Study of vaccinia and cowpox viruses' replication in Rac1-N17 dominant-negative cells.

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

Activation of the PI3K/Akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication.

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to >/=90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.

Differential role played by the MEK/ERK/EGR-1 pathway in orthopoxviruses vaccinia and cowpox biology.

Appropriation of signalling pathways facilitates poxvirus replication. Poxviruses, as do most viruses, try to modify the host cell environment to achieve favourable replication conditions. In the present study, we show that the early growth response 1 gene (egr-1) is one of the host cell factors intensely modulated by the orthopoxviruses VV (vaccinia virus) and CPV (cowpox virus). These viruses stimulated the generation of both egr-1 mRNA and its gene product, throughout their entire replication cycles, via the requirement of MEK [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathway. We showed that, upon VV infection, EGR-1 translocates into the nucleus where it binds to the EBS (egr-1-binding site) positioned at the 5' region of EGR-1-regulated genes. In spite of both viruses belonging to the same genus, several lines of evidence, however, revealed a remarkable contrast between them as far as the roles played by the MEK/ERK/EGR-1 pathway in their biological cycles are concerned. Hence (i) the knocking-down of egr-1 by siRNA (small interfering RNA) proved that this transcription factor is of critical relevance for VV biology, since a decrease of about one log cycle in virus yield was verified, along with a small virus plaque phenotype, whereas the gene silencing did not have a detrimental effect on either CPV multiplication or viral plaque size; (ii) while both pharmacological and genetic inhibition of MEK/ERK resulted in a significant decrease in VV yield, both approaches had no impact on CPV multiplication; and (iii) CPV DNA replication was unaffected by pharmacological inhibition of MEK/ERK, but phosphorylation of MEK/ERK was dependent on CPV DNA replication, contrasting with a significant VV DNA inhibition and VV DNA replication-independence to maintain ERK1/2 phosphorylation, observed under the same conditions.