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Deborah H Spector

Publications and source records attributed to Deborah H Spector.

18 recordsLinked to original sources
PubMed2007

The IE2 60-kilodalton and 40-kilodalton proteins are dispensable for human cytomegalovirus replication but are required for efficient delayed early and late gene expression and production of infectious virus.

The human cytomegalovirus (HCMV) IE2 86-kDa protein is an essential transactivator of viral and cellular gene expression. Additional proteins of 60 and 40 kDa are expressed from the IE2 gene at late times postinfection and are identical to the C terminus of IE2 86. We have constructed HCMV recombinants that express wild-type full-length IE2 86 but do not express the IE2 40- and 60-kDa proteins. Each of these recombinants is viable, indicating that neither the 60-kDa nor the 40-kDa protein is required for virus replication, either alone or in combination. Cells infected with the IE2 60 and IE2 40 deletion mutants, however, exhibit decreased expression of selected viral genes at late times. In particular, expression of the viral DNA replication factor UL84 is affected by the deletion of IE2 40, and expression of the tegument protein pp65 (ppUL83) is affected by the deletion of both IE2 40 and IE2 60. IE2 60 and IE2 40 are also required for the production of normal levels of infectious virus. Finally, IE2 40 appears to function as a repressor of major immediate-early transcription in the infected cell. These results begin to define functions for the IE2 60- and IE2 40-kDa proteins and indicate that these products contribute both to the expression of selected viral genes and to the overall progression of the infection.

Cytomegalovirus↗
PubMed2006

Genome-wide analysis reveals a highly diverse CD8 T cell response to murine cytomegalovirus.

Human CMV establishes a lifelong latent infection in the majority of people worldwide. Although most infections are asymptomatic, immunocompetent hosts devote an extraordinary amount of immune resources to virus control. To increase our understanding of CMV immunobiology in an animal model, we used a genomic approach to comprehensively map the C57BL/6 CD8 T cell response to murine CMV (MCMV). Responses to 27 viral proteins were detectable directly ex vivo, the most diverse CD8 T cell response yet described within an individual animal. Twenty-four peptide epitopes were mapped from 18 Ags, which together account for most of the MCMV-specific response. Most Ags were from genes expressed at early times, after viral genes that interfere with Ag presentation are expressed, consistent with the hypothesis that the CD8 T cell response to MCMV is largely driven by cross-presented Ag. Titration of peptide epitopes in a direct ex vivo intracellular cytokine staining assay revealed a wide range of functional avidities, with no obvious correlation between functional avidity and the strength of the response. The immunodominance hierarchy varied only slightly between mice and between experiments. However, H-2(b)-expressing mice with different genetic backgrounds responded preferentially to different epitopes, indicating that non-MHC-encoded factors contribute to immunodominance in the CD8 T cell response to MCMV.

Acute Disease↗
PubMed2006

Repression of HMGA2 gene expression by human cytomegalovirus involves the IE2 86-kilodalton protein and is necessary for efficient viral replication and inhibition of cyclin A transcription.

Human cytomegalovirus (HCMV) infection results in dysregulation of several cell cycle genes, including inhibition of cyclin A transcription. In this work, we examine the effect of the HCMV infection on expression of the high-mobility group A2 (HMGA2) gene, which encodes an architectural transcription factor that is involved in cyclin A promoter activation. We find that expression of HMGA2 RNA is repressed in infected cells. To determine whether repression of HMGA2 is directly related to the inhibition of cyclin A expression and impacts on the progression of the infection, we constructed an HCMV recombinant that expressed HMGA2. In cells infected with the recombinant virus, cyclin A mRNA and protein are induced, and there is a significant delay in viral early gene expression and DNA replication. To determine the mechanism of HMGA2 repression, we used recombinant viruses that expressed either no IE1 72-kDa protein (CR208) or greatly reduced levels of IE2 86-kDa (IE2 86) protein (IE2 86DeltaSX-EGFP). At a high multiplicity of infection, the IE1 deletion mutant is comparable to the wild type with respect to inhibition of HMGA2. In contrast, the IE2 86DeltaSX-EGFP mutant does not significantly repress HMGA2 expression, suggesting that IE2 86 is involved in the regulation of this gene. Cyclin A expression is also induced in cells infected with this mutant virus. Since HMGA2 is important for cell proliferation and differentiation, particularly during embryogenesis, it is possible that the repression of HMGA2 expression during fetal development could contribute to the specific birth defects in HCMV-infected neonates.

