*E.M. and J.A. contributed equally
We have created a family of mammalian protein expression vectors with cytomegalovirus promoters of differing strengths and shown that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.
Transient transfection of small interfering RNA (siRNA) provides a powerful approach for studying cellular protein functions, particularly when the target protein can be re-expressed from an exogenous siRNA-resistant construct in order to rescue the knockdown phenotype, confirm siRNA target specificity, and support mutational analyses. Rescue experiments often fail, however, when siRNA-resistant constructs are expressed at suboptimal levels. Here, we describe an ensemble of mammalian protein expression vectors with CMV promoters of differing strengths. Using CHMP2A rescue of HIV-1 budding, we show that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.
Small interfering RNAs (siRNAs) are commonly employed, both individually and on a genome-wide scale, to degrade specific mRNAs and test the cellular requirements for their encoded proteins (1-7). The basic siRNA depletion experiment can be extended further using “rescue” experiments in which the target protein is re-expressed from a transiently transfected vector that encodes an altered mRNA resistant to siRNA silencing (8-10). This experiment is useful for confirming siRNA specificity because the exogenously expressed protein should rescue the loss-of-function phenotype. The experiment also enables genetic analyses in cultured mammalian cells because the functional effects of specific mutations can be tested. Phenotypic rescue experiments can fail, however, when the rescuing protein is expressed at such a high level that it dominantly inhibits the pathway of interest. This problem can often be alleviated by reducing the quantity of transfected expression vector, but this approach fails if the overall transfection efficiency is reduced. To address this problem, we created an ensemble of seven mammalian expression vectors designed to allow more precise control of exogenous protein expression levels. These vectors have nested deletions that successively eliminate transcription factor binding sites within the human cytomegalovirus (CMV) intermediate early enhancer/promoter (summarized in Figure 1 and Supplemental Table 1, and see Supplemental Figure 1 for promoter DNA sequences and a summary of the design strategy). The deletions were made in the context of the mammalian expression vector pcDNA 3.1/myc-His(-)A, that contained a custom-designed multiple cloning site (MCS) cassette. These vectors allow optimized expression of siRNA-resistant constructs, while maintaining the high transfection efficiencies necessary for potent phenotypic rescue.
HIV-1 and many other enveloped viruses recruit the cellular endosomal sorting complexes required for transport (ESCRT) pathway to facilitate the final membrane fission step of virus budding (11-14). As is true for many other cellular pathways, siRNA depletion/rescue experiments have contributed to our understanding of the role of the ESCRT pathway in HIV-1 budding (9, 15). We have found, however, that it is often difficult to rescue virus budding to wild type levels following siRNA depletion because many ESCRT proteins, particularly those of the ESCRT-III family, can potently inhibit HIV-1 budding when overexpressed at elevated levels (16-20). The ESCRT-III/HIV-1 system therefore represents an attractive test case for examining the utility of our family of attenuated CMV expression vectors.
HIV-1 budding from cultured 293T cells can be potently inhibited by co-depletion of both members of the human CHMP2 family of ESCRT-III proteins (denoted CHMP2A and CHMP2B) (15). Hence, vector titers were dramatically reduced 48 h after co-transfection of a proviral HIV-1 vector together with siRNAs that targeted both CHMP2 proteins (Figure 2A, 24 ± 5-fold reduction, compare lanes 1 and 2). CHMP2 depletion also blocked virus release into the culture supernatant, as measured by immunoblotting for the virion-associated structural proteins, MA and CA (Figure 2A, panel 2, compare lanes 1 and 2). Western blots of the 293T producing cells demonstrated that both CHMP2A and CHMP2B were depleted efficiently (Figure 2A, panels 4 and 5, compare lanes 1 and 2) and that cellular levels of the structural HIV-1 Gag protein and its MA and CA cleavage products were not altered significantly by CHMP2 protein depletion (Figure 2A, panel 3, compare lanes 1 and 2).
To test for rescue of virus budding, 500 ng of each of the different siRNA-resistant pCMV-CHMP2A expression vectors were co-transfected together with the siRNA and proviral HIV-1 (Figure 2A). As expected, CHMP2A expression levels were highest for the construct that carried the wild type CMV promoter (denoted pCMV(WT)-CHMP2A) and decreased successively over two orders of magnitude as larger and larger promoter deletions were introduced (denoted pCMV(Δ1)-CHMP2A to pCMV(Δ7)-CHMP2A, (Figure 2A, panel 4, compare lanes 3–10)). In contrast, the rescue of virus budding was biphasic: virion release and infectivity were low when CHMP2A levels were highest, increased when CHMP2A was expressed at intermediate levels, and then decreased again at the lowest CHMP2A expression levels (Figure 2A, panels 1 and 2, compare lanes 3–10). Levels of virion release and infectivity generally correlated well, but maximal infectivity occurred at slightly higher CHMP2A levels, perhaps because rapid virus release kinetics contribute more to viral infectivity than to total virion release as measured in the end point release assay. The pCMV(Δ4)-CHMP2A and pCMV(Δ5)-CHMP2A constructs expressed CHMP2A at levels that most closely approximated the normal level of the endogenous protein (Figure 2A, panel 4, compare lanes 7 and 8 to lane 1). These two CHMP2A expression constructs also rescued virus release and infectivity best (Figure 2A, panels 1 and 2). Importantly, the pCMV(Δ4)-CHMP2A construct rescued viral titers very efficiently, to 102 ± 12% of untreated control levels. These data imply that: (i) CHMP2A alone can fully rescue HIV-1 budding, even in the absence of CHMP2B; (ii) CHMP2A functions best when expressed at near-native levels; and (iii) the attenuated pCMV(Δ4)-CHMP2A and pCMV(Δ5)-CHMP2A constructs can express near-native levels of CHMP2A under conditions where transfection efficiencies apparently remain high.