Alterations in NF-kappaB and RBP-Jkappa by arenavirus infection of macrophages in vitro and in vivo.

Pichinde virus is an arenavirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-kappaB and RBP-Jkappa, we compared the activities of NF-kappaB and RBP-Jkappa in P388D1 cells following infection with Pichinde virus. The attenuated P2 virus inhibited NF-kappaB activation and caused a shift in the size of the RBP-Jkappa complex. The virulent P18 virus showed less inhibition of NF-kappaB and failed to alter the size of the RBP-Jkappa complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-kappaB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jkappa. By contrast, P18 induced large amounts of the NF-kappaB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jkappa size. Taken together, these changes suggest that the attenuated viral strain induces an "activation" of macrophages, while the virulent form of the virus does not.

Inhibition of high affinity basic fibroblast growth factor binding by oligonucleotides.

Oligonucleotides can be used to inhibit the binding of basic fibroblast growth factor to cells. Though standard phosphodiester oligonucleotides show a slight inhibition of binding, the oligonucleotides with phosphorothioate internucleoside linkages have inhibition levels equivalent to that of the polyanion heparin. Variations in sequence of the oligonucleotides does lead to differences in the inhibitory action of the oligonucleotides. This inhibition of basic fibroblast growth factor by phosphorothioate oligonucleotides may account for much of the published data on inhibition of various genes by proposed antisense oligonucleotides and needs to be taken into account when considering the mechanism of action of oligonucleotides in biological systems.

Inhibition of herpes simplex virus in culture by oligonucleotides composed entirely of deoxyguanosine and thymidine.

Oligodeoxynucleotides (ODNs) composed entirely of deoxyguanosine and thymidine, but not specifically designed to act as antisense agents, were able to significantly inhibit herpes simplex virus growth in acute infection assay systems. The guanosine/thymidine (GT) ODNs which demonstrated this antiviral activity contained either natural phosphodiester (PO) or phosphorothioate (PS) modified internucleoside linkages. In all experiments, the antiviral activity of the ODNs was enhanced when the backbone was modified to contain the PS linkages. When present during the time of virus addition, the ODNs were able to block the adsorption of virus to Vero cells. In this assay the PS-containing ODNs had ID50 values of approximately 0.020 microM for HSV-2 and of 0.3 microM for HSV-1. When these same PS-containing ODNs were used against HSV-2 in single-cycle viral yield assays, designed to minimize the effects due to external blockage of virus, the ID50 values rose to 0.2 microM. Analysis of viral DNA obtained 14 h post-HSV-2 infections in the single-cycle assay, revealed a decrease in replicated viral DNA in cells treated with PS-ODNs. Analysis of viral mRNA obtained 4 h post-HSV-2 infection revealed, in cells treated with the PS-ODNs, a decrease in measurable HSV-2 alpha- and beta-mRNAs. Although the mechanism of action of the antiviral activity (beyond adsorption blocking) is not fully understood, the toxicity of these compounds was low, giving high therapeutic indices for the GT-rich PS-ODNs. The good therapeutic index of GT-ODNs make this a class of compounds which warrant investigation as therapeutic agents to be used against herpes viruses.

Inhibition of human cytomegalovirus in culture by alkenyl guanine analogs of the thiazolo[4,5-d]pyrimidine ring system.

A series of alkyl and alkenyl guanine analogs containing a thiazolo[4,5-d]pyrimidine ring system were prepared by reaction of the appropriate alkyl halide with the sodium salt of the heterocycle. In preliminary antiviral efficacy evaluations against laboratory strains of both human cytomegalovirus (HCMV) and herpes simplex virus types 1 and 2, it was determined that two of the compounds (T70072 and T01132) were more active and less toxic in stationary-phase cell monolayers than were the other derivatives tested. T01132 and T70072, which have 2-pentenyl and 3-methyl-2-butenyl moieties attached to position 3 of the 5-aminothiazolo[4,5-d]pyrimidine-2,7-dione, respectively, were then more extensively evaluated for anti-HCMV activity. The concentrations of T01132 and T70072 required to inhibit HCMV by 50% in plaque reduction assays were approximately 0.5 and 6.8 microM, respectively. These two compounds inhibited the growth of KB, MRC-5, or Vero cells at concentrations of 75 to 150 microM, depending upon the cell line. In bone marrow progenitor cells T01132 was slightly less toxic than ganciclovir (DHPG). The 50% inhibitory concentrations of T01132 against clinical isolates and DHPG-resistant strains of HCMV were approximately the same as those obtained for laboratory strains of HCMV (approximately 0.5 microM). When tested in combination with DHPG, the resultant antiviral activity was determined to be additive but not synergistic. Experiments performed using variations of the viral multiplicity of infection (MOI) demonstrated that T01132 was more active than DHPG at a low MOI (0.002 or 0.02). However, when a higher MOI (0.2 or 2.0) was used, DHPG was more efficacious than T01132. In experiments in which drug was added at various times post-viral infection, T01132 was most effective when added within the first 24 h post-HCMV infection while DHPG was able to protect cells in this assay system when added up to 48 h postinfection, indicating that T01132 is exerting its antiviral effect on events leading up to and possibly including viral DNA synthesis. The data presented in this report suggest that the antiviral activity of alkenyl-substituted thiazolopyrimidine derivatives may represent a mechanism of action against herpesviruses alternative to that of classical nucleoside analogs such as acyclovir or DHPG.

Expression of human preproapo AI and pre(delta pro)apoAI in a murine pituitary cell line (AtT-20). A comparison of their intracellular compartmentalization and lipid affiliation.

The role of the NH2-terminal propeptide of human apolipoprotein (apo) AI in intracellular transport and lipid-protein interactions was examined by transfecting a murine anterior pituitary cell line (AtT-20) with the human preproapo AI gene and a mutant gene lacking the 18-base pair segment of exon III encoding its hexapeptide prosegment. ProapoAI was not processed to the mature apolipoprotein either prior to or after export from these cells making this an attractive model system for directly assessing structure/activity relationships of its propeptide. ApoAI was sorted into a regulated pathway for protein export. The signal responsible for this trafficking pattern was not contained in the prosegment since both pro- and mature ApoAI exhibited a similar rate of secretion, a similar magnitude of stimulation of export by the secretagogue 8-bromo-cyclic AMP, and similar targeting to dense core granules. This sorting behavior, exhibited by a protein which is not normally targeted to dense core secretory granules in its cells of origin, raises questions about the domains and mechanisms involved in protein sorting into the regulated and constitutive pathways of AtT-20 cells. Density gradient ultracentrifugation, immunoaffinity chromatographic and electron microscopic analysis indicated that approximately 10% of proapoAI and approximately 10% apoAI appeared in AtT-20 culture media in the form of two discrete classes of nascent lipoproteins: a small 6-8 nm spherical particle and a larger discoidal particle. These particles had morphologies identical with those secreted by Hep G2 cells which normally synthesize apolipoproteins. Although these results do not resolve the issue of whether or not a fraction of apoAI can act as an acceptor of cellular lipid during its transport through the secretory pathway, the data do show that this functional capability for lipoprotein assembly is not obviously regulated by its prosegment.