Department of Pediatrics, Laboratory for Molecular Biology, Charité, CCM-Ziegelstr. 5-9, Humboldt-University, Berlin, Germany
Correspondence
Christian Hagemeier
christian.hagemeier{at}charite.de
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ABSTRACT |
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MAIN TEXT |
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To date, no reports have been published describing the use of siRNA technology in human herpesvirus infection. For human cytomegalovirus (HCMV), the prototype of the Betaherpesvirus family and an important pathogen in the immunocompromised host, this may have been due to technical limitations, since cell culture infection systems typically rely on growth-arrested primary fibroblasts, which in turn are difficult to transfect efficiently. We therefore set out to optimize the standard transfection protocol from the Tuschl laboratory (Elbashir et al., 2002) for use in serum-starved primary human foreskin fibroblasts (CRL-2429 from ATCC) employing the transfection reagent Oligofectamine (Invitrogen). Transfections were performed in 12-well plates with cells at 80 % confluence. Three µl 20 µM siRNA and 3 µl Oligofectamine were diluted in 50 µl and 12 µl Optimem (Invitrogen), respectively. After 5 min at room temperature, both solutions were combined. After 20 min, the cell culture medium was aspirated and replaced by the transfection assay, diluted with 400 µl pre-warmed Optimem. At 12 h post-transfection, siRNA-containing medium was removed and cells were washed once with pre-warmed medium.
In order to determine whether RNAi could be used to downregulate HCMV gene expression effectively in primary fibroblasts, we first designed two siRNAs against the viral DNA polymerase, the UL54 gene product, which is essential for virus replication and an established target of antiviral therapy. The selected siRNAs targeted the regions 5'-AACGCAAGGAUGACCUGUCUU-3' [siUL54(1)] and 5'-AAGUACAUCCUCACGCGUCUC-3' [siUL54(2)] corresponding to nt 15321552 and 12431263 of the UL54 coding sequence. To ensure specificity, both sequences were subjected to a BLAST search against human EST libraries. No homology with human polymerases or any other eukaryotic gene was found. A published siRNA targeting the enhanced version of the green fluorescent protein (siEGFP) was chosen as a negative control siRNA (Caplen et al., 2001). In addition, we included an established siRNA against cdk1 gene transcripts as a positive control. This allowed us to monitor the cell-type-specific efficiency of the gene knock-down approach via RNAi. All siRNAs were purchased from Dharmacon. By transfecting the positive control siRNA in primary fibroblasts that had been serum starved for 72 h, we were able to demonstrate that the induction of cdk1 expression after serum restimulation could repeatedly be downregulated by approximately 8090 % indicating a high, albeit not complete, transfection efficiency (data not shown). This enabled us to set up an experimental system that relied on the well-established protocol of infecting cells in G0. Therefore, fibroblasts were first synchronized by serum starvation, then siRNA transfected and finally infected with HCMV (AD169) (Fig. 1
A). Using pre-synchronized cells in G0 is advantageous for several reasons: (i) the replicative cycle of HCMV can start immediately in this cell-cycle phase (Salvant et al., 1998
); (ii) virus replication performance is highest when cells are infected in the state of quiescence (Noris et al., 2002
); and (iii) accumulating viral DNA can easily be distinguished from the peak of unreplicated cellular DNA by flow cytometry (see below).
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To address the question of whether the extent of siUL54(1)-induced RNA interference is sufficient to inhibit viral DNA replication, we next analysed the DNA content of infected cells. Two characteristics of lytic HCMV infection, namely the inhibition of cellular replication licensing in G1 (Biswas et al., 2003; Wiebusch et al., 2003
) and the extraordinarily high replication rate of the viral genome, allow the convenient assessment of newly synthesized viral DNA by propidium iodide staining and flow cytometry. In the absence of cellular DNA synthesis, the newly replicated viral DNA causes the G1 peak to disappear in favour of a pronounced pseudo-S/G2 peak, which results from infected cells containing, besides their normal 2n DNA content, the replicated viral genomes (Bresnahan et al., 1996
; Lu & Shenk, 1996
). Hence, a left to right shift in addition to a broadening of the main DNA peak is the typical finding when G0 cells are infected with HCMV. Using standard protocols as described previously (Wiebusch & Hagemeier, 1999
) this finding becomes most obvious between 48 and 72 h p.i. when the virus replication machinery reaches maximal activity (Fig. 2
A, untreated).
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To confirm further the antiviral potential of UL54-specific siRNAs, we monitored the increase in viral progeny in the supernatants of infected cell cultures. Compared with untreated cells, no significant reduction in the virus titre was observed in control-transfected cells over the time period of 4 days p.i. In contrast, siUL54(1) transfection resulted in a 12-fold reduction in virus titres after 96 h of infection (Fig. 2C). As expected, ganciclovir treatment abolished virus growth. These data are consistent with the results shown in Fig. 2(A)
and (B), since the inefficiently transfected, minor subpopulation of siUL54(1)-treated cells (hatched in Fig. 2B
) is expected to support virus replication and release of progeny into the cell culture medium. Therefore, these data demonstrate the effectiveness of siRNAs in HCMV-infected fibroblasts.
The above data further suggest that by improving the efficiency of siRNA transfection the antiviral effect of siRNAs directed against essential viral gene products could be expected to approximate to that of chemical inhibitors (e.g. ganciclovir) of virus replication. To test this prediction directly, we used an independent cell culture model of HCMV infection that employs fully permissive U373 cells, which can be infected with siRNAs to greater than 99 % (data not shown). In this set of experiments we also targeted a second essential gene product of HCMV, the 86 kDa IE2 protein. The advantage of this was the availability of antibodies directed against IE2 enabling us to follow protein levels of IE2, rather than mRNA levels, as was the case for pUL54 (see Fig. 2). We used an IE2 siRNA directed against a target sequence within exon 5 of the major IE region. Consequently, levels of the IE1 protein, which shares sequences encoded by exons 2 and 3 with IE2, should not be affected. However, at the same time, we were able to use IE1 as a perfect internal control for the specificity of the knock-down approach by employing an antibody directed against exactly those N-terminal sequences that IE1 and IE2 have in common. Fig. 3
(A) shows that the siRNA directed against exon 5 (siIE2) specifically silenced IE2 expression over the entire infection period but left the IE1 protein untouched. In addition, an unrelated siRNA directed against EGFP (siEGFP) did not affect the expression of IE1 or IE2. These results demonstrate that IE2 can be specifically targeted in HCMV-infected U373 cells and, in line with the rationale presented in Fig. 2
, further show that targets (such as IE2) can be completely downregulated in infected cells that have a high transfection efficiency. Consistent with the complete knock-down of IE2 achieved in these cells, we were unable to recover significant amounts of viral progeny from infected U373 cells transfected with siIE2, whereas control-transfected cells, like untreated cells, were not affected (Fig. 3B
). This result is in line with several studies demonstrating that IE2 is an essential viral protein (Azad et al., 1993
; Heider et al., 2002
).
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ACKNOWLEDGEMENTS |
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Received 25 June 2003;
accepted 1 October 2003.