Mouse Adenovirus Type 1 Early Region 1A Is Dispensable for Growth in Cultured Fibroblasts

ABSTRACT Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+,Rb+/−, and Rb−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 andpmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.

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The Role of Mouse Adenovirus Type 1 Early Region 1A in Acute and Persistent Infections in Mice

Journal of Virology ◽

10.1128/jvi.72.7.5699-5706.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5699-5706 ◽

Cited By ~ 23

Author(s):

Kimberley Smith ◽

Corrie C. Brown ◽

Katherine R. Spindler

Keyword(s):

Persistent Infection ◽

Mononuclear Cells ◽

Lethal Dose ◽

Pcr Amplification ◽

Adenovirus Type ◽

Wild Type Virus ◽

Null Mutant ◽

Wild Type ◽

Early Region

ABSTRACT Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 105 PFU of virus, an E1A null mutant,pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant,pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU,pmE109 and pmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.

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E1A-CR3 Interaction-Dependent and -Independent Functions of mSur2 in Viral Replication of Early Region 1A Mutants of Mouse Adenovirus Type 1

Journal of Virology ◽

10.1128/jvi.79.6.3267-3276.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3267-3276 ◽

Cited By ~ 5

Author(s):

Lei Fang ◽

Katherine R. Spindler

Keyword(s):

Viral Replication ◽

Adenovirus Type ◽

Inbred Strains ◽

Wild Type ◽

Embryonic Fibroblasts ◽

Early Region ◽

Inbred Strains Of Mice ◽

Independent Functions ◽

A Subunit

ABSTRACT mSur2, a subunit of the Mediator complex, is required for efficient mouse adenovirus type 1 (MAV-1) replication (L. Fang, J. L. Stevens, A. J. Berk, and K. R. Spindler, J. Virol. 78:12888-12900, 2004). We examined the contributions of early-region 1A (E1A) to mSur2 function in MAV-1 replication with E1A mutant viruses. At a multiplicity of infection (MOI) of 1, viruses containing CR3 replicated better in Sur2+/+ mouse embryonic fibroblasts (MEFs) than in Sur2−/− MEFs. In contrast, viruses lacking CR3 replicated no better in Sur2+/+ than in Sur2−/− MEFs. This result supports the hypothesis that the E1A CR3-mSur2 interaction is important for MAV-1 replication. However, at an MOI of 0.05, viruses lacking CR3 showed replication defects in Sur2−/− MEFs compared to Sur2+/+ MEFs, suggesting an E1A CR3 interaction-independent function of mSur2 in MAV-1 replication in cell culture. Paradoxically, CR1Δ, CR2Δ, and CR3Δ mutant viruses replicated slightly more efficiently than wild-type (wt) MAV-1 and E1A null mutant viruses in Sur2−/− MEFs at an MOI of 0.05. Coinfection of Sur2−/− MEFs with wt MAV-1 and CR1Δ, CR2Δ, or CR3Δ mutant viruses rescued the defects of wt MAV-1 replication. This result suggests that an inhibiting effect on wt E1A protein expression and/or E1A function might account for the severe viral replication defect of MAV-1 in Sur2−/− MEFs at an MOI of 0.05. Moreover, titrations of virus yields from infected brains of inbred strains of mice showed that E1A null and CR3Δ mutant viruses had a significant defect in virus replication compared to wt MAV-1. This result supports the hypothesis that the MAV-1 E1A-mSur2 interaction is important in MAV-1 replication in mice.

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SJL/J Mice Are Highly Susceptible to Infection by Mouse Adenovirus Type 1

Journal of Virology ◽

10.1128/jvi.75.24.12039-12046.2001 ◽

2001 ◽

Vol 75 (24) ◽

pp. 12039-12046 ◽

Cited By ~ 33

Author(s):

Katherine R. Spindler ◽

Lei Fang ◽

Martin L. Moore ◽

Gwen N. Hirsch ◽

Corrie C. Brown ◽

...

Keyword(s):

Inbred Mouse ◽

Adenovirus Type ◽

Wild Type Virus ◽

Mouse Strains ◽

Wild Type ◽

Viral Loads ◽

Sublethal Doses ◽

The Brain ◽

Lethal Doses

ABSTRACT Mouse adenovirus type 1 (MAV-1) targets endothelial and monocyte/macrophage cells throughout the mouse. Depending on the strain of mouse and dose or strain of virus, infected mice may survive, become persistently infected, or die. We surveyed inbred mouse strains and found that for the majority tested the 50% lethal doses (LD50s) were >104.4 PFU. However, SJL/J mice were highly susceptible to MAV-1, with a mean LD50 of 10−0.32 PFU. Infected C3H/HeJ (resistant) and SJL/J (susceptible) mice showed only modest differences in histopathology. Susceptible mice had significantly higher viral loads in the brain and spleen at 8 days postinfection than resistant mice. Infection of primary macrophages or mouse embryo fibroblasts from SJL/J and C3H/HeJ mice gave equivalent yields of virus, suggesting that a receptor difference between strains is not responsible for the susceptibility difference. When C3H/HeJ mice were subjected to sublethal doses of gamma irradiation, they became susceptible to MAV-1, with an LD50 like that of SJL/J mice. Antiviral immunoglobulin G (IgG) levels were measured in susceptible and resistant mice infected by an early region 1A null mutant virus that is less virulent that wild-type virus. The antiviral IgG levels were high and similar in the two strains of mice. Taken together, these results suggest that immune response differences may in part account for differences in susceptibility to MAV-1 infection.