Cells, Cultured↗
PubMed2006

Cyclin-dependent kinase activity is required for efficient expression and posttranslational modification of human cytomegalovirus proteins and for production of extracellular particles.

We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.

Cell Line↗
PubMed2005

Systemic priming-boosting immunization with a trivalent plasmid DNA and inactivated murine cytomegalovirus (MCMV) vaccine provides long-term protection against viral replication following systemic or mucosal MCMV challenge.

We previously demonstrated that vaccination of BALB/c mice with a pool of 13 plasmid DNAs (pDNAs) expressing murine cytomegalovirus (MCMV) genes followed by formalin-inactivated MCMV (FI-MCMV) resulted in complete protection against viral replication in the spleen and salivary glands following sublethal intraperitoneal (i.p.) challenge. Here, we found that following intranasal (i.n.) challenge, titers of virus in the lungs of the immunized mice were reduced approximately 1,000-fold relative to those for mock-immunized controls. We next sought to extend these results and to determine whether similar protection levels could be achieved by priming with a pool of three pDNAs containing three key plasmids (IE1, M84, and gB). We found that the three-pDNA priming elicited IE1- and M84-p65-specific CD8+ T lymphocytes and, following FI-MCMV boost, high levels of virion-specific immunoglobulin G (IgG) and virus-neutralizing antibodies. When mice were i.n. challenged 4 months after the last boost, titers of virus in the lungs of immunized mice were reduced 1,000- to 2,000-fold from those for controls during the peak of viral replication. Additionally, titers of virus were either at or below the detection limits for the salivary glands, liver, and spleen of the majority of the immunized mice. Following sublethal i.p. challenge, virus was undetectable in all of the above target organs of the immunized mice. Virion-specific IgA in the lungs was consistently detected by day 6 post-i.n. challenge for the immunized mice and by day 14 for controls. These results demonstrate the immunity and high levels of protection of the priming-boosting vaccination against both systemic and mucosal challenge.

Administration, Intranasal↗
PubMed2005

Exon 3 of the human cytomegalovirus major immediate-early region is required for efficient viral gene expression and for cellular cyclin modulation.

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Delta30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Delta30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Delta30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Delta30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Delta30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.

Cells, Cultured↗
PubMed2005

Human cytomegalovirus infection induces specific hyperphosphorylation of the carboxyl-terminal domain of the large subunit of RNA polymerase II that is associated with changes in the abundance, activity, and localization of cdk9 and cdk7.

Human cytomegalovirus infection in the presence of the cyclin-dependent kinase (cdk) inhibitor roscovitine leads to changes in differential splicing and the polyadenylation of immediate early IE1/IE2 and UL37 transcripts (V. Sanchez, A. K. McElroy, J. Yen, S. Tamrakar, C. L. Clark, R. A. Schwartz, and D. H. Spector, J. Virol. 78:11219-11232, 2004). To determine if this was associated with specific phosphorylation of the C-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit by cdk7/cyclin H and cdk9/cyclin T1, we examined the expression and localization of these kinases and the various phosphorylated forms of RNAP II. Infection resulted in increased RNAP II CTD phosphorylated on serines 2 and 5 and increased levels of activity of cdk7 and cdk9. At early times, cdk9 localizes with input viral DNA, and aggregates of cdk9 and cdk7 and a subset of Ser2-phosphorylated RNAP II colocalize with IE1/IE2 proteins adjacent to promyelocytic leukemia protein oncogenic domains. Later, cdk9 and Ser2-phosphorylated RNAP II form a nuclear punctate pattern; cdk7 resides in replication centers, and Ser5-phosphorylated RNAP II clusters at the peripheries of replication centers. Roscovitine treatment leads to decreased levels of hyperphosphorylated RNAP II (RNAP IIo) in infected cells and of hypophosphorylated RNAP II in mock-infected and infected cells. The RNAP IIo decrease does not occur if roscovitine is added 8 h postinfection, as was previously observed for processing of IE transcripts. These results suggest that accurate IE gene expression requires specific phosphorylation of the RNAP II CTD early in infection.

