Early Stages Of Replication Research Articles

Potent, selective and cell-mediated inhibition of human herpesvirus 6 at an early stage of viral replication by the non-nucleoside compound CMV423

CMV423 (2-chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1-carboxamide) is a new antiviral agent with potent and selective in vitro activity against the β-herpesvirus human cytomegalovirus (HCMV), but not against α- or γ-herpesviruses. Here we report that its activity also extends to human herpesvirus 6 (HHV-6) and 7 (HHV-7). When compared in vitro to ganciclovir and foscarnet (the standard drugs recommended for treatment of HHV-6 infections), CMV423 showed a superior selectivity, due to its high activity (antiviral ic 50: 53 nM) and low cytotoxicity ( cc 50: 144 μM), both in continuous cell lines and in CBLCs infected with HHV-6. From mechanistic experiments at the level of viral mRNA and protein expression, we learned that CMV423 targets an event following viral entry but preceding viral DNA replication. Its antiviral action was dependent on the cell line used, implying involvement of a cellular component. When compared to a panel of known protein kinase inhibitors, CMV423 was found to share anti-HHV-6 characteristics with herbimycin A, which affects tyrosine kinase activity through heat shock protein 90 (Hsp90) inhibition. We demonstrated that high concentrations of CMV423 have an inhibitory effect on the total cellular protein tyrosine kinase activity, and that CMV423 and herbimycin A, when combined, act synergistically against HHV-6. The activities of cyclin-dependent kinases, protein kinases A and C, and the HHV-6-encoded pU69 kinase were not affected. We, therefore, conclude that CMV423 exerts its activity against HHV-6 through inhibition of a cellular process that is critical at early stages of viral replication and that may affect protein tyrosine kinase activity.

The ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells.

Influenza virus enters cells by endocytosis, and requires the low pH of the late endosome for successful infection. Here, we investigated the requirements for sorting into the multivesicular body pathway of endocytosis. We show that treatment of host cells with the proteasome inhibitors MG132 and lactacystin directly affects the early stages of virus replication. Unlike other viruses, such as retroviruses, influenza virus budding was not affected. The requirement for proteasome function was not shared by two other pH-dependent viruses: Semliki Forest virus and vesicular stomatitis virus. With MG132 treatment, incoming influenza viruses were retained in endosomes that partially colocalized with mannose 6-phosphate receptor, but not with classical markers of early or late endosomes. Colocalization was also observed with Rme-1, which is part of the recycling pathway of endocytosis. In addition, influenza virus entry was dependent on the vacuolar protein sorting pathway, as over-expression of dominant-negative hVPS4 caused arrest of viruses in endosome-like populations that partially colocalized with the hVPS4 protein. Overall, we conclude that influenza virus selectively requires the ubiquitin/vacuolar protein sorting pathway for entry into host cells, and that it must communicate with a specific cellular machinery for intracellular sorting during the initial phase of virus infection.

A novel substance purified from Perilla frutescens Britton inhibits an early stage of HIV-1 replication without blocking viral adsorption.

Pf-gp6, a 6 kDa anti-degranulation glycoprotein purified from the extract of Perilla frutescens, was examined for its antiviral activity against HIV-1 and HIV-2 in vitro. HIV-1-induced cytopathic effect and proviral DNA synthesis were inhibited in the presence of Pf-gp6. The 50% inhibitory concentrations of Pf-gp6 for various HIV-1 strains, including clinical isolates and CCR5-using (R5) HIV-1, ranged between 1.3 and 71.0 microg/ml, depending on the combination of viral strain and host cell. Furthermore, Pf-gp6 did not directly inactivate infectious viral particles. A time-of-addition experiment revealed that Pf-gp6 lost its activity before zidovudine but after the CXCR-4 antagonist AMD3100 during the early stage of viral infection. Although the pinpoint target of Pf-gp6 remains to be elucidated, it may interfere with a step between viral entry and reverse transcription.

Inhibition of cyclin-dependent kinase 1 by purines and pyrrolo[2,3-d]pyrimidines does not correlate with antiviral activity.

