Biomedical subjects

R Schirmbeck

Publications and source records attributed to R Schirmbeck.

At least 73 records · Page 4Linked to original sources

PubMed1994

Vaccination with T cell receptor peptides primes anti-receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL.

We selected three peptides from the germ-line sequence of the V beta 8.2 and J beta 2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative Kd- and Ld-restricted epitopes. Immunization of BALB/c (H-2d) mice with the V beta 8.2(67-90) 23-mer peptide 1 as well as the 15-mer V beta 8.2(95-108)-peptide 2 efficiently primed specific CD8+ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-V beta 8.2 epitopes. V beta 8.2+ T cells were not deleted in TCR peptide-immunized mice because the fractions of V beta 8.2+ CD4+ and V beta 8.2+ CD8+ T cells in spleen and lymph nodes were not altered. The proliferative response of V beta 8.2+ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60-80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the J beta 2.3-derived peptide 3 stimulated a CD8+ CTL response against a class I-restricted epitope within this J beta segment that was also generated during natural "endogenous" processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8+ CTL recognize class I-restricted, natural V beta/J beta-TCR epitopes. Such anti-TCR CTL may, thus, operate in V beta-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.

Amino Acid Sequence↗

PubMed1994

Peptide transporter-independent, stress protein-mediated endosomal processing of endogenous protein antigens for major histocompatibility complex class I presentation.

The peptide transporter-defective cell line RMA-S expressing the wild-type simian virus 40 large T antigen (wtT-Ag) from a transfected gene did not present two well-defined, H-2 class I (Db)-restricted epitopes of T-Ag to cytotoxic T lymphocytes (CTL). Hence, "endogenous" processing and presentation of the wtT-Ag depended on a functional peptide transporter heterodimer. In contrast, both T-Ag epitopes were efficiently presented to CTL by transfected RMA-S cells expressing a truncated, cytoplasmic T-Ag variant (cT-Ag) or a karyophilic, amino-terminal 272-amino acid T-Ag fragment. Transporter-independent "endogenous" processing of mutant T-Ag molecules correlated with their association with the constitutively expressed heat shock protein 73 (hsp73). Class I-restricted presentation of both epitopes processed from these hsp73-associated protein antigens was sensitive to NH4Cl and chloroquine. These data indicate that selected intracellular proteins access an alternative, hsp73-mediated pathway for class I-restricted presentation that operates independent of peptide transporters in an endosomal compartment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Injection of detergent-denatured ovalbumin primes murine class I-restricted cytotoxic T cells in vivo.

Complex adjuvant formulations have been used to introduce soluble protein antigens into the "endogenous" processing pathway and hence to elicit specific, major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) responses. We tested if simple modifications of a model protein antigen, i.e. ovalbumin (OVA), can render it immunogenic for murine class I-restricted CTL when injected into mice in soluble form. Injection of 1-100 micrograms native OVA into C57BL/6 (H-2b) mice did not stimulate a class I-restricted CTL response. In contrast, immunization of mice with 0.5 to 10 micrograms sodium dodecyl sulfate (SDS)- or deoxycholate (DOC)-denatured OVA efficiently primed CD8+ CTL specific for the well-characterized Kb-restricted OVA257-264 epitope. Gel-purified SDS-denatured OVA devoid of protein fragments and excess detergent efficiently stimulated a specific CTL response in vivo. OVA preparations denatured by heat or urea treatment were not immunogenic for murine CTL. Injection of non-treated or detergent-treated, antigenic OVA257-264 peptide into mice did not elicit a CTL response. Thus, denaturation of OVA by simple detergents such as SDS or DOC dramatically enhances its immunogenicity for class I-restricted CTL but not all modes of denaturation are equally effective.

Animals↗

PubMed1994

Antibody and cytotoxic T-cell responses to soluble hepatitis B virus (HBV) S antigen in mice: implication for the pathogenesis of HBV-induced hepatitis.

