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Our previous results have shown that the parainfluenza virus SV5 is a poor inducer of proinflammatory cytokines interleukin-8 (IL-8) and macrophage chemoattractant protein 1 (MCP-1). By contrast, an engineered P/V mutant rSV5-P/V-CPI- and a naturally-occurring variant WF-PIV (Wake Forest-Parainfluenza Virus) are both potent activators of IL-8 and MCP-1. In the present study, we addressed the question of why rSV5-WT is such a poor inducer of host cytokine responses relative to the two SV5 variants, and we used the CC chemokine RANTES as a measure of host responses. Time course experiments showed high-level secretion of IL-6 and RANTES following infections of human A549 lung epithelial cells with the P/V-CPI- mutant and WF-PIV. By contrast, SV5-WT induced very low cytokine responses, with the notable exception of moderate induction of RANTES. The mechanism of RANTES induction by the two SV5 variants shared common properties, since RANTES secretion from infected cells had similar kinetics, depended on virus replication, correlated with increased RANTES mRNA levels and promoter activation, and was reduced by inhibitors of the p38 MAPK, ERK, and PI3K pathways. Despite the similar mechanisms of RANTES induction, the two SV5 variants differed dramatically in their growth and gene expression kinetics. By comparison to the P/V mutant rSV5-P/V-CPI- which has accelerated viral gene expression, WF-PIV infection showed a prolonged delay in viral replication, and infected cells did not show high-level viral RNA and protein expression until approximately 12-24 hpi. Sequence analysis revealed that the N, P, V, and M genes from WF-PIV differed by 3, 8, 5, and 10 amino acids compared to rSV5-WT, respectively. Chimeric viruses harboring the WF-PIV P/V or M genes in the context of the other rSV5 genes had growth properties similar to rSV5-WT but had a RANTES-inducing phenotype similar to that of the bone fide WF-PIV virus. Our data indicate a role for both the P/V and the M gene products as determinants of RANTES induction in response to SV5 infection.

In most tissue culture cell lines tested, infection with the paramyxovirus simian virus 5 (SV5) results in very little cell death. To determine if SV5 could be used as a vector for controlled killing of tumor cells, a recombinant SV5 (rSV5-TK) was constructed to encode the herpes simplex virus thymidine kinase (TK) gene. MDBK cells infected with rSV5-TK showed a time-dependent loss of viability when infected cells were cultured in the presence of the prodrug acyclovir (ACV) or ganciclovir (GCV) while no significant toxicity was observed in the absence of prodrug. Cells infected with a control rSV5 expressing GFP and cultured with prodrug showed only a slight reduction in growth rate and little cell death. Time-lapse video microscopy of rSV5-TK-infected MDBK cells that were cultured in the presence of ACV showed an accumulation of cells with morphological effects characteristic of apoptotic cell death. An MDBK cell line persistently infected with rSV5-TK retained long-term expression of TK and sensitivity to prodrug-mediated cell killing that were comparable to those found in an acute infection. Titration experiments showed that the rSV5-TK plus GCV combination resulted in cell death for all mouse and human cell lines tested, although the kinetics and efficiency of cell death varied between cell types. Our results demonstrating controlled cell killing by a recombinant paramyxovirus support the use of negative-strand RNA viruses as therapeutic vectors for targeted killing of cancer cells.

Short CommunicationExchange of P/V genes between two noncytopathic simian virus 5 variants results in a recombinant virus that kills cells through death pathways that are sensitive to caspase inhibitors The paramyxovirus Simian virus 5 (SV5) is largely non-cytopathic in human epithelial and fibroblast cells. WF-PIV has been described previously as a naturally occurring SV5 variant that encodes P and V proteins differing from the wild-type (WT) SV5 proteins in eight and five amino acid positions, respectively. In this study, it is shown that WF-PIV is like WT SV5 by being largely non-cytopathic in A549 lung epithelial cells. However, substitution of the WF-PIV P/V gene into the background of WT SV5 resulted in a hybrid virus (P/V-WF) that induced apoptotic cell death not seen with either of the parental viruses. The kinetics of HeLa cell killing and induction of apoptosis by the P/V-WF chimera differed from those of the previously described P/V-CPI" chimera by being slower and less extensive. HeLa cell killing by the P/V-WF chimera was effectively reduced by inhibitors of caspase-9, but not of caspase-8. These results demonstrate that an exchange of P/V genes from two non-cytopathic SV5 variants can produce apoptosis-inducing chimeras, and that the role of the SV5 P/V gene products in limiting apoptosis can be dependent on expression in the context of a native viral genome.

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Naturally occurring anthocyanin indicators have been used for many years to introduce science students to acid–base concepts. A classroom activity has been developed that uses naturally occurring anthocyanins to monitor the pH changes associated with the fermentation of red cabbage to sauerkraut by lactic acid bacteria. Through the activity and related discussion, students are introduced to the scientific method, basic laboratory practices, acid–base chemistry, chemical and physical changes, fermentation, and microbiology. Although this activity and accompanying assignments are designed for students ages 6–12, the activity is adaptable to students of all ages and backgrounds.

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