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Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4426 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4426-4435 ◽

Cited By ~ 43

Author(s):

O Kessler ◽

L A Chasin

Keyword(s):

Steady State ◽

Actinomycin D ◽

Chinese Hamster ◽

Nuclear Pore ◽

Mrna Levels ◽

Wild Type ◽

Adenine Phosphoribosyltransferase ◽

Aprt Gene ◽

Steady State Levels ◽

Nonsense Codons

We have analyzed Chinese hamster ovary (CHO) cell mutants bearing nonsense codons in four of the five exons of the adenine phosphoribosyltransferase (aprt) gene and have found a pattern of mRNA reduction similar to that seen in systems studied previously: a decrease in steady-state mRNA levels of 5- to 10-fold for mutations in exons 1, 2, and 4 but little effect for mutations in the 3'-most exon (exon 5). Nuclear aprt mRNA levels showed a similar decrease. Nonsense-containing aprt mRNA decayed at the same rate as wild-type mRNA in these cell lines after inhibition of transcription with actinomycin D. Nonsense-containing aprt mRNA is associated with polysomes, ruling out a model in which stable residual mRNA escapes degradation by avoiding translation initiation. A tetracycline-responsive form of the aprt gene was used to compare the stability of nonsense-containing and wild-type aprt mRNAs without globally inhibiting transcription. In contrast to measurements made in the presence of actinomycin D, after inhibition of aprt transcription with tetracycline, a nonsense-mediated destabilization of aprt mRNA was indeed demonstrable. The increased rate of decay of cytoplasmic aprt mRNA seen here could account for the nonsense-mediated reduction in steady-state levels of aprt mRNA. However, the low levels of nonsense-bearing aprt mRNA in the nucleus suggest a sensibility of mRNA to translation or translatability before it exits that compartment. Quantitation of the steady-state levels of transcripts containing introns revealed no accumulation of partially spliced aprt RNA and hence no indication of nonsense-mediated aberrancies in splicing. Our results are consistent with a model in which translation facilitates the export of mRNA through a nuclear pore. However, the mechanism of this intriguing nucleocytoplasmic communication remains to be determined.

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Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.8.2865-2871.1986 ◽

1986 ◽

Vol 6 (8) ◽

pp. 2865-2871

Author(s):

L McConlogue ◽

S L Dana ◽

P Coffino

Keyword(s):

Steady State ◽

Gene Amplification ◽

Ornithine Decarboxylase ◽

Mrna Levels ◽

Wild Type ◽

Altered Expression ◽

Cells Of Origin ◽

Multistep Process ◽

Steady State Levels ◽

Synthesis And Degradation

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.

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Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells.

Molecular and Cellular Biology ◽

10.1128/mcb.6.8.2865 ◽

1986 ◽

Vol 6 (8) ◽

pp. 2865-2871 ◽

Cited By ~ 41

Author(s):

L McConlogue ◽

S L Dana ◽

P Coffino

Keyword(s):

Steady State ◽

Gene Amplification ◽

Ornithine Decarboxylase ◽

Mrna Levels ◽

Wild Type ◽

Altered Expression ◽

Cells Of Origin ◽

Multistep Process ◽

Steady State Levels ◽

Synthesis And Degradation

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Existence of Transdominant and Potentiating Mutants of UL9, the Herpes Simplex Virus Type 1 Origin-Binding Protein, Suggests that Levels of UL9 Protein May Be Regulated during Infection

Journal of Virology ◽

10.1128/jvi.77.17.9639-9651.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9639-9651 ◽

Cited By ~ 9

Author(s):

Boriana Marintcheva ◽

Sandra K. Weller

Keyword(s):

Steady State ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Viral Infection ◽

