Tumor cell-specific activity of chicken anemia virus viral protein 3 (VP3 or apoptin) is believed to be dependent on its ability to localize in the nucleus of transformed but not of primary or nontransformed cells. The present study characterizes the signals responsible for the novel nucleocytoplasmic trafficking properties of VP3 using two isogenic tumor/ nontumor cell pairs. In addition to the tumor cell-specific nuclear targeting signal, comprising two stretches of basic amino acids in the VP3 COOH terminus which are highly efficient in tumor but not in normal cells, we define the CRM1-recognized nuclear export sequence (NES) within the VP3 tumor cell-specific nuclear targeting signal for the first time. Intriguingly, the NES (amino acids 97-105) is functional in normal but not in tumor cells through the action of the threonine 108 phosphorylation site adjacent to the NES which inhibits its action. In addition, we characterize a leucine-rich sequence (amino acids 33-46) that assists VP3 nuclear accumulation by functioning as a nuclear retention sequence, conferring association with promyelocytic leukemia nuclear bodies. This unique combination of signals is the basis of the tumor cell-specific nuclear targeting abilities of VP3. (Cancer Res 2005; 65(16): 7059-64)

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection.We have previously shown that M localizes in the nucleus through the action of the importin ␤1 nuclear import receptor. Here, we establish for the first time that M's ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.The Pneumovirus respiratory syncytial virus (RSV) within the Paramyxoviridae family is the most common cause of lowerrespiratory-tract disease in infants (7). The negative-sense single-strand RNA genome of RSV encodes two nonstructural and nine structural proteins, comprising the envelope glycoproteins (F, G, and SH), the nucleocapsid proteins (N, P, and L), the nucleocapsid-associated proteins (M2-1 and M2-2), and the matrix (M) protein (1, 7, 11). Previously, we have shown that M protein localizes in the nucleus at early stages of infection, but later in infection it is localized mainly in the cytoplasm, in association with nucleocapsid-containing cytoplasmic inclusions (13,16). The M proteins of other negative-strand viruses, such as Sendai virus, Newcastle disease virus, and vesicular stomatitis virus (VSV), have also been observed in the nucleus at early stages of infection (32,40,48). Interestingly, the M proteins of all of these viruses, including RSV, play major roles in virus assembly, which take place in the cytoplasm and at the cell membrane (11,12,24,34,36,39), but the mechanisms by which trafficking between the nucleus and cytoplasm occurs are unknown.The importin ␤ family member Crm1 (exportin 1) is known to mediate nuclear export of proteins bearing leucine-rich nuclear export signals (NES) (8,9,18,19,37,42,43), such as the human immunodeficiency virus type 1 Rev protein (4). In the case of the influenza virus matrix (M1) protein, binding to the influenza virus nuclear export protein, which possesses a Crm1-recogn...

The matrix (M) protein of respiratory syncytial virus (RSV) plays an important role in virus assembly through specific interactions with RSV nucleocapsids and envelope glycoproteins in the cytoplasm as well as with the host cell membrane. We have previously shown that M localizes to the nucleus of infected cells at an early stage in the RSV infection cycle, where it may be instrumental in inhibiting host cell processes. The present study uses transient expression of M as well as a truncated green fluorescent protein (GFP) fusion derivative to show for the first time that M is able to localize in the nucleus in the absence of other RSV gene products, through the action of amino acids 110-183, encompassing the nucleic acid binding regions of the protein, that are sufficient to target GFP to the nucleus. Using native PAGE, ELISA-based binding assays, a novel Alphascreen assay, and an in vitro nuclear transport assay, we show that M is recognized directly by the importin beta1 nuclear import receptor, which mediates its nuclear import in concert with the guanine nucleotide-binding protein Ran. Retention of M in the nucleus through binding to nuclear components, probably mediated by the putative zinc finger domain of M, also contributes to M nuclear accumulation. This is the first report of the importin binding and nuclear import properties of a gene product from a negative sense RNA virus, with implications for the function of RSV M and possibly other viral M proteins in the nucleus of infected cells.