Cyclin-Dependent Kinase 9↗
PubMed2004

Cyclin-dependent kinase activity is required at early times for accurate processing and accumulation of the human cytomegalovirus UL122-123 and UL37 immediate-early transcripts and at later times for virus production.

Human cytomegalovirus (HCMV) infection leads to dysregulation of multiple cell cycle-regulatory proteins. In this study, we examined the effects of inhibition of cyclin-dependent kinase (cdk) activity on viral replication. With the drug Roscovitine, a specific inhibitor of cyclin-dependent kinases 1, 2, 5, 7, and 9, we have shown that during the first 6 h of infection, cyclin-dependent kinase-dependent events occurred that included the regulated processing and accumulation of the immediate-early (IE) UL122-123 transcripts and UL36-37 transcripts. Altered processing of UL122-123 led to a loss of IE1-72 and an increase in IE2-86. The ratio of spliced to unspliced UL37 transcripts also changed. These effects did not require de novo protein synthesis or degradation of proteins by the proteasome. Addition of Roscovitine at the beginning of the infection was also associated with inhibition of expression of selected viral early gene products, viral DNA replication, and late viral gene expression. When Roscovitine was added after the first 6 h of infection, the effects on IE gene expression were no longer observed and viral replication proceeded through the late phase, but viral titers were reduced. The reduction in viral titer was observed even when Roscovitine was first added at 48 h postinfection, indicating that cyclin-dependent kinase activity is required at both IE and late times. Flavopiridol, another specific inhibitor of cyclin-dependent kinases, had similar effects on IE and early gene expression. These results underscore the importance of accurate RNA processing and reiterate the significant role of cell cycle-regulatory factors in HCMV infection.

Base Sequence↗
PubMed2004

Multiple epitopes in the murine cytomegalovirus early gene product M84 are efficiently presented in infected primary macrophages and contribute to strong CD8+-T-lymphocyte responses and protection following DNA immunization.

We previously demonstrated that after vaccination of BALB/c mice with DNA encoding murine cytomegalovirus (MCMV) IE1 or M84, a similar level of protection against MCMV infection was achieved. However, the percentage of antigen-specific CD8(+) T cells elicited by IE1 was higher than that by M84 as measured by intracellular cytokine staining when splenocytes were stimulated with an epitope peptide (M. Ye at al., J. Virol. 76:2100-2112, 2002). We show here that after DNA vaccination with M84, a higher percentage of M84-specific CD8(+) T cells was detected when splenocytes were stimulated with J774 cells expressing full-length M84. When the defined M84 epitope 297-305 was deleted, the mutant DNA vaccine was still protective against MCMV replication and induced strong M84-specific CD8(+)-T-cell responses. The M84 gene was subsequently subcloned into three fragments encoding overlapping protein fragments. When mice were immunized with each of the M84 subfragment DNAs, at least two additional protective CD8(+)-T-cell epitopes were detected. In contrast to strong responses after DNA vaccination, M84-specific CD8(+)-T-cell responses were poorly induced during MCMV infection. The weak M84-specific response after MCMV infection was not due to poor antigen presentation in antigen-presenting cells, since both J774 macrophages and primary peritoneal macrophages infected with MCMV in vitro were able to efficiently and constitutively present M84-specific epitopes starting at the early phase of infection. These results indicate that antigen presentation by macrophages is not sufficient for M84-specific CD8(+)-T-cell responses during MCMV infection.

Animals↗
PubMed2004

Small internal deletions in the human cytomegalovirus IE2 gene result in nonviable recombinant viruses with differential defects in viral gene expression.