Herpes Simplex Virus Type ◽

Mutant Protein ◽

Wild Type ◽

Steady State Levels ◽

Simplex Virus

ABSTRACT UL9 is a multifunctional protein required for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. We have previously shown that mutations in the conserved helicase motifs of UL9 can have either a transdominant or potentiating effect on the plaque-forming ability of infectious DNA from wild-type virus (A. J. Malik and S. K. Weller, J. Virol. 70:7859-7866, 1996). In this paper, the mechanisms of transdominance and potentiation are explored. We show that the motif V mutant protein containing a G to A substitution at residue 354 is unstable when expressed by transfection and is either processed to a 38-kDa N-terminal fragment or degraded completely. The overexpression of the MV mutant protein is able to influence the steady-state protein levels of wild-type UL9 and to override the inhibitory effects of wild-type UL9. Potentiation correlates with the ability of the UL9 variants containing the G354A mutation to be processed or degraded to the 38-kDa form. We propose that the MV mutant protein is able to interact with full-length UL9 and that this interaction results in a decrease in the steady-state levels of UL9, which in turn leads to enhanced viral infection. Furthermore, we demonstrate that inhibition of HSV-1 infection can be obtained by overexpression of full-length UL9, the C-terminal third of the protein containing the origin-binding domain, or the N-terminal two-thirds of UL9 containing the conserved helicase motifs and the putative dimerization domain. Our results suggest that transdominance can be mediated by overexpression, origin-binding activity, and dimerization, whereas potentiation is most likely caused by the ability of the UL9 MV mutant to influence the steady-state levels of wild-type UL9. Taken together, the results presented in this paper suggest that the regulation of steady-state levels of UL9 may play an important role in controlling viral infection.

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Stimulation of Osteoprotegerin Ligand and Inhibition of Osteoprotegerin Production by Glucocorticoids in Human Osteoblastic Lineage Cells: Potential Paracrine Mechanisms of Glucocorticoid-Induced Osteoporosis1

Endocrinology ◽

10.1210/endo.140.10.7034 ◽

1999 ◽

Vol 140 (10) ◽

pp. 4382-4389 ◽

Cited By ~ 351

Author(s):

Lorenz C. Hofbauer ◽

Francesca Gori ◽

B. Lawrence Riggs ◽

David L. Lacey ◽

Colin R. Dunstan ◽

...

Keyword(s):

Steady State ◽

Bone Resorption ◽

Conditioned Medium ◽

Northern Analysis ◽

Protein Synthesis Inhibitor ◽

Mrna Levels ◽

Trap Activity ◽

Osteoblastic Lineage ◽

Steady State Levels

Abstract Osteoporosis is a serious complication of systemic glucocorticoid use. However, while glucocorticoids increase bone resorption in vitro and in vivo, the mechanism(s) of this effect are at present unclear. Recent studies have identified the osteoprotegerin (OPG) ligand (OPG-L) as the final effector of osteoclastogenesis, an action that is opposed by the soluble neutralizing receptor, OPG. Thus, we assessed glucocorticoid regulation of OPG and OPG-L in various human osteoblastic lineage cells using Northern analysis, RT-PCR, and ELISA. Dexamethasone inhibited constitutive OPG messenger RNA (mRNA) steady-state levels by 70–90% in primary (MS) and immortalized stromal cells (hMS), primary trabecular osteoblasts (hOB), immortalized fetal osteoblasts (hFOB), and osteosarcoma cells (MG-63). In hFOB cells, dexamethasone inhibited constitutive OPG mRNA steady-state levels in a dose- and time-dependent fashion by 90%, and also suppressed cytokine-stimulated OPG mRNA steady-state levels. Dexamethasone-induced inhibition of OPG mRNA levels was not affected by the protein synthesis inhibitor, cycloheximide, and was shown to be due to inhibition of OPG gene transcription using a nuclear run-on assay. Moreover, dexamethasone also dose dependently (10−10m–10−7m) inhibited constitutive OPG protein concentrations in the conditioned medium of hFOB cells from 2.59 ± 0.02 ng/ml (control) to 0.30 ± 0.01 ng/ml (88% inhibition; P < 0.001 by ANOVA). Concurrently, dexamethasone stimulated OPG-L mRNA steady-state levels in MS and hFOB cells by 2- and 4-fold, respectively. Treatment of murine marrow cultures with conditioned medium harvested from dexamethasone-treated MG-63 cells increased tartrate-resistant acid phosphatase (TRAP) activity by 54% (P < 0.005) compared with medium harvested from control-treated cells (in the presence of OPG-L and macrophage colony-stimulating factor). Moreover, dexamethasone (10−8m) promoted osteoclast formation in vitro, as assessed by a 2.5-fold increase of TRAP activity in cell lysates (P < 0.001) and the appearance of TRAP-positive multinucleated cells. Our data are thus consistent with the hypothesis that glucocorticoids promote osteoclastogenesis by inhibiting OPG and concurrently stimulating OPG-L production by osteoblastic lineage cells, thereby enhancing bone resorption.

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Lack of evidence of phenotypic complementation of E1A/E1B-deleted adenovirus type 5 upon superinfection by wild-type virus in the cotton rat.

Journal of Virology ◽

10.1128/jvi.69.10.6518-6524.1995 ◽

1995 ◽

Vol 69 (10) ◽

pp. 6518-6524 ◽

Cited By ~ 11

Author(s):

W Oualikene ◽

P Gonin ◽

M Eloit

Keyword(s):

Adenovirus Type ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Cotton Rat ◽

Adenovirus Type 5

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The critical role of T cells in glucocorticoid-induced osteoporosis

Cell Death and Disease ◽

10.1038/s41419-020-03249-4 ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Lin Song ◽

Lijuan Cao ◽

Rui Liu ◽

Hui Ma ◽

Yanan Li ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Steady State ◽

Side Effects ◽

Critical Role ◽

Wild Type ◽

Receptor Activator ◽

Steady State Levels ◽

Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

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