The Host Factor Early Growth Response Gene 1 (Egr-1) Is Regulated By Both Orthopoxviruses VV And CPV But Exerts Distinct Roles In Viral Biology
Brasil, B. S. A. F. 1,2, Oliveira, L. C.1,2, Ferreira, P. C. P. 2, Kroon, E. G.2, Bruna-Romero, O. & Bonjardim, C. A.1,2
Grupo de Transdução de Sinal1, Laboratório de Vírus2, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 - Belo Horizonte, Minas Gerais, Brazil. brunosafb@gmail.com
Host cell factors strongly influence the outcome of viral infections. Poxviruses, as most viruses, modify the host cell environment to achieve favorable replication conditions. Here we show that the early growth response gene (egr-1) is a host cell factors intensely modulated by the orthopoxviruses Vaccinia virus (VV) and Cowpox virus (CPV). The 82-KDa phosphoprotein early growth response factor (EGR-1) is equipped with a 3 C2H2 zinc fingers and belongs to a family of transcription factors that includes EGR-2-4 and NGFI-B. In this study we show that VV and CPV stimulate EGR-1 protein expression, from 1 hour post-infection (hpi) up to 12 hpi, via the MEK/ERK pathway. Insights on how VV and CPV benefits from the interaction with its host were obtained by one-step growth of VV and CPV performed with the egr-1 -/- MEFs. Unexpectadly, while a decrease of about one log in VV yield was verified, no difference in CPV yield was observed. This difference between VV and CPV was not observed on plaque size phenotype, since both viruses produced smaller plaques in egr-1 -/- MEFs. In order to detail the role played by egr-1 gene product on the biology of both VV and CPV viral DNA accumulation was performed during the course of infection in egr-1 -/- MEFs by dot-blot analysis. We also analyzed the expression of the late viral genes H3L and F13L in these cells by western blot and immunofluorescence microscopy, respectively. The results revealed a larger accumulation of VV DNA and of the late proteins H3 and F13 in egr-1 -/- MEFs during VV infection, while no differences were observed for CPV infection. In contrast, the absence of EGR-1 does not affect the expression of the VV timidine kinase, an early viral gene, tracing the requirement of EGR-1 to intermediate/late times during VV infection. Since orthopoxviruses use host factors for the expression of theirs intermediate/late genes, and as VV was shown to recruit host cell nuclear transcription factors to the cytoplasm, we analyzed the nucleus/cytoplasm compartimentalization of EGR-1 upon VV and CPV infection. While infection carried out with VV showed a nucleus/cytoplasm distruibution of EGR-1, CPV infection caused EGR-1 to concentrate at the nucleus only. The analysis were based on both subcellular fractionation and immunofluorescence microscopy. Taken together these results suggest that EGR-1 may act as an intermediate/late transcription factor during VV infection, but not during CPV infection.
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