ANP32B-dependent precipitation of HeV M.

<p>(A) Western Blot detection of ANP32B and HeV M in cell lysates (input) and after purification of Strep-ANP32B (eluate). (B) silver gel analysis of purified protein samples. The identities of abundant signals at 40 and 35 kDa were confirmed by mass spectrometry as HeV M and ANP32B, respectively.</p

ANP32B-dependent nuclear retention of M in Nipah virus infected cells.

<p>After infection of mCherry-ANP32B expressing A549 cells at an MOI of 0.5, nuclear accumulation of NiV M was observed (arrows). In contrast, no accumulation was detectable in nuclei (DAPI-stained; blue) without mCherry-ANP32B (arrowheads). Shown are composites of two images. Scale bar: 10 µM.</p

Over-expression of ANP32B results in specific nuclear accumulation of HeV M.

<p>(a–c) No nuclear accumulation was detectable in HEK 293T cells in the absence of over-expressed ANP32B. (d–f) At ANP32B over-expression, HeV M accumulated in the nucleus. (g–i) Leptomycin B led to nuclear accumulation of HeV M without over-expression of ANP32B. (j–l) Leptomycin B led to nuclear accumulation of rabies virus P. (m–o) In the absence of Leptomycin B, rabies virus P (RABV P) was not detected in the nucleus. (p–r) ANP32B over-expression did not induce nuclear accumulation of RABV P.</p

ANP32B-dependent precipitation of HeV M.

<p>(A) Western Blot detection of ANP32B and HeV M in cell lysates (input) and after purification of Strep-ANP32B (eluate). (B) silver gel analysis of purified protein samples. The identities of abundant signals at 40 and 35 kDa were confirmed by mass spectrometry as HeV M and ANP32B, respectively.</p

Virus release is not inhibited by shRNA knock-down of ANP32B expression.

<p>(A) Western Blots confirmed knock down of ANP32B protein in HEK-293T cells that stably express shRNAs against ANP32B mRNA (shRNA 765 and 767). Control cells shRNA 782 and 784 expressed irrelevant shRNAs and in authentic HEK293T cells ((−) shRNA) no shRNAs were expressed. (B) Growth curves for Nipah virus revealed that Nipah virus production was not affected by the shRNA knock down.</p

Strep-tagged HeV M proteins and expression in plasmid transfected HEK293T cells.

<p>(A) Schematic drawing of non-tagged HeV M and N- and C-terminally tagged HeV M proteins. The Strep-tag sequence is indicated by dark boxes. (B) Western Blot with HeV M specific serum confirming HeV M expression in plasmid transfected cells. (C) 16 h after transfection the cells were fixed and conducted to indirect immunofluorescence with HeV M specific serum and confocal laser scan analysis. No immunostaining was detectable in empty vector transfected cells (not shown). Nuclei were stained with Hoechst 33342 (blue). Scale bar: 5 µm.</p