Immune responses to components of hepatitis B virus (HBV) are assumed to play an essential role not only in the elimination of the virus but also in the pathogenesis of HBV-induced hepatitis. Protective humoral immunity to HBV is mediated by immune responses to HBV surface antigen (HBsAg). It is important to know which HBsAg preparations induce which type of cellular and humoral immune responses under which immunization conditions. We studied in BALB/c mice the humoral (antibody) response and the class I-restricted cytotoxic T-lymphocyte (CTL) response to different preparations of HBsAg particles: recombinant, small protein particles; plasma-derived, mixed particles formed by large, medium, and small surface proteins; and different preparations of recombinant, mixed particles formed by large and small surface proteins. Specific antibody levels appeared in the sera of immunized mice 2 to 3 weeks after immunization and were correlated with the antigen dose used for priming. HBsAg-specific antibody levels were enhanced by boost injections or by adsorbing the antigen to aluminum hydroxide. Injected in particulate form without adjuvants in the dose range of 0.1 to 10 micrograms per mouse, all HBsAg preparations tested efficiently primed specific CD8+ CTL of defined restriction and epitope specificity. Specific CTL reactivity was detectable from 5 days to more than 4 months postimmunization. In the dose range tested, it was independent of the antigen dose used for immunization and not enhanced by repeated boost injections. CTL were not elicited by HBsAg adsorbed to aluminum hydroxide. We have thus defined conditions under which HBsAg induced preferentially either a cellular immune response or a humoral immune response. These findings may be relevant for the interpretation of HBV-associated immunopathologic phenomena.

Adjuvants, Immunologic↗

PubMed1994

Alternative processing pathways for MHC class I-restricted epitope presentation to CD8+ cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTL) mediate protective immunity against many intracellular pathogens. New generation vaccines that exploit the potential of recombinant protein technology have to meet the challenge to identify immunodominant antigen systems of the pathogen of interest, to produce these antigens in recombinant form, and to find ways to selectively deliver them to the compartments of the specific immune system that mediate the protective responses. Immunization with soluble protein antigens usually primers CD4+ T cells but not CD8+ T cells because of the stringent requirements for 'endogenous processing' for major histocompatibility complex (MHC) class I-restricted epitope presentation to CD8+ CTL. This rule is not absolute because priming of class I-restricted CTL by soluble, recombinant viral protein antigens injected without adjuvants has been reported in various antigen systems. An understanding of the cell biology of alternative pathways of protein antigen processing for MHC class I-restricted epitope presentation is emerging. We describe subcellular sites, alternative peptide transport mechanisms, and alternative modes of MHC class I molecule recycling that allow different modes of peptide loading of class I molecules. Experimental evidence for some of these novel pathways of 'endogenous' processing for class I-restricted epitope presentation is discussed. Some pathways are still hypothetical. The issue of alternative modes of 'endogenous' processing for class I-restricted antigen presentation is of theoretical and practical relevance. Immunologists are interested in the question from which source protein antigens are derived that are potentially accessible to specific recognition by different T cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

A self-reactive class I-restricted T-cell response of H-2b mice to determinants of the V beta 8.2 domain of the T-cell receptor for antigen.

We studied the induction of a self-reactive cytotoxic T lymphocyte (CTL) response to determinants of the variable V beta 8.2 region of the beta-chain of the T-cell receptor (TCR) for antigen in C57BL/6 (H-2b) mice. A CTL response was elicited in vivo by TCR peptide vaccination, and detected in vitro using syngeneic transfectants expressing a rearranged V beta 8.2+ TCR beta-chain. The first series of experiments used a 15-mer peptide representing residues 68-82 of the V beta 8.2 domain and containing Kb and Db allele-specific motifs. Immunization with this peptide stimulated an autoreactive CTL response that cross-reacted with V beta 8.2 epitopes presented by transfectants endogenously processing a V beta 8.2+ TCR beta-chain. These transfectants expressed a construct derived from a murine, rearranged V beta 8.2/D beta 2/J beta 2.3/C beta 2 TCR beta-chain cDNA. The V beta 8.2+ T-cell subset of peptide-primed mice was not deleted but its proliferative response to stimulation by the V beta 8.2-specific monoclonal antibody (mAb) F23.2 was suppressed. In a second series of experiments we immunized mice with a 23-mer peptide representing residues 41-63 of the V beta 8.2 domain that does not contain putative, allele-specific H-2b class I-restricted motifs. This TCR peptide vaccination stimulated a CD8+ CTL response reacting against syngeneic, peptide-pulsed targets but not cross-reacting against transfectants processing/presenting epitopes of the beta-chain. V beta 8.2+ T cells of these peptide-primed mice were not anergized. These data demonstrate that vaccination with an immunogenic peptide representing a naturally processed epitope of the V beta 8.2 domain of the TCR beta-chain induces a self-reactive CD8+ CTL specific for this V beta 8.2 epitope; and anergizes (but does not delete) V beta 8.2+ T cells.

Amino Acid Sequence↗

PubMed1994

Targeting of exogenous protein antigens to a novel endosomal processing pathway for class I-restricted presentation.

