Nuclear localization of dengue virus nonstructural protein 5 through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection.

Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin alpha/beta and importin beta1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartite-type aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K(387)K(388)K(389)); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

Offspring produced from heterotopic ovarian allografts in male and female recipient mice.

Studies on human ovarian xenografts and mouse allografts indicate that the male hormonal milieu and exogenous gonadotrophin administration stimulate antral follicle growth. However, it is not known whether oocytes produced under these conditions are developmentally competent. The objective of our study was to evaluate the developmental competence of oocytes produced in heterotopic mouse ovarian grafts placed in male and female recipient mice. Gonadotrophins were 7.5 IU pregnant mare serum gonadotrophin (PMSG) alone or 7.5 IU PMSG and 7.5 IU human chorionic gonadotrophin or were not given prior to oocyte collection. The developmental competence of oocytes was assessed by performing in vitro fertilisation and embryo transfer to recipients. When no gonadotrophins were given the cleavage rate was similar for oocytes collected from ovarian grafts in male and female recipients. Gonadotrophin treatment significantly (P < 0.05) increased two-cell formation by oocytes grown in female graft recipients but not in male recipients. Implantation rates, fetal development and the birth of live young were unaffected by the sex of the graft recipient or gonadotrophin treatment. Live offspring were produced from oocytes collected from ovarian grafts in male and female recipients treated with or without gonadotrophins. In conclusion, this work has shown that the hormonal environment of male mice can support the growth of oocytes in ovarian allografts and that these oocytes can produce live offspring.