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. 2009 Nov;83(21):10869-76.
doi: 10.1128/JVI.01271-09. Epub 2009 Aug 26.

Mutational analysis reveals a noncontractile but interactive role of actin and profilin in viral RNA-dependent RNA synthesis

Mary Harpen  1 , Tiasha Barik, Alla Musiyenko, Sailen Barik
Affiliations

Mutational analysis reveals a noncontractile but interactive role of actin and profilin in viral RNA-dependent RNA synthesis

Mary Harpen et al. J Virol. 2009 Nov.

Abstract

As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role.

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Figures

FIG. 1.
FIG. 1.
Mutationally targeted actin and profilin residues selected based on structure-function relationship. Relevant portions of an actin-profilin cocrystal structure (NCBI accession no. 2BTF) (43) are rendered by PyMOL in a ribbon diagram. (A) Actin (green) and profilin (cyan) are labeled, and the selected mutually interacting residues of the two proteins are shown in pink. N61 in profilin is a representative noninteracting control, situated far away from actin. (B) Selected divalent-cation-binding (red) and nucleotide-binding (blue) residues of actin are shown. The divalent cation and the nucleotide (ADP/ATP) fit into the visible groove surrounded by these residues. Profilin is irrelevant for panel B but is still visible (light cyan) in the background. Overall, these residues form the basis of our mutagenesis studies.
FIG. 2.
FIG. 2.
Recombinant expression and ATP-binding activities of actin mutants. The actin mutants, generated by site-directed mutagenesis, have the same numbers used in Table 1. His-tagged recombinant actins were expressed in bacteria, after which the tag was removed by thrombin digestion. (A) Selected proteins with mutations in each functional category were resolved by SDS-PAGE, and the Coomassie blue-stained bands are shown. The actin band intensities were densitometrically quantified to use equal amounts of the mutant proteins in all our subsequent studies, including the ATP-binding assay for which results are shown in panel B. The two molecular weight markers (Mr, 1,000) in lane M are identified to the left. (B) Autoradiograph of selected actin mutants cross-linked to [α-32P]ATP and subjected to SDS-PAGE. In both panels, “C” indicates the vector-only E. coli control (with no actin) processed similarly.
FIG. 3.
FIG. 3.
Binding properties of actin and profilin mutants. (A) Actin mutants were tested for RSV N-RNA binding in the presence (bottom) or absence (top) of recombinant profilin. The bound actin and N protein were detected by immunoblotting (Western) using the respective antibodies. C, vector-only E. coli control (with no actin) processed similarly. (B) Reciprocally, the profilin mutants were tested for N-RNA binding in the presence of various mutant actins by using various combinations. Profilin and N were detected by immunoblotting as described in panel A. (C) Direct actin-profilin interaction was tested using the tagged versions of the proteins, in the absence of N-RNA. IP was done with FLAG antibody (α-FLAG) to pull down actin, and the bound profilin was detected by immunoblotting (IB). α-His, His antibody.
FIG. 4.
FIG. 4.
Model for the roles of actin and profilin in transcription. The model is schematic only and not drawn to scale. The viral genomic RNA (wavy line) is wrapped with the nucleocapsid protein N. It is copied by viral RdRP, made up of L (large circle), P (rhomboid), and M2-1 (ellipsoid) proteins. Although the exact placement and number of these proteins are currently unknown, we have tried to accommodate all interactions known to date: P-P, P-N, actin-N-RNA, actin-profilin, and profilin-P (see the text for details). Actin (gray) is shown to bind to the N-RNA with the help of the divalent cation M2+. Actin can recruit profilin without any help from P; however, it is possible that P, when present, makes some contribution. Both actin and profilin may have activation domains (striped) that interact with RdRP, and reciprocally, this interaction may stabilize the binding of both proteins.
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