S-phase-dependent enhancement of dengue virus 2 replication in mosquito cells, but not in human cells.

Dengue virus (DEN) is the most prevalent cause of arthropod-borne viral illness in humans. We determined the influence of cellular growth state on DEN type 2 (DEN2) replication in mosquito and human cells, based on the hypothesis that manipulation of cellular growth state will facilitate identification of viral and cellular determinants of productive infection. Comparison of density-arrested and cycling C6/36 Aedes albopictus cells infected with a low-passage DEN2 isolate revealed that cycling cells generated higher virus titers per cell. When C6/36 cells were stalled in S-phase via a thymidine (THY) block, titers of low-passage DEN2 isolates and a high-passage strain, 16681, were increased approximately 30-fold and 10-fold, respectively. Moreover, virus release was earlier in THY-treated cells than in asynchronously cycling cells. Adsorption, entry, genome uncoating, and translation were not responsible for increased titers of virus from S-phase C6/36 cells. In contrast to the 30-fold increase in virus titers, intracellular levels of viral RNA were increased approximately 2-fold, suggesting that the S-phase-responsive step is late in the DEN2 replication cycle. Analysis of viral RNA and protein released from the cells indicated that enhanced DEN2 assembly is largely responsible for increased virus titers produced during S-phase. In contrast to C6/36 cells, DEN2 titers from S-phase human hepatoma cells or primary human fibroblasts were not increased. These results demonstrate a differential response of DEN2 to the mosquito and human cell cycle and provide a framework for detailed studies into the mechanisms mediating virus assembly.

Dendritic cell precursors are permissive to dengue virus and human immunodeficiency virus infection.

CD14(+) interstitial cells reside beneath the epidermis of skin and mucosal tissue and may therefore play an important role in viral infections and the shaping of an antiviral immune response. However, in contrast to dendritic cells (DC) or blood monocytes, these antigen-presenting cells (APC) have not been well studied. We have previously described long-lived CD14(+) cells generated from CD34(+) hematopoietic progenitors, which may represent model cells for interstitial CD14(+) APC. Here, we show that these cells carry DC-SIGN and differentiate into immature DC in the presence of granulocyte-macrophage colony-stimulating factor. We have compared the CD14(+) cells and the DC derived from these cells with respect to dengue virus and human immunodeficiency virus type 1 (HIV-1) infection. Both cell types are permissive to dengue virus infection, but the CD14(+) cells secrete the anti-inflammatory cytokine interleukin 10 and no tumor necrosis factor alpha. Regarding HIV, the CD14(+) cells are permissive to HIV-1, release higher p24 levels than the derived DC, and more efficiently activate HIV Pol-specific CD8(+) memory T cells. The CD14(+) DC precursors infected with either virus retain their DC differentiation potential. The results suggest that interstitial CD14(+) APC may contribute to HIV-1 and dengue virus infection and the shaping of an antiviral immune response.

Mechanisms by which DNA tumor virus oncoproteins target the Rb family of pocket proteins.

Small DNA tumor viruses have evolved different mechanisms to abrogate the function of the retinoblastoma tumor suppressor (pRb). Studies of these viruses have been invaluable in uncovering the central role of the Rb family of pocket proteins in cell cycle control. While the molecular mechanisms by which the viral oncoproteins inactivate the Rb family are still being elucidated, it is clear that targeting of this family is required both for viral replication and for virus-induced transformation of mammalian cells. This review compares and contrasts the approaches DNA tumor viruses have evolved to antagonize Rb family members--ranging from relatively simple equilibrium dissociation of pRb from cellular pRb-binding factors to chaperone-mediated alterations in pocket protein stability and phosphorylation levels. The review will focus on the viral oncoproteins adenovirus E1A, human papillomavirus E7 and the large T antigens of several polyomaviruses. An understanding of these mechanisms may provide further insight into the regulation and functions of Rb family members as well as uncover new targets for the development of novel anti-viral agents, particularly against human papillomavirus, which is a significant cause of human cancer.

Inactivation of both the retinoblastoma tumor suppressor and p21 by the human papillomavirus type 16 E7 oncoprotein is necessary to inhibit cell cycle arrest in human epithelial cells.

The human papillomavirus (HPV) type 16 E7 oncoprotein must inactivate the retinoblastoma tumor suppressor (Rb) pathway to bypass G(1) arrest. However, E7 C-terminal mutants that were able to inactivate Rb were unable to bypass DNA damage-induced G(1) arrest and keratinocyte senescence, suggesting that the E7 C terminus may target additional G(1) regulators. The E7 C-terminal mutant proteins E7 CVQ68-70AAA and E7 Delta79-83 (deletion of positions 79 through 83) were further tested in several models of cell cycle arrest associated with elevated levels of p21. C-terminal mutations rendered E7 unable to induce S phase and endoreduplication in differentiated keratinocytes and rendered it less efficient in delaying senescence of human mammary epithelial cells. Interestingly, when cell cycle arrest was induced with a peptide form of p21, the E7 C-terminal mutants were deficient in overcoming arrest, whereas a mutant defective in Rb binding was competent in inhibiting G(1) arrest. These results suggest that the inactivation of both p21 and Rb by E7 contributes to subversion of cell cycle control in normal human epithelia but that neither p21 nor Rb inactivation alone is sufficient.