Herpesviral transcription, DNA synthesis, and capsid assembly occur within the infected cell nucleus. To further define the spatial relationship among these processes, we have examined the intranuclear distributions of viral DNA replication, gene regulatory, and capsid proteins using dual label immunofluorescence and confocal microscopy. We observed that several of the viral DNA replication proteins localize preferentially to punctate structures within replication compartments while the major transcriptional activator, ICP4, and the ICP27 regulatory protein show a more diffuse distribution within replication compartments. The viral proteins that show a punctate distribution in replication compartments redistribute from these compartments to prereplicative sites when viral DNA replication is inhibited, whereas viral proteins that show a diffuse distribution remain within replication compartments when viral DNA replication is inhibited. Thus the sites of viral DNA replication and late transcription appear to be distinct but codistribute within the boundaries of replication compartments. The major capsid protein, ICP5, also localizes initially to a diffuse distribution within replication compartments, but during the time of maximal progeny virus assembly, ICP5 becomes localized to punctate structures within replication compartments that are often near the punctate structures occupied by viral DNA replication proteins. Hence the processes of viral DNA replication, late transcription, and capsid assembly show a general overlapping distribution within replication compartments but appear to be located at distinct sites within these regions of the infected cell nucleus.
Herpes simplex virus DNA replication proteins localize in characteristic patterns corresponding to viral DNA replication structures in the infected cell nucleus. The intranuclear spatial organization of the HSV DNA replication structures and the factors regulating their nuclear location remain to be defined. We have used the HSV ICP8 DNA-binding protein and bromodeoxyuridine labeling as markers for sites of herpesviral DNA synthesis to examine the spatial organization of these structures within the cell nucleus. Confocal microscopy and three-dimensional computer graphics reconstruction of optical series through infected cells indicated that viral DNA replication structures extend through the interior of the cell nucleus and appear to be spatially separate from the nuclear lamina. Examination of viral DNA replication structures in infected, binucleate cells showed similar or virtually identical patterns of DNA replication structures oriented along a twofold axis of symmetry between many of the sister nuclei. These results demonstrate that HSV DNA replication structures are organized in the interior of the nucleus and that their location is defined by preexisting host cell nuclear architecture, probably the internal nuclear matrix.
Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defmed gene family of C3-and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2.The C3d/Epstein-Barr virus (EBV) receptor (complement receptor type 2, CR2) of human B lymphocytes is a Mr 145,000 glycoprotein (1-3) comprised of a single polypeptide of Mr 110,000 and 8-11 N-linked oligosaccharides (4). It is expressed by B lymphocytes (5, 6) and follicular dendritic cells of the spleen (7). CR2 recognizes the d region of the C3 component of complement (5, 6) and also serves as the membrane receptor for the human herpesvirus EBV (8, 9), which induces polyclonal activation and immortalization of B lymphocytes and is associated with Burkitt lymphoma and nasopharyngeal carcinoma. CR2 may have a role in physiologic B-cell activation (10, 11) and is phosphorylated when B cells are treated with phorbol 12-myristate 13-acetate or antibody to surface immunoglobulin (12).The recent purification of CR2 permitted the demonstration (13) of significant similarities between its amino acid composition and that of three other proteins of the complement system: factor H, C4-binding protein, and the C3b receptor (complement receptor type 1, CR1). Factor H and C4-binding protein are plasma proteins and CR1 is a membrane glycoprotein found on erythrocytes, most leukocytes, and glomerular podocytes (14). These proteins share certain functions, s...
Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process. A small number of integral membrane proteins speci®c to the envelope have been identi®ed in budding yeast, however, none has been reported to affect transport. We have identi®ed an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein. Notably, the brr6-1 mutant speci®cally affects transport of mRNA and a protein reporter containing a nuclear export signal. In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores. BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green¯uorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore. However, Brr6-GFP fails to redistribute in a Dnup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain. We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery. Keywords: integral membrane protein/mRNA export/ nuclear envelope/nuclear transport IntroductionThe presence of a nuclear envelope (NE) in eukaryotes divides the cell into nuclear and cytoplasmic compartments, greatly enhancing its capacity to control gene expression. Transport of proteins and ribonucleoprotein particles (RNPs) between the nucleus and cytoplasm through the nuclear pore complex (NPC) is central to this ability (reviewed in Go Èrlich and Kutay, 1999;Nakielny and Dreyfuss, 1999). The structure of the NPC is conserved from yeast to humans, consisting of a highly symmetrical core structure organized around a central pore channel as well as peripheral cytoplasmic ®laments and nucleoplasmic basket structures thought to play key roles in initiation and termination of transport pathways (reviewed in Franke and Scheer, 1974;Davis, 1995;Stof¯er et al., 1999;Wente, 2000). Transport of different types of protein and RNP cargoes through the NPC is mediated by speci®c protein carriers, including members of the karyopherin b family and a number of unrelated transport factors such as Ntf2 and Mex67 (reviewed in Pemberton et al., 1998;Wozniak et al., 1998;Go Èrlich and Kutay, 1999).Interactions between carriers and nucleoporins lining the channel of the pore are thought to be important for transport (reviewed in Fabre and Hurt, 1997;Pemberton et al., 1997;Wozniak et al., 1998;Go Èrlich and Kutay, 1999;Wente, 2000), although the precise roles of particular nucleoporins are not well understood. Our knowledge of the functional architecture of the yeast nuclear pore has bene®ted greatly from a detailed study of isolated NPCs carried out recently by Rout et al. (2000). The study produced a working model of the NPC, con®rming the presence of~30 yeast nucleoporins at the pore and placing them spat...
The maturation and export of mRNA from the nucleus through the nuclear pore complex is critical for maintaining an appropriate proteome in all eukaryotic cells. Here we summarize a previously unpublished screen in S. cerevisiae that utilized an established dT50 in situ hybridization assay to identify cold-sensitive mutants that accumulated bulk poly A RNA in the nucleus. The screen identified seven mutants in six complementation groups, including the brr6-1 strain that we described previously. In addition to brr6-1, we identified novel alleles of the key transport gene GLE1 and NUP188, a component of the Nic96 nucleoporin complex. Notably, we show that the nup188-brr7 allele causes defects in select protein import pathways as well as mRNA export. Given recent structural and functional evidence linking the Nic96 complex to transport components, this mutant may be particularly useful to the transport community.
Correlation between transcriptional regulation and positioning of genes at the nuclear envelope is well established in eukaryotes, but the mechanisms involved are not well understood. We show that brr6-1, a mutant of the essential yeast envelope transmembrane protein Brr6p, impairs normal positioning and expression of the PAB1 and FUR4- GAL1,10,7 loci. Similarly, expression of a dominant negative nucleoplasmic Brr6 fragment in wild-type cells reproduced many of the brr6-1 effects. Histone chromatin immunoprecipitation (ChIP) experiments showed decreased acetylation at the key histone H4K16 residue in the FUR4-GAL1,10,7 region in brr6-1. Importantly, blocking deacetylation significantly suppressed selected brr6-1 phenotypes. ChIPseq with FLAG-tagged Brr6 fragments showed enrichment at FUR4 and several other genes that showed striking changes in brr6-1 RNAseq data. These associations depended on a Brr6 putative zinc finger domain. Importantly, artificially tethering the GAL1 locus to the envelope suppressed the brr6-1 effects on GAL1 and FUR4 expression and increased H4K16 acetylation between GAL1 and FUR4 in the mutant. Together these results argue that Brr6 interacts with chromatin, helping to maintain normal chromatin architecture and transcriptional regulation of certain loci at the nuclear envelope.
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