The human cytomegalovirus (HCMV) IE2 86-kDa protein is a key viral transactivator and an important regulator of HCMV infections. We used the HCMV genome cloned as a bacterial artificial chromosome (BAC) to construct four HCMV mutants with disruptions in regions of IE2 86 that are predicted to be important for its transactivation and autoregulatory functions. Three of these mutants have mutations that remove amino acids 356 to 359, 427 to 435, and 505 to 511, which disrupts a region of IE2 86 implicated in the activation of HCMV early promoters, a predicted zinc finger domain, and a putative helix-loop-helix motif, respectively, while the fourth carries three arginine-to-alanine substitution mutations in the region of amino acids 356 to 359. The resulting recombinant viruses are not viable, and by using quantitative real-time reverse transcription-PCR and immunofluorescence we have determined the location of the block in their replicative cycles. The IE2 86 Delta 356-359 mutant is able to support early gene expression, as indicated by the presence of UL112-113 transcripts and UL112-113 and UL44 proteins in cells transfected with the mutant BAC. This mutant does not express late genes and behaves nearly indistinguishably from the IE2 86R356/7/9A substitution mutant. Both exhibit detectable upregulation of major immediate-early transcripts at early times. The IE2 86 Delta 427-435 and IE2 86 Delta 505-511 recombinant viruses do not activate the early genes examined and are defective in repression of the major immediate-early promoter. These two mutants also induce the expression of selected delayed early (UL89) and late genes at early times in the infection. We conclude that these three regions of IE2 86 are necessary for productive infections and for differential control of downstream viral gene expression.

Cytomegalovirus↗
PubMed2003

Mechanisms governing maintenance of Cdk1/cyclin B1 kinase activity in cells infected with human cytomegalovirus.

Previous work has demonstrated dysregulation of key cell cycle components in human cytomegalovirus (HCMV)-infected human fibroblasts, resulting in cell cycle arrest (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. L. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). The activation of the mitotic kinase Cdk1/cyclin B, which was detected as early as 8 h postinfection (p.i.) and maintained throughout the time course, was particularly interesting. To understand the mechanisms underlying the induction of this kinase activity, we have examined the pathways that regulate the activation of Cdk1/cyclin B1 complexes. The accumulation of the cyclin B1 subunit in HCMV-infected cells is the result of increased synthesis and reduced degradation of the protein. In addition, the catalytic subunit, Cdk1, accumulates in its active form in virus-infected cells. The decreased level of the Tyr15-phosphorylated form of Cdk1 in virus-infected fibroblasts is due in part to the down-regulation of the expression and activity of the Cdk1 inhibitory kinases Myt1 and Wee1. Increased degradation of Wee1 via the proteasome also accounts for its absence at 24 h p.i. At late times, we observed accumulation of the Cdc25 phosphatases that remove the inhibitory phosphates from Cdk1. Interestingly, biochemical fractionation studies revealed that the active form of Cdk1, a fraction of total cyclin B1, and the Cdc25 phosphatases reside predominantly in the cytoplasm of infected cells. Collectively, these data suggest that the maintenance of Cdk1/cyclin B1 activity observed in HCMV-infected cells can be explained by three mechanisms: the accumulation of cyclin B1, the inactivation of negative regulatory pathways for Cdk1, and the accumulation of positive factors that promote Cdk1 activity.

CDC2 Protein Kinase↗
PubMed2003

Human cytomegalovirus infection leads to accumulation of geminin and inhibition of the licensing of cellular DNA replication.

Previous studies have shown that infection of G(0)-synchronized human fibroblasts by human cytomegalovirus (HCMV) results in a block to cellular DNA synthesis. In this study, we have examined the effect of viral infection on the formation of the host cell DNA prereplication complex (pre-RC). We found that the Cdc6 protein level was significantly upregulated in the virus-infected cells and that there was a delay in the expression of the Mcm family of proteins. The loading of the Mcm proteins onto the DNA pre-RC complex also appeared to be defective in the virus-infected cells. This inhibition of DNA replication licensing was associated with the accumulation of geminin, a replication inhibitor. Cdt1, which participates in the loading of the Mcm proteins, was also downregulated and modified differentially in the infected cells. Early viral gene expression was sufficient for the virus-induced alteration of the pre-RC, and the immediate-early protein IE1 was not required. These studies show that the inhibition of replication licensing in HCMV-infected cells is one of the multiple pathways by which the virus dysregulates the host cell cycle.

