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Figure 1. MVA expression of NiV proteins results in protein release. (A) Vero cells were infected with rMVAs expressing NiV N, M, F, or G, along with MVAGKT7 and then metabolically labeled. Lysates were immunoprecipitated with either rabbit anti-NiV polyclonal serum (N, M, G) or rabbit anti-F polyclonal serum. (B) Vero cells were infected with rMVAs and MVAGKT7 and metabolically labeled. Released protein was purified by centrifugation through a 10% sucrose cushion followed by flotation in a sucrose gradient. Proteins derived from cell lysates (L) or culture supernatants (S) were immunoprecipitated and analyzed as described above and in Methods. Lysate bands represent 1/20 of total lysate. (C) Supernatant from radiolabeled cells expressing N, M, F, and G was clarified and released protein was loaded onto a 5–45% sucrose gradient followed by centrifugation for 16 h as described in Methods. Fractions were obtained and proteins were analyzed by immunoprecipitation. A portion of each fraction was used to determine density.
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Figure 2. Quantitated release of MVA-expressed NiV proteins. Cells were infected with rMVAs in order to express NiV proteins alone or in combination. Cells and supernatants were treated as in Fig. 1B. Membrane-associated protein release of (A) N, (B) M and (C) F and G was quantitated by densitometry. A representative experiment is shown for A, and the mean and standard deviation of three or more experiments are shown in B and C.
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Figure 3. NiV proteins are released in the absence of MVA. 293T cells were transfected with pCAGGS constructs containing NiV genes. (A) Protein released into the supernatant was purified by gradient centrifugation as described in Methods. Proteins were immunoprecipitated with MAbs F45G6 (anti-N), F45G5 (anti-M) or rabbit polyclonal anti-serum against NiV F or HeV G. (B) Protein released as in (A) was quantitated by densitometry. The mean and standard deviation of three or more experiments are shown. ND = not detectable. Release of protein from cells expressing combinations of F and G (C) N, M, F, and G (D) or N and M (E) are shown along with the percent protein released. N was not immunoprecipitated in D to allow visualization of F0. Quantitation for C was performed on a lighter exposure. Proteins derived from cell lysates (L) or culture supernatants (S) are indicated. Lysate bands represent 1/6 of total lysate. Representative experiments are shown.
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Figure 4. Comparison of NiV M release with SV5 M and kinetics of NiV M release. (A) Culture supernatants were analyzed for release of either NiV or SV5 M. Released protein was treated as described in Methods and proteins were immunoprecipitated using either rabbit anti-NiV polyclonal serum or MAb M-h (anti-SV5 M). Image shown is over-exposed to show contrast. (B) Cells expressing NiV M were starved for 45 min, 35S-pulsed for 15 min, then chased with DMEM-10 for times indicated. Released protein was purified and protein derived from lysate or supernatant was immunoprecipitated with MAb F45G5. Percent protein release was quantitated by densitometry. Proteins derived from cell lysates (L) or culture supernatants (S) are indicated. Lysate bands represent 1/6 of total lysate.
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Figure 5. Immunoelectron microscopy shows particles consistent in size with authentic NiV virions. VLPs released from transfected 293T cells were purified by gradient centrifugation, labeled by immuno-gold, negatively stained, and viewed by electron microscopy as described in Methods. VLPs were derived from cells that expressed M (A to C) or N, M, F, and G (D and E). Immunolabeling against M was done using MAb F45G5 (A to D) and G was detected with mouse anti-HeV G polyclonal serum (E). Bars represent 100 nm. Specificity of primary and secondary antibodies was demonstrated by their failure to stain negative controls (not shown).
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Figure 6. Density analysis of VLPs released from transfected cells expressing NiV proteins. (A) Clarified culture supernatants from cells expressing different combinations of NiV proteins were layered onto 5–45% sucrose gradients and centrifuged. Fractions were immunoprecipitated with MAbs F45G6 (ant-N), F45G5 (anti-M) or rabbit polyclonal anti-serum against NiV F or HeV G. A portion of each fraction was used to determine density. Densitometry analysis of the gels shown in (A) was used to determine the quantitative distribution of proteins (B). Where more than one protein was expressed, the protein analyzed by densitometry is indicated by parentheses. (C) Supernatant from cells infected with NiV was layered onto a 5–45% sucrose gradient and centrifuged. RNA was extracted from fractions and used for real-time PCR. The threshold cycle (Ct) value was used as a proxy for virus concentration.
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