Decades of research on the human papillomavirus oncogenes, E6 and E7, have given us huge amounts of data on their expression, functions and structures. We know much about the very many cellular proteins and pathways that they influence in one way or another. However, much of this information is quite discrete, referring to one activity examined under one condition. It is now time to join the dots to try to understand a larger picture: how, where and when do all these interactions occur... and why? Examining these questions will also show how many of the yet obscure cellular processes work together for cellular and tissue homeostasis in health and disease.
During the late stages of rotavirus morphogenesis the surface proteins VP4 and VP7 are assembled onto the previously structured double-layered virus particles to yield a triple-layered, mature infectious virus. The current model for the assembly of the outer capsid is that it occurs within the lumen of the endoplasmic reticulum. However, it has been shown that VP4 and infectious virus associate with lipid rafts, suggesting that the final assembly of the rotavirus spike protein VP4 involves a post-endoplasmic reticulum event. In this work, we found that the actin inhibitor jasplakinolide blocks the cell egress of rotavirus from non-polarized MA104 cells at early times of infection when there is still no evidence of cell lysis. These findings are in contrast with the traditional assumption that rotavirus is released from non-polarized cells by a non-specific mechanism when the cell integrity is lost. Inspection of the virus present in the extracellular media by density flotation gradients revealed that a fraction of the released virus is associated with low-density membranous structures. Furthermore, the intracellular localization of VP4, its interaction with lipid rafts and its targeting to the cell surface were shown to be prevented by jasplakinolide, implying a role for actin in these processes. Finally, the VP4 present at the plasma membrane was shown to be incorporated into the extracellular infectious virus, suggesting the existence of a novel pathway for the assembly of the rotavirus spike protein. Rotavirus is a major etiological agent of infantile acute severe diarrhea. It is a non-enveloped virus formed by three concentric layers of protein. The early stages of rotavirus replication, including cell attachment and entry, synthesis and translation of viral mRNAs, replication of the genomic dsRNA, and the assembly of double-layered viral particles, have been widely studied. However, the mechanism involved in the later stages of infection, i.e, viral particle maturation and cell exit, have been less characterized. It has been historically assumed that rotavirus exits non-polarized cells following cell lysis. In this work, we show that the virus exits cells by a non-lytic, actin-dependent mechanism and, most importantly, we describe that VP4, the spike protein of the virus, is present on the cell surface and is incorporated into mature, infectious virus, indicating a novel pathway for the assembly of this protein.
A B S T R A C TNumerous host factors are required for the efficient replication of rotavirus, including the activation and inactivation of several cell signaling pathways. One of the cellular structures that are reorganized during rotavirus infection is the actin cytoskeleton. In this work, we report that the dynamics of the actin microfilaments are important at different stages of the virus life cycle, specifically, during virus internalization and viral RNA synthesis at 6 h post-infection. Our results show that the actin-binding proteins alpha-actinin 4 and Diaph, as well as the Rho-family small GTPase Cdc42 are necessary for an efficient virus entry, while GTPase Rac1 is required for maximal viral RNA synthesis.
This is the first demonstration that E6AP can directly help stabilize the HPV E7 oncoprotein, in a manner similar to that observed with HPV E6. This redefines how E6 and E7 can cooperate and potentially modulate each other’s activity and further highlights the essential role played by E6AP in the viral life cycle and malignancy.
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