Neonatal maternal separation alters immune, endocrine, and behavioral responses to acute Theiler's virus infection in adult mice.

Previous studies have established a link between adverse early life events and subsequent disease vulnerability. The present study assessed the long-term effects of neonatal maternal separation on the response to Theiler's murine encephalomyelitis virus infection, a model of multiple sclerosis. Balb/cJ mouse pups were separated from their dam for 180-min/day (180-min MS), 15-min/day (15-min MS), or left undisturbed from postnatal days 2-14. During adolescence, mice were infected with Theiler's virus and sacrificed at days 14, 21, or 35 post-infection. Prolonged 180-min MS increased viral load and delayed viral clearance in the spinal cords of males and females, whereas brief 15-min MS increased the rate of viral clearance in females. The 15-min and 180-min MS mice exhibited blunted corticosterone responses during infection, suggesting that reduced HPA sensitivity may have altered the immune response to infection. These findings demonstrate that early life events alter vulnerability to CNS infection later in life. Therefore, this model could be used to study gene-environment interactions that contribute to individual differences in susceptibility to infectious and autoimmune diseases of the CNS.

Alterations in chemokine expression following Theiler's virus infection and restraint stress.

Restraint stress (RS) applied to mice during acute infection with Theiler's virus causes corticosterone-induced immunosuppression. This effect was further investigated by measuring chemokine changes in the spleen and central nervous system (CNS) using an RNase Protection Assay. mRNAs for lymphotactin (Ltn), interferon-induced protein-10 (IP-10), MIP-1 beta, monocyte chemoattractant protein-1 (MCP-1) and TCA-3 were detected in the spleen at day 2 pi, but not in the brain of CBA mice infected with Theiler's virus. Ltn, IP-10 and RANTES were elevated in both the spleen and the brain at day 7 pi, and were significantly decreased by RS in the brain. RS also resulted in decreased inflammation within the CNS.

The nucleocapsid protein of murine hepatitis virus type 3 induces transcription of the novel fgl2 prothrombinase gene.

Using a set of parental and recombinant murine hepatitis virus strains, we demonstrate that the nucleocapsid protein induces transcription of the novel fgl2 prothrombinase gene and elevated procoagulant activity in those strains that produce fulminant hepatitis. Chinese hamster ovary cells cotransfected with a construct expressing nucleocapsid protein from susceptible strains and with a luciferase reporter construct containing the fgl2 promoter showed a 6-fold increase in luciferase activity compared with nontransfected cells or cells cotransfected with a construct expressing nucleocapsid protein from resistant strains. Two deletions found at coding sites 111-123 and 1143-1145 of structural domains I and III, respectively, of the nucleocapsid gene may account for the differences between pathogenic and nonpathogenic strains. Preliminary mapping of the fgl2 promoter has defined a region from -372 to -306 upstream from the ATG translation initiation site to be responsive to nucleocapsid protein. Hence, mapping of genetic determinants in parental and recombinant strains demonstrates that the nucleocapsid protein of strains that induce fulminant hepatitis is responsible for transcription of the fgl2 prothrombinase gene. These studies provide new insights into the role of the nucleocapsid gene in the pathogenesis of viral hepatitis.

The pattern of induction of apoptosis during infection with MHV-3 correlates with strain variation in resistance and susceptibility to lethal hepatitis.

In the present study we have investigated the possibility that strain specific differences in the induction of apoptosis in macrophages could play a role in the resistance of strain A/J mice to MHV-3 induced hepatitis. MHV-3 infected macrophages from Balb/c and A/J mice were analyzed at various time points after infection. Apoptosis in A/J macrophages could be detected at 8 h post infection and increased significantly by 12 h, when almost 50-70% of the infected cells were undergoing apoptosis. In Balb/c macrophages, apoptotic changes were less pronounced and were observed in only 5-10% of the cells. MHV-3 induced apoptosis was inversely correlated with the ability of this virus to induce expression of fgl-2 prothrombinase protein and syncytia formation. Infected macrophages from A/J mice did not express fgl-2 protein and did not form syncytia. In contrast, infection of Balb/c derived macrophages resulted in fgl-2 expression and extensive syncytia formation. These data fit a model in which apoptosis of virally infected cells is a protective response which eliminates cells whose survival might be harmful for the whole organism.

The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.

Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.

Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase.

