We next tested whether HIV-1 budding could be rescued to comparable levels simply by varying the quantity of pCMV(WT)-CHMP2A used in the transfection reaction. 3-fold dilutions over a range of 500–0.69 ng of pCMV(WT)-CHMP2A were tested for rescue of HIV-1 budding from cells that lacked endogenous CHMP2 proteins. CHMP2A expression levels correlated well with the quantity of pCMV(WT)-CHMP2A vector used (Figure 2B, panel 4, lanes 3–9), and CHMP2A levels most closely approximated normal endogenous protein levels when 56 and 19 ng of pCMV(WT)-CHMP2A were used (compare lane 1 to lanes 6 and 7). Rescue of HIV-1 budding again followed a biphasic curve, with optimal rescue observed when CHMP2A was expressed at intermediate levels (170–19 ng pCMV(WT)-CHMP2A, lanes 5–7). In this case, however, HIV-1 titers never exceeded 26% of control levels, even when the bulk levels of exogenous CHMP2A approximated endogenous control levels (Figure 2B, panel 1, compare lane 1 to lanes 6 and 7). In a parallel control experiment, HIV-1 release was again rescued to nearly wild type levels upon co-transfection of 500 ng of the pCMV(Δ4)-CHMP2A construct (lane 10). We therefore conclude that although optimizing pCMV(WT)-CHMP2A vector levels improved HIV-1 budding, overall rescue levels were never as high as could be achieved with the attenuated pCMV(Δ4)-CHMP2 expression construct.
We hypothesized that the pCMV(Δ4)-CHMP2A and pCMV(Δ5)-CHMP2A vectors worked well in the rescue experiment because they could be used at concentrations that coupled high transfection efficiencies with restricted protein expression. To test this idea, we created pCMV(WT)-YFP, pCMV(Δ4)-YFP and pCMV(Δ5)-YFP expression vectors and used YFP fluorescence as a measure of protein expression in 293T cells. This approach allowed us to use flow cytometry to quantify transfection efficiencies and relative protein expression levels at the single-cell level. Titrations were again performed to determine the quantity of pCMV(WT)-YFP required to express YFP at levels comparable to those produced by transfections with 500 ng of pCMV(Δ4)-YFP or pCMV(Δ5)-YFP. This was achieved with 19 ng of pCMV(WT)-YFP, in reasonable agreement with the analogous CHMP2A titration experiments (Figure 3A, compare total mean fluorescence levels for 500 ng of pCMV(Δ4)-YFP or pCMV(Δ5)-YFP DNA with 19 ng of pCMV(WT)-YFP). As shown in Figure 3B, overall transfection efficiencies under these three conditions were: 94 ± 1% for 500 ng of pCMV(Δ4)-YFP, 90 ± 2% for 500 ng of pCMV(Δ5)-YFP DNA and 36 ± 8% for 19 ng pCMV(WT)-YFP (compare lanes 2, 3 and 5). Thus, overall transfection efficiencies dropped off significantly when the quantity of vector was reduced from 500 to 19 ng. We also quantified the mean fluorescence intensity (MFI) in the subsets of cells that were actually transfected in each reaction (i.e., now excluding cells in which YFP expression was undetectable). As shown in Figure 3C, transfected cells in the 19 ng pCMV(WT)-YFP reaction had a MFI of 11 ± 2, whereas transfected cells in the 500 ng pCMV(Δ4)-YFP and pCMV(Δ5)-YFP reactions had MFI of 5.4 ± 0.4 and 3.4 ± 0.6. These data demonstrate that although bulk YFP expression levels were comparable for the three conditions, this was achieved in different ways: the pCMV(Δ4)-YFP and pCMV(Δ5)-YFP vectors supported low-level YFP expression in nearly all of the cells, whereas the pCMV(WT)-YFP vector supported higher expression levels per cell, but in fewer than half of the cells. Thus, the attenuated vectors appear to work better in rescue experiments because, unlike the wild type pCMV(WT) vector, they can be used at sufficiently high concentrations to maintain high overall transfection efficiencies, yet they express low levels of the target protein in each cell. It is possible that varying HIV-1 vector levels could also affect the degree of rescue, but our experiments did not test this parameter.
In summary, we have created mammalian expression vectors that allow tunable expression of siRNA-resistant constructs and demonstrated their utility in rescuing HIV-1 budding from cells that lacked endogenous CHMP2 proteins. We have also used this system successfully in other experiments, for example to achieve high-level rescue of retrovirus budding from cells depleted of endogenous ALIX and CHMP4 proteins (although the relative advantages of using the attenuated CMV vector system were somewhat less pronounced in these two cases, data not shown). The optimal CMV vector must, of course, be determined empirically for each new system because the correct choice will be influenced by differences in endogenous protein levels, protein expression efficiencies, and the degree to which the specific pathway and cell type can tolerate protein overexpression. Although, we are not aware of previous studies that have employed the approach described here, related approaches such as the use of inducible promoters to optimize the expression of siRNA-resistant rescue constructs have been described (21). In principle, this is an elegant approach that can also be used to maximize phenotypic rescue, but it requires the creation of stable cell lines and is therefore less convenient than transient transfection, particularly when the functions of multiple mutant proteins are being screened. Hence, our system is likely to be most useful in cases where levels of the rescue protein must be tightly controlled and where the creation of stable cell lines is overly time consuming or problematic. Our vectors should also be useful in other applications where it is desirable to attenuate protein expression while maintaining high transfection levels.
This work was supported by National Institutes of Health grant AI051174 (W.I.S) and research fellowships from the Japanese Herpesvirus Infections Forum (J.A.) and the Deutsche Forschungsgemeinschaft (J.V., VO 1836/1-1). This paper is subject to the NIH Public Access Policy.
The authors declare no competing interests.
Address correspondence to: Wesley I. Sundquist, Department of Biochemistry, 15 N. Medical Drive, Room 4100, University of Utah School of Medicine, Salt Lake City, UT, USA. Email: