The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association - PubMed (original) (raw)
The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association
N Zou et al. J Virol. 2000 Apr.
Abstract
The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein. It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication.
Figures
FIG. 1
Schematic representations of the GFP fusion proteins. The full-length HPV-11 E2 protein consists of the N, H, and C domains, each denoted by a different boxed symbol. The amino acid residues of the E2 protein expressed in each fusion protein are indicated. Mutations of the H domain are also illustrated. The GFP moiety is placed at the amino terminus of each fusion protein and is represented by an oval. The calculated molecular masses (kilodaltons) are given at the right; ∗ signifies the dimeric form of the protein when the C domain is present.
FIG. 2
Regulatory functions of gfpE2 and gfpE2bm. (A) Transient replication of HPV-11 origin (ori) plasmid p7730-99 in the presence of HPV-11 E1 plus E2 (lanes 1 and 2), gfpE2 (lanes 3 and 4), gfpE2bm (lane 5 and 6), or GFP (lanes 7 and 8) expression vectors. An autoradiogram of one of two experiments reveals _Hin_dIII-linearized, total origin-containing DNA prior to _Dpn_I digestion (lanes 1, 3, 5, and 7) and replicated, origin-containing DNA after _Dpn_I digestion (lanes 2, 4, 6, and 8). (B) Repression of the HPV-11 URR-E6 promoter-driven lacZ reporter by E2, gfpE2, or gfpE2bm in transfected cells. The results shown were averages of two separate experiments, each performed in duplicate. Both functional assays were conducted in 293 cells as described in Materials and Methods.
FIG. 3
Localization of GFP and GFP fusion proteins as visualized microscopically via GFP fluorescence. (A and C) Subcellular distribution of GFP and GFP fusion proteins in whole cells as indicated in each panel. COS cells were transfected with various expression vectors; 24 h posttransfection, cells were fixed with paraformaldehyde and treated with a mounting medium containing DAPI to reveal their nuclei. (B) Typical patterns of GFP fluorescence which remained in transfected COS-7 cells after in situ fractionation as described in Materials and Methods. (D and E) Typical COS-7 cells cotransfected with an origin-containing plasmid and plasmids expressing EE-E1 and gfpE2 (D) or gfpE2bm (E). E2 was directly visualized via GFP (left). The EE-E1 protein was revealed by the Texas red through indirect immunofluorescence after staining with a monoclonal antibody against the EE epitope (center). Merged images reveal colocalization (right). The images were acquired through a 40× (A and C) or 100× (B, D, and E) objective lens of a monochromatic camera, pseudo-colored, and digitally processed. Relative to panels A and C, images in panels B, D, and E have been enlarged to reveal detailed subnuclear structures.
FIG. 4
Subcellular distribution of GFP and GFP fusion proteins as detected by immunoblotting. COS-7 cells were fractionated by an in situ protocol. The whole cell extracts (WC) as well as the sequentially collected cytoplasm (Cyt) and nucleoplasm (NP) or, alternatively, pooled cytoplasm and nucleoplasm (Ext), chromatin (Chr), and nuclear matrix (NM) fractions were solubilized in PAGE loading buffer, separated by SDS-PAGE, and Western blotted with a monoclonal antibody against H1 (A, left), lamins A and C (A, right), or GFP (B to D).
FIG. 5
Alignment of a portion of the E2 H domain of 15 mucosotropic human and animal papillomaviruses spanning a cluster of basic amino acids residues. Pcpv, pigmy chimp papillomavirus; Rpv, rhesus monkey papillomavirus. Dashes represent gaps introduced for maximal alignment. The cluster of basic amino acids is marked by dots. The inner bracket denotes the pentapeptide mutated to alanine in the HPV-11 E2 fusion proteins. Note that the region is flanked by helix-breaking glycine and proline residues in many of the viruses (outer bracket).
FIG. 6
A model for the structure and function of the dimeric HPV-11 E2 protein. The N domain (oval) binds to focal NM-associated proteins, while the flexible hinge (H; bold zig zag lines) helps anchor the protein by association with the fibrous matrix scaffold (thin lines). The C domain (fans) secures the supercoiled viral DNA (ribbon). The E2 protein and the viral DNA then interact with additional proteins on NM or recruit proteins to the matrix-bound DNA, leading to the regulation of viral mRNA transcription or DNA replication. The cluster of basic residues in the hinge is an important element for both nuclear localization and for NM association.
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