We have previously shown that a series of nonnucleoside pyrrolo[2,3-d]pyrimidines selectively inhibit the replication of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). These compounds act at the immediate-early or early stage of HCMV replication and have antiviral properties somewhat similar to those of roscovitine and olomoucine, specific inhibitors of cyclin-dependent kinases (cdks). In the present study we examine the hypothesis that pyrrolo[2,3-d]pyrimidines exert their antiviral effects by inhibition of cellular cdks. Much higher concentrations of a panel of pyrrolo[2,3-d]pyrimidine nucleoside analogs with antiviral activity were required to inhibit recombinant cdk1/cyclin B compared to the submicromolar concentrations required to inhibit HCMV and HSV-1 replication. 4,6-Diamino-5-cyano-7-(2-phenylethyl)pyrrolo[2,3-d]pyrimidine (compound 1369) was the best inhibitor of cdk1 and cyclin B, with a 50% inhibitory concentration (IC(50); 14 microM) similar to that of roscovitine; it was competitive with respect to ATP (K(i) = 14 microM). The potency of compound 1369 against cdk1 and cyclin B was similar to its cytotoxicity (IC(50)s, 32 to 100 microM) but not its antiviral efficacy (IC(50)s, 0.02 to 0.3 microM). Thus, our results indicated the null hypothesis. In contrast, roscovitine was only weakly active against HSV-1 (IC(50), 38 microM) and HCMV (IC(50), 40 microM). These values were similar to those derived by cytotoxicity and cell growth inhibition assays, thereby suggesting that roscovitine is not a selective antiviral. Therefore, we propose that inhibition of cdk1 and cyclin B is not responsible for selective antiviral activity and that pyrrolo[2,3-d]pyrimidines constitute novel pharmacophores which compete with ATP to inhibit cdk1 and cyclin B.

DNA replication initiation is required for mid‐cell positioning of FtsZ rings in Caulobacter crescentus

Polymerization of the GTPase FtsZ to form a structure called the Z-ring is the earliest known step in bacterial cell division. Mid-cell Z-ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus. Z-ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells. New Z-rings can only form in the replicative stalked cell. Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z-ring assembly at different stages in the cell cycle. Z-ring assembly occurred even when early stages of DNA replication were blocked; however, the Z-rings were localized at a subpolar region of the cell. Z-rings only assembled at the proper mid-cell location if DNA replication had initiated. Z-ring assembly coincided with areas containing little or no DNA, and Z-rings could not form over an unreplicated chromosome. Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid-cell Z-ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid. These results indicate that the state of chromosome replication is a major determinant of Z-ring localization in Caulobacter.

ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase.

Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication-proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase-DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.

Advances in HIV molecular biology

Advances in HIV molecular biology

Effects of inactivation of the coat protein and movement genes of Tomato bushy stunt virus on early accumulation of genomic and subgenomic RNAs

Accumulation of RNA of Tomato bushy stunt virus (TBSV) was examined within the first few hours after infection of Nicotiana benthamiana protoplasts to determine the influence of the coat protein (CP), the movement-associated proteins P22 and P19 and RNA sequences at very early stages of replication. The results showed that P19 had no effect on early RNA replication, whereas the absence of CP and/or P22 expression delayed RNA accumulation only marginally. Removal of CP-coding sequences had no added negative effects, but when the deletion extended into the downstream p22 gene, it not only eliminated synthesis of subgenomic RNA2 but also delayed accumulation of genomic RNA by 10 h. At times beyond 20 h post-transfection, RNA accumulated to normal high levels for all mutants. This illustrates that TBSV RNA sequences that have negligible impact on overall RNA levels observed late in infection can actually have pronounced effects at very early stages.

Transgenically expressed T-Rep of tomato yellow leaf curl Sardinia virus acts as a trans-dominant-negative mutant, inhibiting viral transcription and replication.