CD8+ cytotoxic T lymphocytes (CTL) mediate protective effector functions against many viral, bacterial and parasitic infections. CTL specifically recognize peptides presented by major histocompatibility complex (MHC) class I molecules on the surface of cells. Usually, CTL are stimulated by peptides from intracellularly synthesized, 'endogenous' protein antigens processed in the cytosol or endoplasmic reticulum. Because of this 'endogenous' processing pathway, immunization with 'exogenous' proteins rarely induces class I-restricted CTL responses. We have found that immunization of mice without adjuvants with a single low dose of, either different lipoprotein particles, or various denatured protein antigens by various routes efficiently primes class I-restricted CTL responses of defined epitope and restriction specificity. Priming of CTL requires processing of S-protein and is not inducible by the respective immunogenic peptides. These data reveal a novel immunogenic property of these two types of 'exogenous' protein antigens. The observation is relevant for the development of subunit vaccines designed to specifically stimulate T cell responses.

Animals↗

PubMed1994

Induction of a MHC class I-restricted, CD8 positive cytolytic T-cell response by chimeric HIV-1 virus-like particles in vivo: implications on HIV vaccine development.

New insights into HIV-pathogenesis suggest that the cell mediated immune response might play a crucial role in controlling HIV infection by suppressing HIV-replication in CD4-positive cells by a lymphokine-like soluble factor and by killing HIV-infected cells via classical CTL mediated lysis. This type of a cellular immune response rather than an antibody response seems to be most promising to protect if not from infection, so at least from disease. Therefore rationally designed candidate vaccines should be capable of inducing a cell mediated immunity in addition to a humoral immune response. In order to avoid adverse side effects upon immunization, carefully selected antigens and epitopes should be presented in a favourable manner to the immune system. In previous experiments, we could demonstrate that the gag-polyprotein precursor, known to include a series of T-helper and CTL epitopes, assembles to highly immunogenic, complete noninfectious HIV-1 virus-like particles (VLP). Based on these VLP we developed a novel antigen presentation system, which allows the presentation of selected epitopes derived from HIV reading frames other than gag to the immune system. Alternatively complete derivatives of the HIV-1 external glycoprotein can be presented by the VLP. Immunological analysis of different VLP preparations in a BALB/c mouse model revealed the induction of a strong CTL response. The significance of these observations for future vaccine strategies is discussed.

AIDS Vaccines↗

PubMed1993

Immunization of mice with the N-terminal (1-272) fragment of simian virus 40 large T antigen (without adjuvants) specifically primes cytotoxic T lymphocytes.

Immunization of C57BL/6 (B6) mice (H-2b) with the "large tumor antigen" (T-Ag) of simian virus 40 (SV40) in its soluble form without adjuvants primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. CD8+ CTL primed in vivo by this non-structural 708-amino acid (aa) viral protein, and specifically restimulated in vitro, lysed H-2b target cells, either transfected with an SV40 T-Ag-encoding vector, or transformed by SV40 infection. H-2b RMA-S transfectants expressing the complete 708 aa T-Ag (which fail to transport peptides through the endoplasmic reticulum membranes) were not lysed. CTL were also efficiently primed in vivo by injection of the N-terminal 272 aa fragment of the T-Ag. Hence, this fragment contains the structure(s) required for a soluble protein to enter the "endogenous" class I-restricted antigen processing and presentation pathway for CD8+ CTL activation. In soluble form, the complete T-Ag or the N-terminal T-Ag fragment sensitized in vitro RBL5 cells for lysis by T-Ag-specific CTL lines and clones. This in vitro sensitization was blocked by brefeldin A. In contrast, specific recognition of RBL5 cells pulsed in vitro with synthetic, immunogenic nonapeptides (derived from N-terminal T-Ag epitopes) by CTL lines was insensitive to brefeldin A. Hence, T-Ag and its 272-aa N-terminal fragment can enter the "endogenous" processing pathway and prime CD8+ CTL in vivo and in vitro.

Adjuvants, Immunologic↗

PubMed1993

In vitro expansion distorts the detectable pattern of specific recognition of in vivo primed cytotoxic T lymphocyte populations.