Cell Cycle Proteins↗
PubMed2002

Development of a vaccine against murine cytomegalovirus (MCMV), consisting of plasmid DNA and formalin-inactivated MCMV, that provides long-term, complete protection against viral replication.

We previously demonstrated that immunization of mice with plasmid DNAs (pDNAs) expressing the murine cytomegalovirus (MCMV) genes IE1-pp89 and M84 provided synergistic protection against sublethal viral challenge, while immunization with plasmids expressing putative virion proteins provided no or inconsistent protection. In this report, we sought to augment protection by increasing the breadth of the immune response. We identified another MCMV gene (m04 encoding gp34) that provided strong and consistent protection against viral replication in the spleen. We also found that immunization with a DNA pool containing 10 MCMV genes that individually were nonprotective elicited reproducible protection against low to intermediate doses of challenge virus. Moreover, inclusion of these plasmids into a mixture with gp34, pp89, and M84 DNAs provided even greater protection than did coimmunization with pp89 and M84. The highest level of protection was achieved by immunization of mice with the pool of 13 pDNAs, followed by formalin-inactivated MCMV (FI-MCMV). Immunization with FI-MCMV elicited neutralizing antibodies against salivary gland-derived MCMV, and of greatest importance, mice immunized with both the combined pDNA pool and FI-MCMV had undetectable levels of virus in the spleen and salivary glands after challenge. Intracellular cytokine staining of splenocytes from pDNA- and FI-MCMV-immunized mice showed that pDNA immunization elicited high levels of pp89- and M83-specific CD8(+) T cells, whereas both pDNA and FI-MCMV immunizations generated strong CD8(+)-T-cell responses against virion-associated antigens. Taken together, these results show that immunization with pDNA and inactivated virus provides strong antibody and cell-mediated immunity against CMV infection.

Amino Acid Sequence↗
PubMed2002

Infection of cells with human cytomegalovirus during S phase results in a blockade to immediate-early gene expression that can be overcome by inhibition of the proteasome.

Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G(0) using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE(+) in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE(+) cells that gradually accumulated within the S and G(2)/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU(+) cells were IE(-) and had progressed into G(2)/M or back to G(1). The majority of the IE(+) cells in S and G(2)/M were BrdU(-). Only a few cells were IE(+) BrdU(+), and they resided in G(2)/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.

Cells, Cultured↗
PubMed2002

Strong CD8 T-cell responses following coimmunization with plasmids expressing the dominant pp89 and subdominant M84 antigens of murine cytomegalovirus correlate with long-term protection against subsequent viral challenge.

We previously showed that intradermal immunization with plasmids expressing the murine cytomegalovirus (MCMV) protein IE1-pp89 or M84 protects against viral challenge and that coimmunization has a synergistic protective effect (C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 74:3696-3708, 2000). Using an intracellular gamma interferon cytokine staining assay, we have now characterized the CD8+ T-cell response after DNA immunization with pp89, M84, or pp89 plus M84. The pp89- and M84-specific CD8+ T-cell responses peaked rapidly after three immunizations. DNA immunization and MCMV infection generated similar levels of pp89-specific CD8+ T cells. In contrast, a significantly higher level of M84-specific CD8+ T cells was elicited by DNA immunization than by MCMV infection. Fusion of ubiquitin to pp89 enhanced the CD8+ T-cell response only under conditions where vaccination was suboptimal. Three immunizations with either pp89, M84, or pp89 plus M84 DNA also provided significant protection against MCMV infection for at least 6 months, with the best protection produced by coimmunization. A substantial percentage of antigen-specific CD8+ T cells remained detectable, and they responded rapidly to the MCMV challenge. These results underscore the importance of considering antigens that do not appear to be highly immunogenic during infection as DNA vaccine candidates.

