cells and viral particles shown in figure 3.5C suggested that most transcripts were contained in lower relative amounts in viral particles than in gp350+ cells, with exception of LMP1, LMP2AB and EBER transcripts. This finding might reflect their preferential packaging together with transcripts of the BMRF1 gene pointing to a preferential incorporation of certain transcripts.

Whether the observed differences resulted from a selective packaging of BMRF1, LMP1, LMP2AB and EBER transcripts to virions is currently unclear. It will be essential to verify a reliable reference transcript for relative transcript comparisons. Analyses on the basis of absolute transcript levels remain error-prone because of a different compositions of cellular and particle RNAs, as 1µg of cellular RNA contains more than 90% of ribosomal and transfer RNA that is unlikely to be present to the same extent in viral particles.

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Figure 3.5:

Viral particles and producer cells differ in their transcript levels.

A. The fraction of spontaneously virus-producing B95.8 cells was assessed by FACS analysis. Gp350+ and gp350- cells were separated by FACS sorting B. Levels of different transcripts were normalized to BMRF1 and compared for viral particles and gp350+ cells. C. The ratio of the tran- script levels was calculated from data in B.

2.4 The RNA shuttle-protein EB2 of EBV is present in virions

The BMLF1/BSLF1-encoded EBV protein EB2 is a candidate for the selective packaging of RNAs into viral particles. This hypothesis builds on previous analyses of capsid assembly and particle function (Gruffat et al., 2002; Batisse et al., 2005), and the in vitro interaction of EB2 with RNA (Hiriart et al., 2003a). EB2 has been shown to be essential for the formation of progeny virus in producer cells and mediates the export of non-spliced transcripts during the lytic phase. If EB2 is a mediator of the selective packaging of RNAs, it might become incorporated and be itself part of virions. I addressed the presence of EB2 in virions in the recombinant system for EBV production. Because an EB2 antibody is not available, I took use of a FLAG-tagged wildtype EB2 (wtEB2-FLAG) and FLAG-tagged mutant EB2 (mutEB2-FLAG), with mutEB2 being incapable of RNA binding (Hiriart et al., 2003b). Expression plasmids for these constructs were transfected into the EBV producer cells 293/2089 (rec.wt.) (Delecluse et al., 1998) together with an expression plasmid for BZLF1, which induces the lytic phase and the synthesis of progeny virus. The supernatants were harvested and parts of them used to infect Raji cells. The number of infected GFP+ Raji cells was assessed by FACS on day three after infection. The virus titers in the supernatants generated with the FLAG-tagged wildtype EB2 or mutant EB2 were comparable (figure 3.6A).

Figure 3.6: EB2 protein is packaged into virions.

Wildtype recombinant EBV producer cells (293/2089) were induced for virus production by transfecting a BZLF1 expression plasmid. WtEB2-FLAG or mutEB2-FLAG expression vectors were co-transfected. A. Raji cells were infected with the respective supernatant. Equal percentages of GFP+ Raji cells in FACS analyses 3dpi confirmed equal virus production for both samples. B. Lysates from producer cells (left) and produced particles (right) were assessed by Western blot for FLAG-tagged EB2 proteins, BZLF1 and BRLF1. Actin signals served as loading control.

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Lysates from the producer cells were analyzed by Western blot immunodetection and confirmed successful transfection of wtEB2-FLAG, mutEB2-FLAG as well as BZLF1. BRLF1, a viral gene downstream of BZLF1 was also readily induced and indicated the initiation of the lytic cycle in the cells (Amon et al., 2004; Yuan et al., 2006). Viral particles were purified and the protein lysates analyzed for the presence of wtEB2-FLAG and mutEB2-FLAG protein by Western blot immunodetection. Distinct signals for wtEB2- FLAG, but not EB2mut, indicated that EB2 is contained within EBV particles suggesting that it might mediate RNA incorporation. The binding motif must be essential for incorporation, ARM-motif deleted “mutEB2” protein could not be detected in viral particles (figure 3.6B).

In analogy to findings with HSV1 (Sciortino et al., 2002), the incorporation of EB2 into viral particles points to its role as a mediator of transcript selection serving as a transfer matrix. The general presence of EB2 in particles gives room to speculate on its role as a mediator of directed transcript incorporation into particles.