Injection of the 708-amino-acid (aa) viral protein "large tumor antigen" (T-Ag) of simian virus 40 (SV40) or its N-terminal 272-aa fragment into C57BL/6 (B6) mice (H-2b) primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. Surprisingly, injection of this nonstructural viral protein (or its N-terminal fragment) in soluble form (without adjuvants) was as efficient in priming CD8+ CTL in vivo as the infection of B6 mice with the virus SV40. CTL activated in vivo by immunization with T-Ag proteins or SV40 infection specifically lysed syngeneic RBL5 cells transfected with a T-Ag-encoding vector; these RBL5/M7 transfectants efficiently presented N- and C-terminal T-Ag epitopes in association with H-2 class I restriction elements. N- and C-terminal T-Ag epitopes were recognized by CTL primed in vivo by immunization with the complete T-Ag protein or by infection with SV40, and (as expected) only N-terminal T-Ag epitopes were recognized by CTL primed in vivo by the soluble N-terminal T-Ag fragment. In CD8+ CTL populations primed in vivo by immunization with the complete T-Ag protein or by SV40 infection and restimulated in vitro with RBL5/M7 transfectants in a mixed tumor cell-lymphocyte culture (MTLC), CTL with specificity for C-terminal T-Ag epitopes were selectively expanded in vitro for months. Hence, the in vitro expansion of CTL population with heterogenous recognition specificities can dramatically distort the picture of its specific recognition repertoire primed in vivo.

Animals↗

PubMed1993

Structural requirements for simian virus 40 replication and virion maturation.

The nuclear matrix plays an important role in simian virus 40 (SV40) DNA replication in vivo, since functional replication complexes containing large T and replicating SV40 minichromosomes are anchored to this structure (R. Schirmbeck and W. Deppert, J. Virol. 65:2578-2588, 1991). In the present study, we have analyzed the course of events leading from nuclear matrix-associated replicating SV40 minichromosomes to fully replicated minichromosomes and, further, to their encapsidation into mature SV40 virions. Pulse-chase experiments revealed that newly replicated SV40 minichromosomes accumulated at the nuclear matrix and were directly encapsidated into DNase-resistant SV40 virions at this nuclear structure. Alternatively, a small fraction of newly replicated minichromosomes left the nuclear matrix to associate with the cellular chromatin. During the course of infection, progeny virions continuously were released from the nuclear matrix to the cellular chromatin and into the cytoplasm-nucleoplasm. The bulk of SV40 progeny virions, however, remained at the nuclear matrix until virus-induced cell lysis.

Cell Compartmentation↗

PubMed1993

Diversity of functional T-cell receptor delta-chain transcripts from bone marrow cells of athymic nude mice.

CD3+ cells are detectable in bone marrow of athymic mice homozygous for the nude mutation. As previously shown, cells expressing the gamma delta T-cell receptor (TcR) represent 30-40% of this T-cell population. Using V delta-specific, V alpha 4-specific, and C delta-specific primers, TcR delta-chain transcripts were reverse transcribed and polymerase chain reaction (PCR)-amplified from total RNA prepared from bone marrow cells (BMC) of 6-month-old NMRI nu/nu mice. Amplified TcR delta-chain cDNA was cloned, and 49 randomly selected clones derived from seven amplification reactions were sequenced. Sequence analyses showed: (1) more than 80% of the sequenced clones represented in-frame transcripts of the TcR delta-chain; (2) in-frame transcripts containing V delta 1-, V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6- and V alpha 4-gene segments were detectable in nude BMC; (3) V delta 2-, V delta 4- and V delta 5-containing transcripts were more abundant and more diverse than V delta 1- and V delta 3-containing transcripts; (4) extensive N-region diversity was present in the V delta-D delta 2 (N1), D delta 2-D delta 1 (N2) and D delta 1-J delta 1 (N3) junctional regions; (5) P nucleotide additions were present in many transcripts; and (6) unusual truncated, in-frame transcripts with deleted D- and J-region genes were detected. A large potential TcR delta-chain repertoire is thus present in nude BMC.

Amino Acid Sequence↗

PubMed1992

Immunization with soluble simian virus 40 large T antigen induces a specific response of CD3+ CD4- CD8+ cytotoxic T lymphocytes in mice.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.

Animals↗

PubMed1992

Bone marrow cells of athymic nude mice express functional T cell receptor alpha chain transcripts rearranged to V delta 2, 3, 4, 5, 6 genes.

From bone marrow cells (BMC) of athymic nude mice, T cell receptor (TcR) alpha chain transcripts were selectively amplified by polymerase chain reaction (PCR) using V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6- and C alpha-specific primers. Amplified DNA fragments were cloned, and 32 randomly selected clones from 5 PCR were sequenced. Twenty-three distinct rearrangement events were detected, of which 87% (20/23) were in-frame. All five tested V delta genes (V delta 2, 3, 4, 5, 6) rearranged in-frame to J alpha-C alpha. N-region diversity in V delta-J alpha junctions present in most clones was limited to two to five nucleotides. P-nucleotide additions in this region were also detected. The V delta 5 gene located 3' of C delta in reversed transcriptional orientation was rearranged to J alpha by inversion. The J alpha usage pattern of the sequenced clones was strongly biased towards rearrangement of the most 5' genes (located nearest to C delta) of the J alpha cluster: the most 5' J alpha (J alpha TA1) was used by 30% of all clones, and 78% of all J alpha rearranged to V delta were located in the 5' 12 kb of the 60-kb J alpha cluster. As distinct V delta/C delta and V alpha/C alpha TcR usage patterns are prevalent in peripheral T cell populations, our data suggest that these TcR usage patterns results from repertoire selections operating in alpha beta and gamma delta T cell lineages, but not from preferential V delta-C delta and V alpha-C alpha rearrangement patterns.