We have previously shown that transgenic expression of a truncated C1 gene of Tomato yellow leaf curl Sardinia virus (TYLCSV), expressing the first 210 amino acids of the replication-associated protein (T-Rep) and potentially coexpressing the C4 protein, confers resistance to the homologous virus in Nicotiana benthamiana plants. In the present study we have investigated the role of T-Rep and C4 proteins in the resistance mechanism, analyzing changes in virus transcription and replication. Transgenic plants and protoplasts were challenged with TYLCSV and the related TYLCSV Murcia strain (TYLCSV-ES[1]). TYLCSV-resistant plants were susceptible to TYLCSV-ES[1]; moreover, TYLCSV but not TYLCSV-ES[1] replication was strongly inhibited in transgenic protoplasts as well as in wild-type (wt) protoplasts transiently expressing T-Rep but not the C4 protein. Viral circular single-stranded DNA (cssDNA) was usually undetectable in transgenically and transiently T-Rep-expressing protoplasts, while viral DNAs migrating more slowly than the cssDNA were observed. Biochemical studies showed that these DNAs were partial duplexes with the minus strand incomplete. Interestingly, similar viral DNA forms were also found at early stages of TYLCSV replication in wt N. benthamiana protoplasts. Transgenically expressed T-Rep repressed the transcription of the GUS reporter gene up to 300-fold when fused to the homologous (TYLCSV) but not to the heterologous (TYLCSV-ES[1]) C1 promoter. Similarly, transiently expressed T-Rep but not C4 protein strongly repressed GUS transcription when fused to the C1 promoter of TYLCSV. A model of T-Rep interference with TYLCSV transcription-replication is proposed.

Deoxythioguanosine triphosphate impairs HIV replication: a new mechanism for an old drug.

Inhibition of HIV-1 reverse transcriptase (RT) and HIV protease are effective mechanisms for anti-retroviral agents, and the combined use of mechanistically different medications has markedly improved the treatment of HIV infected patients. The active metabolite of mercaptopurine and thioguanine (TG), deoxythioguanosine triphosphate, was shown to be incorporated into DNA by the polymerase function of HIV-1 RT and then to abrogate RNA cleavage by HIV-1 RNaseH. Treatment of human lymphocyte cultures with thioguanine produced substantial inhibition of HIV replication (IC(50)=0.035 microM, IC(95)=15.4 microM), with minimal toxicity to host lymphocytes (<10% at 15.4 microM TG, P<0.000005). Furthermore, low concentrations of TG and zidovudine were synergistic in inhibiting HIV replication in human lymphocytes (synergy volume=19 microM(2)%), without additive cytotoxicity to host lymphocytes. Thus, thiopurines are novel anti-retroviral agents that alter the DNA-RNA substrates for HIV RNaseH, thereby abrogating early stages of HIV replication.