3T3 Cells↗
PubMed2002

Viable human cytomegalovirus recombinant virus with an internal deletion of the IE2 86 gene affects late stages of viral replication.

Using bacterial artificial chromosome (BAC) technology, we have constructed and characterized a human cytomegalovirus recombinant virus with a mutation in the exon specific for the major immediate-early region 2 (IE2) gene product. The resulting IE2 86-kDa protein (IE2 86) has an internal deletion of amino acids 136 to 290 and is fused at the carboxy terminus to enhanced green fluorescent protein (EGFP). The deletion also removes the promoter and initiator methionine for the p40 form of IE2 and initiator methionine for the p60 form of the protein, and therefore, these late gene products are not produced. The mutant virus IE2 86 Delta SX-EGFP is viable but exhibits altered growth characteristics in tissue culture compared with a full-length wild-type (wt) IE2 86-EGFP virus or a revertant virus. When cells are infected with the mutant virus at a low multiplicity of infection (MOI), there is a marked delay in the production of infectious virus. This is associated with slower cell-to-cell spread of the virus. By immunofluorescence and Western blot analyses, we show that the early steps in the replication of the mutant virus are comparable to those for the wt. Although there is significantly less IE2 protein in the cells infected with the mutant, there is only a modest lag in the initial accumulation of IE1 72 and viral early proteins, and viral DNA replication proceeds normally. The mutation also has only a small effect on the synthesis of the viral major capsid protein. The most notable molecular defect in the mutant virus infection is that the steady-state levels of the pp65 (UL83) and pp28 (UL99) matrix proteins are greatly reduced. In the case of UL83, but not UL99, there is also a corresponding decrease in the amount of mRNA present in cells infected with the mutant virus.

Cells, Cultured↗
PubMedDate not supplied

Viral induction of site-specific chromosome damage.

The advent of advanced cell culture and cytogenetics techniques in the 1950s opened a new avenue for research on the pathogenic interactions between animal viruses and their hosts. Studies of many viruses revealed their ability to nonspecifically induce cytogenetic damage to their host cell's chromosomes. However, only three viruses, the oncogenic adenoviruses, herpes simplex virus (HSV) and human cytomegalovirus (HCMV), have been found to cause non-random, site-specific chromosomal damage. Adenovirus (Ad) type 12 induces fragility at four distinct loci (RNU1, RNU2, RN5S and PSU1) in many different types of human cells. A common feature of these loci is that they contain a repeated array of transcriptionally active genes encoding small structural RNAs. Site-specific induction of breaks also requires the virally encoded E1B protein of M(r) 55000 and the C-terminus of the cellular p53 protein. Analysis of the induction of damage by HSV and HCMV necessitates consideration of several factors, including the strain of virus used, the timing of infection, the type of cell used, and the multiplicity of infection. Both HSV strains 1 and 2 are cytotoxic, although the former seems to be more proficient at inducing damage. At early times post infection, HSV induces breaks and specific uncoiling of the centromeres of chromosomes 1, 9 and 16. This is followed at later times by a more complete severing of all of the chromosomes, termed pulverisation. Damage by HSV requires viral entry and de novo viral protein synthesis, with immediate early viral proteins responsible for the induction of breaks and uncoiling and early gene products (most likely nucleases) involved in the extensive pulverisation seen later. HCMV has been studied primarily in permissive human fibroblasts. Its ability to induce specific damage in chromosome 1 at two loci, 1q21 and 1q42, was only recently revealed as the cells must be in S-phase when they are infected for the breaks to be observed. In contrast to adenovirus and HSV, HCMV induction of specific breakage requires only viral entry into the cell and not de novo viral protein expression. This latter point may be a factor in its ability to cause damage in the developing fetal brain, where the most severe clinical manifestations of congenital infection are observed.

Adenovirus Infections, Human↗