ANP32B is a nuclear target of henipavirus M proteins.

Membrane envelopment and budding of negative strand RNA viruses (NSVs) is mainly driven by viral matrix proteins (M). In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV) M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV) M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed

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

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

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

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