Cardiac Myosin and the TH1/TH2 Paradigm in Autoimmune Myocarditis

Myocarditis and dilated cardiomyopathy may represent acute and chronic stages of a progressive organ-specific autoimmune disease of the myocardium. 1-3 Myocarditis is an inflammatory disease of the heart that is characterized by a cellular infiltrate in the myocardium, and dilated cardiomyopathy is a chronic heart muscle disease characterized by ventricular hypertrophy. 1-4 Dilated cardiomyopathy is a primary cause of severe heart failure with subsequent transplantation or death within several years after diagnosis. 1,5 The direct result of cellular infiltration of the myocardium is necrosis and loss of myocytes leading to the development of contractile dysfunction and ventricular dilatation. The loss of myocytes and formation of scar tissue in the myocardium would lead to loss of contractile function and ventricular enlargement. In a small percentage of individuals, loss of myocyte function and development of dilated cardiomyopathy can result from mutations in or viral proteolytic digestion of dystrophin or dystrophin-associated glycoproteins that lead to cytoskeletal disruption and loss of overall contractile function in the heart. 4,6-8 However, the most common cause of myocarditis is viral infection. An exhaustive list of myocarditis-inducing agents including microbial pathogens and toxins is provided by Brown and O’Connell 2 in a review of myocarditis and dilated cardiomyopathy. Among the most common viral causes of human myocarditis are coxsackieviruses. 2,9-11 Mouse models of coxsackievirus-induced myocarditis were studied by Woodruff and colleagues 12-14 who demonstrated that lymphocytes from mouse models of myocarditis could destroy cardiomyocytes in culture. Gauntt and colleagues 15 also investigated the role of virulent myocarditic versus nonvirulent amyocarditic coxsackieviruses in mouse models of myocarditis. In their studies, there appeared to be several stages of disease in their mouse model. The first early stage of viral replication and cell lysis in the heart showed no evidence of heart failure or cellular inflammation, however, with the onset of specific immune responses, cellular infiltration of the myocardium was observed in susceptible mice. The cellular infiltrates in the myocardium were minimal to severe and led to the loss of myocytes by necrosis. The development of dilated cardiomyopathy is thought to represent the third and more chronic stage of heart disease that develops after the cellular inflammation. 16 It would follow that once the damage has been done to the cardiomyocyte function either by elimination of large numbers of myocytes because of inflammation, necrosis, and scarring or by disruption of the cytoskeleton so that the myocyte is dysfunctional, then anti-inflammatory therapy would not be effective. Anti-inflammatory therapies would only be efficacious during the time of inflammatory onset. In fact, immunosuppressive therapy was not effective in the Myocarditis Trial. 2 By the time myocarditis symptoms present, many cases may have already advanced to a stage in which necrosis and scarring have already led to ventricular dilatation and contractile dysfunction. For comparison, in rheumatic carditis, the valve is insidiously and permanently damaged by autoimmune attack after streptococcal pharyngitis. In rheumatic carditis, valve injury may not be evident until a heart murmur is perceived that reflects the scarred valve. In myocarditis, the end-stage of disease may represent a physical, structural defect as a result of autoimmune-mediated damage during the inflammatory stage of the disease.

Bovine immunodeficiency virus in experimentally infected rabbit: tropism for lymphoid and nonlymphoid tissues

The bovine immunodeficiency virus (BIV)/New Zealand ( Oryctolagus cuniculus) rabbit model was used to study events that underlie the early and chronic stages of viral replication, routes and time course of viral dissemination and the distribution of the virus in the lymphoid, nonlymphoid and mucosa associated tissues. The results indicated that BIV, a lentivirus with genetic relatedness to the HIV, induced changes of clinical (anorexia, weight loss, muscular wasting, diarrhea, hypoalgesia, torticollis), immunological (recurrent T- and B-cell dysfunctions) and histopathological (lymphadenopathy, splenomegaly) nature that closely parallels those described for cat (FIV), monkey (SIV) and human (HIV) lentiviral diseases. These findings showing that BIV induces both splenomegaly and lymphadenopathy syndromes with associated fatal immune dysfunctions and the ability of the virus to replicate productively at the mucosal surfaces in rabbits, emphasize the importance of the BIV/rabbit system as a good small-animal model for the study of retrovirus-induced AIDS and offers the opportunity to evaluate prophylactic and therapeutic anti-retroviral agents of relevance to HIV-1 as well as the opportunity to study mechanisms of drug resistance phenomena.

Lytic but not latent replication of epstein-barr virus is associated with PML and induces sequential release of nuclear domain 10 proteins.