Animals↗

PubMed1991

Structural topography of simian virus 40 DNA replication.

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.

Animals↗

PubMed1989

Nuclear subcompartmentalization of simian virus 40 large T antigen: evidence for in vivo regulation of biochemical activities.

Simian virus 40 large T antigen (large T) in the early and the late phases of infection differs significantly in its sequence-specific DNA-binding and ATPase activities, indicating that different large-T populations participate in virus-specific events at various stages of the infectious cycle. To further characterize these large-T populations, we have analyzed nuclear subclasses of large T, isolated from their in vivo location, for their biochemical activities. We show that chromatin- and nuclear matrix-associated large-T molecules exhibit different simian virus 40 control region (ORI) DNA-binding and ATPase activities. The association of large T with a certain nuclear substructure, therefore, subcompartmentalizes large-T molecules exerting different biochemical activities. Nuclear subcompartmentalization thus may provide a higher-order level for the regulation of biochemical activities of large T in vivo.

Adenosine Triphosphatases↗

PubMed1988

Analysis of mechanisms controlling the interactions of SV40 large T antigen with the SV40 ORI region.

We have characterized the interactions of simian virus 40 (SV40) large tumor antigen (large T) with the control region of the SV40 genome, the SV40 ORI, by analyzing the specific binding of large T antigen to SV40 wild-type origin DNA and to isolated binding sites I and II, respectively. DNA binding affinities of large T antigen were determined under standardized conditions and DNA excess, using a target-bound DNA binding assay (M. Hinzpeter, E. Fanning, and W. Deppert, 1986, Virology 148, 159-167). Our results show that large T antigen exhibits similar affinities for isolated binding sites I and II and for combined sites I and II on wild-type ORI DNA. When the fraction of large T antigen molecules (calculated per large T antigen monomers) able to bind specifically to these sites was determined (DNA binding activity of large T antigen) we found that only 2% of large T antigen molecules present in extracts of lytically infected cells were able to bind to isolated site II, whereas about 50% bound to isolated site I. However, only about 10% of large T antigen molecules bound to the complete wild-type ORI, containing combined binding sites I and II. Thus, a much larger proportion of large T antigen molecules is capable of binding specifically to site I as is suggested by analysis of large T antigen binding to combined sites I and II on the SV40 wild-type ORI. These findings indicate that the interaction of large T antigen with the SV40 wild-type ORI is restricted on one hand by the ability of large T antigen to bind to site II, and on the other hand by the spatial arrangement of binding sites I and II on the SV40 wild-type ORI.

Adenosine Triphosphate↗

PubMed1987

Specific interaction of simian virus 40 large T antigen with cellular chromatin and nuclear matrix during the course of infection.

We analyzed the subnuclear distribution of the simian virus 40 (SV40) large tumor (large T) antigen during the course of viral infection. Three distinct nuclear subclasses were detected in SV40 lytically infected TC7 cells (large T antigen in the nucleoplasm, at the cellular chromatin, and at the nuclear matrix). During the course of infection the relative subnuclear distribution of large T antigen changed significantly at about the switch from the early to late phase of infection: at early times postinfection, large T antigen was present mainly in the nucleoplasm and at the cellular chromatin, and nuclear-matrix-associated large T antigen was barely detectable. Concomitant with the onset of viral DNA replication, the amount of nuclear-matrix-associated large T antigen increased drastically. During the further course of infection large T antigen accumulated at the cellular chromatin and nuclear matrix, paralleling the increase in viral DNA synthesis. The biological significance of this correlation was corroborated by analysis of cells infected with the SV40 mutant tsA58 at permissive (32 degrees C) and restrictive (39 degrees C) temperatures. tsA58 large T antigen failed to initiate viral DNA replication in infected cells kept at the restrictive temperature and also failed to associate with the cellular chromatin and nuclear matrix. By blocking viral DNA synthesis with aphidicolin, an inhibitor of DNA polymerase alpha, we were able to show that the accumulation of large T antigen at these structures does not result from the binding of large T antigen to viral chromatin but reflects an association with cellular components of the chromatin and nuclear matrix of infected cells.

Animals↗