Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3′-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes.

Vaccinia virus gene expression is temporally regulated, and three gene classes have been identified: early, intermediate, and late. Several virus-encoded proteins and an activity designated VLTF-X are required for maximum transcription in vitro of a template containing a late promoter. VLTF-X is present in both cytoplasmic and nuclear extracts prepared from uninfected mammalian cells and co-purifies with a late promoter DNA-binding activity. Here, extensive purification of VLTF-X has revealed that heterogeneous nuclear ribonucleoproteins A2/B1 and RBM3 co-purified with in vitro late transcription stimulation. Overexpression and purification of these proteins from Escherichia coli demonstrated that they both complemented for VLTF-X activity in in vitro transcription reactions. These studies identify two host cell factors potentially contributing to poxvirus replication in vivo.The heterogeneous nuclear ribonucleoproteins (hnRNPs) 1 are a family of single-stranded nucleic acid-binding proteins involved in a variety of cellular functions including mRNA splicing, transport, and turnover (1). Approximately 20 major hnRNPs are known, and they are designated hnRNP A1 (the smallest at 34 kDa) to hnRNP U (the largest at 120 kDa). The most well studied member of this family, hnRNP A1, has been implicated in determining splice site selection of cellular mRNAs. The protein is predominantly nuclear but has been shown to shuttle between the nucleus and the cytoplasm, presumably as a chaperone for mRNA export from the nucleus (2). In addition to its role in cellular mRNA biogenesis, hnRNP A1 has been shown to bind to mouse hepatitis virus template RNA and has been implicated in the replication of this RNA virus (3), although its role in the mouse hepatitis virus life cycle has recently been challenged (4). hnRNP A2 is a closely related member of this family and, similar to hnRNP A1, is a modular protein containing two N-terminal RNA binding domains (RBDs) and a C-terminal glycine-rich domain implicated in protein-protein interactions (2XRBD-gly). hnRNP A1 and A2 are ϳ80% identical in the N-terminal 2XRBD domain (5), and the genes encoding these proteins presumably arose from a gene duplication event (6). The genes for both proteins encode RNAs that can be alternatively spliced; the alternative product to hnRNP A2 is designated hnRNP B1 and is identical to hnRNP A2 but with 12 additional amino acids at the extreme N terminus (5). RBM3 is a more recently described hnRNP closely related to hnRNP G but having only one RBD and a glycinerich tail (7).The poxviruses are DNA-containing viruses that replicate in the cytoplasm of eukaryotic cells and are pathogenic to many animal species. Gene expression in vaccinia virus, the prototypic member of the poxvirus family, is temporally regulated and can be divided into early, intermediate, and late phases. All three phases of gene expression rely on virally encoded factors and a viral multisubunit RNA polymerase with homology to eukaryotic RNA polymerase II. Transcription of the late genes ...

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The viral proteins A1L, A2L, G8R, and H5R positively modulate vaccinia virus late gene expression. Host-encoded proteins hnRNP A2 and RBM3 may also interact with these viral factors to influence late gene expression. In these studies, a yeast two-hybrid screen and in vitro pulldown and crosslinking experiments were used to investigate protein--protein interactions among these factors. These studies confirmed a previous observation that G8R interacts with itself and A1L. However, self-interactions of A1L and H5R, and interactions between A2L and G8R, A2L and H5R, and H5R and G8R were also observed. In addition, the proteins hnRNP A2 and RBM3 both showed some interaction with A2L. Illustration of these interactions is a step toward understanding the architecture of the late gene transcription complex as it occurs in poxviruses.