Baculovirus infection results in the induction of membrane structures within the nucleoplasm of the host cells. The source of these membranes is unclear; however, using the normal dynamics of cellular membranes and the nuclear envelope as a model, it is possible that the cell cycle might play a role in the regulation of formation of these intranuclear membranes. Therefore, one goal of this study was to investigate the effect of baculovirus infection on the cell cycle of Sf9 host cells. Since few data are available on the cell cycle of insect cells, the first task was to define Sf9 cell cycle kinetics. The cell cycle phase distribution of Sf9 cells grown in suspension culture was determined to be evenly distributed (29% of the cells in G1, 33% in S, and 36% in G2/M phase), with the duration of G1 and S phases both being about 6 h and the combined duration of G2/M phase being about 8 h. When Sf9 cells were infected with AcMNPV (Autographa californica nuclear polyhedrosis virus), approximately 84% of the cells were arrested in G2/M phase by 18-24 h p.i. Concomitant with the viral-induced arrest in G2/M phase, high levels of both cdc2-associated histone H1 kinase activity and cyclin B protein were detected. By 24 h p.i. cyclin B was no longer detected; however, cdc2-associated histone H1 kinase activity remained throughout the infection. These data suggested that early in infection, cyclin B/cdc2 complex may be used to regulate the transition from G2 to M phase, but prolonged arrest may be due to a protein(s) encoded by AcMNPV. DNA hybridization analysis showed that the maximal rate of viral DNA replication occurred before G2/M arrest. We noted that viral DNA replication still occurred late in infection, when the majority of the cells were arrested in G2/M phase. Since cellular DNA replication normally does not occur during G2 or M phase, experiments were designed to determine if viral DNA replication could occur even when host cell DNA replication was arrested. Sf9 cells were arrested and "frozen" at the boundary of G1/S phase using 5-fluoro-2'deoxyuridine (FdUrd) treatment and then infected with AcMNPV In the blocked, infected cells, viral DNA replication was detected; however, cellular DNA remained at steady-state levels. These results suggested that cellular DNA replication was not necessary for viral DNA replication and show that viral DNA replication was not significantly inhibited by FdUrd treatment. It was a surprise to detect viral DNA replication when the host cells were "frozen" at G1/S phase. We wanted to determine if the viral infection was progressing to the stage of progeny virus production. Our data showed that progeny budded virus (BV) and virus-induced intranuclear microvesicles were produced in the frozen, infected cells; however, the intranuclear microvesicles had an unusual structure. They were irregular in shape and thickened compared to those observed in a normal infection. Very few enveloped nucleocapsids were visible in the nucleus of the frozen, infected cells and the occluded-derived virus envelope proteins, ODV-E66 and ODV-EC27, were not detected by Western blot analyses. Since the cells were sustained at the boundary of G1 and S phases for the duration of this experiment, the decreased amount of enveloped ODV in the nucleus could be due to several factors, including decreased levels of proteins expressed from late genes, aberrant microvesicles, or the necessity of G2/M phasing of the infected cell for efficient production and maturation of intranuclear microvesicles. These data indicate that AcMNPV infection results in cell cycle arrest in G2/M phase and this arrest may be due to a viral-encoded protein(s) that has cdc2-associated kinase activity. (ABSTRACT TRUNCATED)

Perturbation of the host cell cycle and DNA replication by the bovine papillomavirus replication protein E1.

A stable cell line expressing the bovine papillomavirus E1 protein (C2E1) was compared with an E1 minus control line (CNEO) to study the effects of E1 protein on host cell growth. C2E1 and CNEO cells were synchronized either at mitosis or at the G1/S boundary by the cell cycle inhibitors nocodazole and mimosine, respectively. After release from the drug-induced cell cycle block, the progression through the succeeding stages of the cell cycle was temporally monitored using flow cytometry. In addition, incorporation of bromodeoxyuridine (BrdUrd) was used to determine precisely the time of initiation of DNA synthesis in C2E1 and CNEO cells after release from drug-induced cell cycle arrest. Expression of E1 protein decreased the duration of G1 phase and increased S and G2 phase durations without affecting the overall cell doubling time. In conjunction with the increase in G2 phase duration, histone H1 kinase activity was prolonged during the G2 to M phase transition in C2E1 cells, which suggested that E1 protein may affect the mechanisms which ensure proper timing of kinase inactivation. During the G1 to S phase transition in C2E1 cells, the timing of appearance and abundance of cyclin D1 were altered compared to CNEO cells, while cyclin E levels were unaffected. Consequently, E1 protein may affect G1 phase duration through a cyclin D1-dependent pathway. Finally, a subpopulation of cells with a greater than G2 DNA content (>G2 DNA), and which was still capable of incorporating BrdUrd, was shown to exist only in the E1-expressing cell line. These combined results demonstrate that the viral replication protein E1 has the potential to influence the host cell environment significantly, which may contribute to pathogenesis and viral persistence.

BPV E1 protein alters the kinetics of cell cycle entry of serum starved mouse fibroblasts.

A stable bovine papillomavirus E1 expressing cell line (C2E1) was used to investigate the effects of E1 protein on the requirement for growth factors during serum-induced reentry from quiescence to proliferation. Flow cytometric bivariate DNA/PCNA analysis was utilized to study the expression of proliferating cell nuclear antigen (PCNA) concomitant with this transition. C2E1 cells, unlike the control cells (CNEO), were able to reenter the cell cycle when stimulated with low serum (1%). Stimulation with 10% serum revealed that C2E1 cells entered the first cell cycle faster than CNEO, indicating that E1 protein decreased the time of progression from G0 stage upon serum activation. It was also shown that PCNA expression started earlier in C2E1 cells than in CNEO cells after quiescent cells were stimulated with 10% serum. Addition of 1% serum was able to induce PCNA expression in C2E1 but not in CNEO cells in the first 24 h after stimulation. Using Triton X-100 treatment, it was found that the distribution between bound and unbound forms of PCNA was altered in E1-expressing cells compared to CNEO cells. Based on these results, it is suggested that E1 might possess mitogen-like properties.