Nuclear domains called ND10 (nuclear domain 10) are discrete nuclear protein aggregations characterized by a set of interferon-upregulated proteins including Sp100 and PML, where papova-, adeno-, and herpesviruses begin their transcription and DNA replication. Both the alpha- and betaherpesvirus subfamilies disrupt ND10 upon infection by dispersing and/or destroying ND10-associated proteins. We studied the effect of the gammaherpesvirus Epstein-Barr virus (EBV) on ND10 and its spatial distribution in the nucleus of cells during latency and lytic reactivation. In latently infected Burkitt's lymphoma, lymphoblastoid, and D98/HR1 cells, ND10 were intact, as judged by immunofluorescence localization of PML, Sp100, NDP55, and Daxx. Fluorescent in situ hybridization revealed no association between viral episomes and ND10 during latency, implying that the maintenance replication of EBV, which depends on host cell proliferation, occurs independent of ND10. As in mitosis, the EBV genomes were attached to interphase chromosomes, suggesting that they are unable to move freely within the interchromosomal space and thus unable to associate with the interchromosomally located ND10 or other nuclear domains. Upon lytic activation, ND10 became dispersed in cells expressing lytic proteins. Redistribution of ND10 proteins occurred sequentially at different stages of the lytic cycle, with Sp100, Daxx, and NDP55 dispersed before and PML dispersed after the onset of lytic replication. ND10 remnants were retained until the early stages of lytic replication, and replicating EBV genomes were frequently found beside this nuclear domain; the number of replication domains was usually lower than the average latent virus frequency. Thus, latency does not require or induce interaction of EBV with ND10 for transcription and replication, whereas lytic replication triggers dispersion of ND10 proteins and occurs in close association with PML aggregates. The required movement of chromosome-attached latent EBV episomes to ND10 after reactivation from latency might include physical release of the chromosome-bound episomes. Only episomes contacting ND10 after such a release might be able to begin lytic replication.

Disruption of PML-Associated Nuclear Bodies by IE1 Correlates with Efficient Early Stages of Viral Gene Expression and DNA Replication in Human Cytomegalovirus Infection

In human cytomegalovirus (HCMV) infection, both of the major immediate-early proteins IE1(IE68, UL123) and IE2(IE86, UL122) target to PML protein-associated nuclear bodies known as PODs or ND10 at very early times after infection. IE1 causes a redistribution of both PML and IE1 from the PODs into a nuclear diffuse form, whereas IE2 initially localizes adjacent to PODs but later associates with viral DNA replication compartments. The peripheries of PODs are also believed to be sites for initiation of both viral IE transcription and DNA replication. However, because IE1 is nonessential at high multiplicity of infection (m.o.i.) in HF cells, the exact role of these processes in viral infection has been enigmatic. Therefore, we investigated the effects of overexpression of PML in the presence or absence of IE1 on the intranuclear distribution of IE2 and formation of viral DNA replication compartments, as well as on the levels of delayed-early and late viral transcription and protein accumulation. Infection with wild-type HCMV(Towne) and the IE1-deleted derivative HCMV(CR208), which fails to disrupt PODs, was compared in a pair of related astrocytoma/glioblastoma cell lines, the U373-Neo control and a variant U373-PML that constitutively overexpresses PML(560) in much larger than normal PODs. IFA studies on the localization patterns for IE1, IE2, and PML showed that, although the numbers of IE2-positive cells were not significantly reduced in either the wild-type virus-infected U373-PML cell line or in ΔIE1-infected control cells, POD disruption by IE1 in wild-type virus infection was delayed by up to 6 h in U373-PML cells compared to control cells. Furthermore, there was considerable enhancement of IE2 colocalization with PODs in ΔIE1-infected U373-PML cells. Formation of viral DNA replication compartments in the U373-PML cell line was also greatly delayed, measured at fivefold lower after wild-type virus infection and 12-fold lower after infection with ΔIE1 than in the control cell line at 48 h at an m.o.i. of 1.0. The levels of representative early and late viral proteins detected by Western blotting were suppressed by fivefold and 22-fold at 24 and 72 h, respectively, in the U373-PML cell line, even with high m.o.i. wild-type HCMV infection. Decreased viral protein levels also occurred when control cells were infected with the ΔIE1 virus and these two effects were additive in the U373-PML cell line. Similarly, when U373-PML cells were infected with recombinant HCMV expressing an extragenic luciferase reporter gene under the control of viral early (Pol) or late (pp28) promoters, their transcriptional activation was reduced up to fivefold at both high and low m.o.i. compared to that of the control cells. Overall, these results suggest that POD disruption by IE1 and subsequent redistribution of both PML and IE1 at very early times after infection may play an important role in the efficient utilization of cellular transcription and replication machinery by HCMV and contribute to rapid progression of the HCMV lytic cycle.

Antiviral properties of a series of 1,6-naphthyridine and 7, 8-dihydroisoquinoline derivatives exhibiting potent activity against human cytomegalovirus.

