Viviane Bibor-Hardy

The nuclear matrix is involved in herpes simplex virogenesis.

Abstract Nuclear matrices have been purified from BHK cells infected with herpes simplex virus type 1 (HSV-1) and compared with nuclear matrices from uninfected BHK cells. Both showed typical structure with residual nucleolus, peripheral lamina with pore complexes, and a nonchromatin fibrillar network. Numerous viral capsids were seen stuck to that framework in matrices from infected cells. SDS-PAGE and fluorography analysis showed that polypeptides normally found in the noninfected BHK nuclear matrix were still present in the infected matrices. Nine additional virus-induced polypeptides were detected 15 hr after infection by labeling with [ 35 S]methionine. This suggests that the nuclear matrix is involved in the formation of the capsid or in the encapsidation process. Twelve DNA-binding proteins were also detected in infected matrices.

DNA-binding Proteins of Herpes Simplex Virus Type 1-infected BHK Cell Nuclear Matrices

The nuclear matrix is involved in the replicative cycle of herpes simplex virus type 1 (HSV-1) and in at least some cases viral DNA has been shown to be closely associated with this structure. In this communication, we report the presence of five DNA-binding proteins in the nuclear matrix of HSV-1-infected BHK cells. These proteins (p114, p89, p77, p37 and p29) were detected by probing with 32P-labelled HSV DNA after Western blotting of nuclear matrix proteins. Three were identified as virion components: p89 as VP12, p77 as VP13 and p37 as the capsid protein VP22a. These polypeptides were detected in cells and nuclei and found to be associated with the nuclear matrix late during the lytic cycle, long after the onset of viral DNA replication. The nuclear matrix-binding capacity of VP22a depended on viral DNA replication, since after DNA polymerase inhibition it was still synthesized and transported into the nucleus but was no longer associated with the nuclear matrix. After inhibition of viral DNA synthesis, VP13 was no longer found in cells, nuclei or nuclear matrices. These results suggest a possible involvement in anchoring viral progeny DNA to the nuclear matrix.

Characterization of lamin proteins in BHK cells

Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.

Detection in BHK cells of a precursor form for lamin A

Lamins are structural proteins found in the fibrous lamina underlining the nuclear envelope. In vitro translation of polyadenylated RNA or polysomes followed by immunoprecipitation with a serum raised against BHK nuclear matrix proteins showed that lamin A (72 kD) is synthesized as a high molecular weight precursor (74 kD) (Laliberté et al., J Cell Biol 98 (1984) 980) [23]. We have thus investigated the presence in BHK cells of this putative precursor by in vivo labelling with [35S]methionine and immunoprecipitation of lamin proteins. Short labelling times, ranging from 5 to 60 min reveal the presence of the 74 kD protein. Pulse-chase experiments indicate that the half-life of the precursor is about 60 min. On two-dimensional gel, the 74 kD protein is resolved in a cluster of isovariants between pH 7.4 and 6.6, which are generally slightly more alkaline than their counterparts in lamin A. These results indicate that lamin A is synthesized as a precursor of 74 kD; the long half-life further suggests that pre-lamin A might accumulate in some sort of cellular pool before undergoing post-transcriptional modification(s) to give the mature form of lamin A.

In situ localization of the major capsid protein during lytic infection by herpes simplex virus.

The intracellular localization of the major capsid protein (ICP5) of herpes simplex virus was studied during virogenesis. Except for a brief period at the onset of synthesis, this protein was found almost exclusively inside the nucleus. Its localization was not at random since 80% was tightly bound to the nuclear matrix as early as 4 h after infection. Discrete modifications of the fluorescence pattern occurred in an orderly fashion during the progression of the infection. Immunoelectron microscopic studies using Protein A-gold labelling demonstrated that this protein is synthesized on cytoskeleton-bound polyribosomes and accumulates in the central part of the nucleus where formation of viral capsids occurs; no gold particles were found in association with the peripheral chromatin or with the nucleolus.

Synthesis of nuclear lamins in BHK-21 cells synchronized with aphidicolin

Lamins A, B and C are the major proteins of mammalian nuclear lamina and have been well studied in BHK-21 cells. By synchronizing BHK-21 cells with aphidicolin, a potent inhibitor of DNA alpha-polymerase, we were able to detect a differential pattern of synthesis for nuclear lamins during the cell cycle. Lamin B starts to be synthesized only in S phase up to mitosis while synthesis of lamins A and C remain stable throughout the cell cycle. The precursor of lamin A see its half-life increase from a reported 63 min in interphase cells to 103 min in G2/M cells.

Modifications of the Nuclear Envelope of BHK Cells After Infection with Herpes Simplex Virus Type 1

Numerous discrete lesions, which we have termed blebs, appeared in the nucleus of BHK cells 10 to 15 h after infection with herpes simplex virus type 1 (HSV-1). They were formed in the inner portion of the nuclear envelope by the apposition of two thickened lamellae overlying a vacuole. As demonstrated by electron microscopic studies, blebs were regular and associated with the peripheral lamina in the nucleus, averaging 3.5 blebs per micron2. They appeared to be associated with an enrichment of the 155K major capsid protein in the nuclear membrane subfractions as compared with the protein composition of nuclei and plasma membrane fractions. We propose that blebs represent the site of assembly of capsid proteins before DNA insertion and eventual envelopment.

Herpes specific and α DNA polymerase in nuclear envelope of BHK infected cells

Summary Nuclear envelopes of normal and Herpes simplex virus infected BHK cells were isolated and separated in two fractions by a step gradient procedure. α, β and Herpes-specific DNA polymerases activity were tested in the chromatin and nuclear envelope fractions of these cells. We found that both α and Herpes-specific DNA polymerases activity were associated with only one of the two nuclear envelope fractions and were released by Triton X-100 treatment. On the contrary, β DNA polymerase activity was associated with the chromatin fraction. More endonucleotylic activity and particularly apurinic and UV specific endonucleases were found associated with the same nuclear envelope fraction. These findings suggest an active role of the nuclear envelope in the replication of both cellular and Herpes simplex viral DNA.

Role of the nuclear matrix in adenovirus maturation

The nuclear matrix has been implicated in several important cellular processes. In this paper, we investigate the role of the nuclear matrix in adenovirus type 2 assembly. Electron microscopic examination of nuclear matrices isolated from adenovirus infected Hep-2 cells clearly reveals that late in the lytic cycle, adenovirus capsids are intimately associated with the nuclear matrix. SDS-PAGE analysis showed that the viral core polypeptides V, PVII and 11 kDa were enriched in the nuclear matrix fraction. After a 3 h chase period a constant high ratio of PVII to VII prevailed in the nuclear matrix suggesting that mostly young virions and viral cores are bound to this structure. Most of the virus maturation endoproteinase activity co-purified with the nuclear matrix and the data suggest that the enzyme may be released from fragile young virions or assembly intermediates. Together these experiments suggest that the nuclear matrix is the site of adenovirus assembly and that mature virions may be released from the matrix by the viral endoproteinase.

cDNA encoding a novel TCP1-related protein

We report the cloning of a mouse cDNA encoding a novel protein which has significant homology with the t-complex protein 1b (TCP1b). In addition, this protein has high sequence identity with tryptic peptides from the bovine P5 subunit of the TCP1-ring complex. We named this novel protein mTRiC-P5 for mouse TCP1-Ring Complex Protein #5. Results indicate that mTRiC-P5 is a new member of the TCP1-TF55 family and is part of the TCP1-ring complex.