The bovine papillomavirus E1 protein alters the host cell cycle and growth properties.

A stable E1-expressing cell line, CE1, was developed to investigate the effect of bovine papillomavirus E1 protein on host cell growth. Expression of E1 in CE1 cells is under control of the dexamethasone-inducible mouse mammary tumor virus LTR promoter. Flow cytometry was used to determine the intracellular levels of E1 in CE1 cells in parallel with cell cycle distributions and cell growth parameters. CE1 cells expressed a basal level of E1 that was increased following exposure to increasing concentrations of dexamethasone. As E1 expression increased, the following effects on cell growth were observed: (1) the overall cell generation time increased and (2) the duration of G1 + G0 phases increased slightly while the duration of G2 + M phases increased twofold. The appearance of a cell subpopulation with a greater than G2 phase DNA content was also observed in E1-expressing cells. Removal of dexamethasone resulted in a return of E1 levels to the basal state with a concomitant restoration of both cell growth properties and cell cycle phase distributions. All of the observed effects were seen in the absence of both other papillomaviral proteins and papillomaviral DNA replication. We conclude that E1 is sufficient to negatively affect host cell growth, with a prominent effect observed in the G2 + M phases.

Bovine papillomavirus E1 protein affects the host cell cycle phase fractions.

C127 murine fibroblast cells were electroporated with a bovine papillomavirus E1 protein expression vector and examined by flow cytometry. E1 expressing cells (E1+) within the total cell population were distinguished from nonexpressing cells (E1-) by immunofluorescent staining with anti-E1 serum and a fluorescein-conjugated second antibody. Under conditions of saturation with the first and second antibodies, the specific green fluorescence reflected the level of intracellular E1 protein. Simultaneous staining with a DNA-specific dye, propidium iodide (PI), enabled the cell cycle distributions for the E1+ and E1- cell populations to be determined. It was found that the E1+ subpopulation had a reduced percentage of cells in G1 phase and an increased percentage of G2+M phase cells, compared to the E1- subpopulation. There was no significant difference in overall doubling time or percentage of noncycling cells in the E1+ vs. E1- populations, indicating that the change in cell cycle distribution was not due to a general activation or inhibition of cell growth by E1. Direct measurement of cell cycle phase fractions confirmed that the G1 phase was decreased and the G2+M phase was increased in E1 expressing cells. As these observations were made in the absence of other viral proteins or viral DNA replication, it suggests that the E1 protein exerts an effect on the host cell independent of its direct role in viral DNA replication. Thus, E1 may interact directly with the host cell cycle regulatory machinery.

Research note: differential chemotaxis of Physarum polycephalum to Salmonella gallinarum and Salmonella pullorum.

The chemotactic and chemotropic responses of the plasmodial stage of the slime mold Physarum polycephalum were used to distinguish Salmonella gallinarum and Salmonella pullorum from 10 Salmonella serovars that are commonly isolated from domestic poultry. Utilizing an in vitro plasmodium agar plate assay method, P. polycephalum was attracted to S. gallinarum and S. pullorum, but the organism was repelled by Salmonella derby, Salmonella dublin, Salmonella enteritidis, Salmonella heidelberg, Salmonella minnesota, Salmonella montevideo, Salmonella newington, Salmonella newport, Salmonella reading, and Salmonella typhimurium.

Intrinsic fluctuations of phosphoinositol levels are involved in the progression of the naturally synchronous cell cycle in Physarum polycephalum.

Using [3H]myo-inositol incorporation, changes in phosphoinositol (PI) metabolism at different cell cycle stages in the myxomycete Physarum polycephalum were examined by column chromatography. Two base levels for the inositol trisphosphate (IP3) fraction were determined: a low one for S-phase and a higher one for G2 phase. Two transient increases of IP3 were also observed, one in S-phase, 70 min after mitosis (no G1 phase in the Physarum cell cycle) and another in G2 phase, 90 min before mitosis. It is concluded that the fluctuations in IP3 levels reflect endogenous events in the cell cycle of Physarum, because they occurred in the absence of any exogenous signals. Pulse treatment with Li+ (10 mM) at the points of the cell cycle, characterized by the IP3 transients, had opposite effects: in early S-phase it caused an acceleration while in late G2 phase it caused a prolongation of the cell cycle duration. The pattern of Li(+)-induced changes in PI turnover is also antagonistic: in most cases the IP3 level would decrease, however, Li+ prevents the cell cycle-dependent reduction in IP3 concentration when applied at early S-phase. The possible implications of the autogenous fluctuations in the IP3 fraction on the progression of the cell cycle through several distinct checkpoints are discussed.