The Bla VLP assay can also be adapted to a fluorometric plate reader format, allowing its use in high-throughput screening applications

The Bla VLP assay can also be adapted to a fluorometric plate reader format, allowing its use in high-throughput screening applications. determined as in (B). (D) Protease treatment of VLPs. Supernatants from BlaM1 alone (no env) or BlaM1-HA/NA transfected cells were left untreated (untr) or were incubated with Proteinase K only (+P), or (+)-Phenserine with both Proteinase K and Triton X-100 (+P, +Tx-100). Samples were lysed in SDS sample buffer and separated by SDS-PAGE (4-15% gradient). Proteins were detected by immunoblotting using an anti-beta-lactamase antibody. (E) Analysis of purified VLPs. Supernatants from BlaM1 alone (no env) or BlaM1-HA/NA transfected cells were purified over a 30% sucrose cushion. BlaM1-HA/NA-transfected cell supernatants were left untreated (+)-Phenserine or incubated with 3 g/ml TPCK-trypsin for 30 min at 37 C. The samples were lysed in SDS sample buffer and separated by SDS-PAGE (4-15% gradient). Proteins were detected by immunoblotting with an anti-WSN polyclonal antibody. To generate the BlaM1 construct, which upon expression would be incorporated as a structural component into influenza VLPs, Bla was fused to the N terminus of the M1 protein from the pandemic 1918 (A/BrevigMission/1/18) influenza virus. BlaM1 was cotransfected with 1918 HA and 1918 NA expression constructs into 293T cells. Following transfection, Bla activity in the 293T cells was evident and their supernatants contained an appreciable hemagglutination (HA) titer (data not shown and Fig. 1B), suggesting the generation of BlaM1 VLPs. There was no detectable HA titer in the supernatants of the BlaM1 and HA-transfected cells (Fig. 1B). However, when exogenous bacterial neuraminidase was added during the transfection, HA titer within the cell supernatants was recovered, albeit to a lesser extent than when both HA and NA were present (Fig. 1C). These data indicate a requirement for NA enzymatic activity in releasing the VLPs, but also suggest that the influenza NA protein may contribute to VLP budding. Finally, despite there being MRC1 no HA or NA in the BlaM1 alone control transfections, BlaM1 protein was detectable in the supernatants of these cells, although it was sensitive to Proteinase K treatment (Fig. 1D). These data suggest that BlaM1 is nonspecifically secreted into the supernatant following its overexpression in 293T cells, rather than incorporated into a VLP containing BlaM1 alone. In contrast, a significant proportion of BlaM1 protein within the supernatants from BlaM1, HA, and NA-containing transfections was protected (+)-Phenserine from protease treatment, presumably by a lipid membrane, unless detergent was also present (Fig. 1D). It was next examined using flow cytometry whether BlaM1-containing VLPs could transfer Bla activity to na?ve MDCK cells. A prerequisite for influenza virus membrane fusion is the proteolytic cleavage of the HA precursor HA0 into HA1 and HA2, liberating the hydrophobic fusion peptide located at the amino terminus of HA2 (Klenk et al., 1975; Lazarowitz et al., 1973). Here this phenomenon was mimicked by the addition of TPCK-trypsin to the VLP preparation. Examination of purified VLPs by immunoblotting supported the incorporation of BlaM1, HA and NA into particles and the cleavage of HA after trypsin addition (Fig. 1E). Only cells that had been incubated with TPCK-trypsin-treated BlaM1 VLPs bearing both HA and NA (influenza VLPs) demonstrated Bla activity in MDCK cells, indicated by the shift in fluorescence from the green to the blue channel (Fig. 2A). Serial dilution of the VLP inoculum resulted in a linear decrease of Bla-positive cells (Fig. 2B). Low levels of background Bla activity, similar to that from mock-treated cells, were observed after incubation of MDCK cells with supernatants from cells transfected with BlaM1 alone or cells transfected with only HA and BlaM1, indicating a requirement for HA and NA in the transfer of BlaM1 to na?ve cells (Fig. 2A). Variable levels of VLP-mediated BlaM1 delivery were observed between experiments, most likely due to the multiplicity of infection or differential incorporation of BlaM1 into the VLPs from preparation to preparation. Influenza VLPs containing the HA and NA from A/PuertoRico/8 and A/WSN/33 (WSN) H1N1 viruses demonstrated similar properties, except that for WSN VLPs, TPCK-trypsin was not required for BlaM1 transfer, consistent with the trypsin-independent activation of the WSN HA in the presence of.