A series of 1,6-naphthyridine (L. Chan, H. Jin, T. Stefanac, J. F. Lavallee, G. Falardeau, W. Wang, J. Bedard, S. May, and L. Yuen, J. Med. Chem. 42:3023-3025, 1999) and isoquinoline (L. Chan, H. Jin, T. Stefanac, W. Wang, J. F. Lavallee, J. Bedard, and S. May, Bioorg. Med. Chem. Lett. 9:2583-2586, 1999) analogues exhibiting a high level of anti-human cytomegalovirus (HCMV) activity were investigated in a series of studies aimed at better understanding the mechanism of action of some representatives of this class of compounds. In vitro antiviral profiling revealed that these compounds were active against a narrow spectrum of viruses, essentially the human herpesviruses and type 2 rhinovirus. In HCMV assays, a 39- to 223-fold lower 50% inhibitory concentration was obtained for compound A1 than for ganciclovir against strains AD 169 and Towne. In addition, ganciclovir, foscarnet, cidofovir, and BDCRB (2-bromo-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole)-resistant HCMV strains remained susceptible to 1,6-naphthyridines and 7, 8-dihydroisoquinolines tested in this study, supporting the view that a novel mechanism of action could be involved. Drug combination studies showed a small but significant synergistic antiviral effect between compound B2 and ganciclovir. Cytotoxicity profiling of representative compounds under various cell growth conditions indicated a generally similar cytotoxic effect, relative to ganciclovir, in log-phase growing cells. However, in stationary cells, a relatively higher level of toxicity was observed than that for control compound. Effect of time of drug addition showed that the anti-HCMV activity of compound A1, ganciclovir, and cidofovir was lost at approximately the same time (72 h postinfection), indicating that the compound was affecting events at the early and late stage of virus replication. This interpretation is also supported by reduction of de novo synthesis of pp65 tegument protein and lack of any effect of the compound on viral adsorption. A reduction of the HCMV enhancer-promoter-directed luciferase expression was also observed in a stably transfected cell line when compound A1 was present at relatively high concentrations.

124 The dendrimer BRI2999 inhibits the early stages of herpes simplex viruses replication : Y. Gong, C. Luscombe, D. Leung, P. Crosson, G. Holan, and S. L. Sacks, Viridae Clinical Sciences, Inc. and Starpharma Limited, Vancouver, BC, Canada

124 The dendrimer BRI2999 inhibits the early stages of herpes simplex viruses replication : Y. Gong, C. Luscombe, D. Leung, P. Crosson, G. Holan, and S. L. Sacks, Viridae Clinical Sciences, Inc. and Starpharma Limited, Vancouver, BC, Canada

Replication of lengthened Moloney murine leukemia virus genomes is impaired at multiple stages.

It has been assumed that RNA packaging constraints limit the size of retroviral genomes. This notion of a retroviral "headful" was tested by examining the ability of Moloney murine leukemia virus genomes lengthened by 4, 8, or 11 kb to participate in a single replication cycle. Overall, replication of these lengthened genomes was 5- to 10-fold less efficient than that of native-length genomes. When RNA expression and virion formation, RNA packaging, and early stages of replication were compared, long genomes were found to complete each step less efficiently than did normal-length genomes. To test whether short RNAs might facilitate the packaging of lengthy RNAs by heterodimerization, some experiments involved coexpression of a short packageable RNA. However, enhancement of neither long vector RNA packaging nor long vector DNA synthesis was observed in the presence of the short RNA. Most of the proviruses templated by 12 and 16 kb vectors appeared to be full length. Most products of a 19. 2-kb vector contained deletions, but some integrated proviruses were around twice the native genome length. These results demonstrate that lengthy retroviral genomes can be packaged and that genome length is not strictly limited at any individual replication step. These observations also suggest that the lengthy read-through RNAs postulated to be intermediates in retroviral transduction can be packaged directly without further processing.

The human autoantigen MCP1 is required during early stages of DNA replication.

Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen that is associated with condensed chromosomes throughout mitosis. During interphase, this antigen shows a speckle distribution in the nucleus, excluding the nucleolus. Additionally, MCP1 binds tightly to the scaffold/matrix component of nuclei and isolated chromosomes. In order to determine the in-vivo localization of the antigen, we have expressed MCP1 fused to EGFP in tissue culture cells. The results demonstrate that MCP1 is located in the nucleus during interphase and during mitosis associates tightly to condensed chromosomes. Furthermore, microinjection of specific antibody confirms these results. We have used a specific monoclonal antibody (mAb 402) against MCP1 to assess the function of this antigen during cell cycle progression. HeLa and Ptk-2 cells that were microinjected into the nucleus and/or cytoplasm at G1/S and very early S phase were not able to progress and complete DNA replication. However, injection of mAb 402 at mid or late S phase does not prevent completion of DNA replication and subsequent progression into mitosis. Microinjection of mAb 402 in Ptk-2 cells synchronized in mitosis did not interfere with progression of mitosis and cells divided. Our results suggest that MCP1 is required at the G1/S transition and during early S phase.

Fatty-acid substrate interactions with cyclo-oxygenases.

Prostaglandin endoperoxide H synthases 1 and -2 (PGHS-1 and -2) convert arachidonic acid and O2 (along with two reducing equivalents) to PGH2 — the committing step in the formation of prostanoids (Smith and DeWitt 1996; Smith et al. 1996). PGHS-1 is often referred to as the constitutive enzyme, whereas PGHS-2 is known as the inducible isoform. They differ from one another mainly with respect to their temporal patterns of expression. The reason for the existence of the two PGHS isozymes is still unknown. One possibility is that PGHS-2 is induced and then functions at relatively low fatty-acid substrate and hydroperoxide-activator concentrations to generate prostanoid products during early stages of cell replication or differentiation, whereas PGHS-1 forms products that are involved in “housekeeping” functions when circulating hormones act on cells acutely to cause the release of higher concentrations of arachidonate (Capdevila et al. 1995; Kulmacz and Wang 1995; Kulmacz 1998; So et al. 1998).

Developmental cholinotoxicants: nicotine and chlorpyrifos.

The stimulation of cholinergic receptors in target cells during a critical developmental period provides signals that influence cell replication and differentiation. Accordingly, environmental agents that promote cholinergic activity evoke neurodevelopmental damage because of the inappropriate timing or intensity of stimulation. Nicotine evokes mitotic arrest in brain cells possessing high concentrations of nicotinic cholinergic receptors. In addition, the cholinergic overstimulation programs the expression of genes that evoke apoptosis and delayed cell loss. Effects of cholinesterase inhibitors exhibit many similarities to those of nicotine. Chlorpyrifos administered to developing rats in doses that do not evoke signs of overt toxicity decreased DNA synthesis and caused shortfalls in cell numbers in brain regions enriched in cholinergic innervation. In embryo cultures, chlorpyrifos also evoked apoptosis during neurulation. However, chlorpyrifos also evokes noncholinergic disruption of cell development by interfering with cell signaling via adenylyl cyclase, leading to widespread disruption that is not limited to cholinergic systems. We have tested this hypothesis in vitro with PC12 cells, which lack the enzymes necessary to produce chlorpyrifos oxon, the metabolite that inhibits cholinesterase. Chlorpyrifos inhibited DNA synthesis in undifferentiated PC12 cells, which have relatively few cholinergic receptors. Furthermore, chlorpyrifos was more effective than nicotine and its effects were not blocked by cholinergic antagonists. When cells were allowed to differentiate in the presence of chlorpyrifos, cell replication was inhibited even more profoundly and cell acquisition was arrested. At higher concentrations, chlorpyrifos also inhibited neuritic outgrowth. Thus, chlorpyrifos elicits damage by both noncholinergic and cholinergic mechanisms extending from early stages of neural cell replication through late stages of axonogenesis and terminal differentiation. Accordingly, the window of developmental vulnerability to chlorpyrifos is likely to extend from the embryonic period into postnatal life.