Presence of Intact Hepatitis B Virions in Exosomes.

BACKGROUND & AIMS: Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome biogenesis. In light of this, it was studied whether intact virions wrapped in exosomes are released by HBV-producing cells. METHODS: Robust methods for efficient separation of exosomes from virions were established. Exosomes were subjected to limited detergent treatment for release of viral particles. Electron microscopy of immunogold labeled ultrathin sections of purified exosomes was performed for characterization of exosomal HBV. Exosome formation/release was affected by inhibitors or Crispr/Cas-mediated gene silencing. Infectivity/uptake of exosomal HBV was investigated in susceptible and non-susceptible cells. RESULTS: Exosomes could be isolated from supernatants of HBV-producing cells, which are characterized by the presence of exosomal and HBV markers. These exosomal fractions could be separated from the fractions containing free virions. Limited detergent treatment of exosomes causes stepwise release of intact HBV virions and naked capsids. Inhibition of exosome morphogenesis impairs the release of exosome-wrapped HBV. Electron microscopy confirmed the presence of intact virions in exosomes. Moreover, the presence of large hepatitis B virus surface antigen on the surface of exosomes derived from HBV expressing cells was observed, which conferred exosome-encapsulated HBV initiating infection in susceptible cells in a , large hepatitis B virus surface antigen/Na+-taurocholate co-transporting polypeptide-dependent manner. The uptake of exosomal HBV with low efficiency was also observed in non-permissive cells. CONCLUSION: These data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV.

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy.

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

Structure and assembly mechanism of virus-associated pyramids.

Viruses have developed intricate molecular machines to infect, replicate within and escape from their host cells. Perhaps one of the most intriguing of these mechanisms is the pyramidal egress structure that has evolved in archaeal viruses, such as SIRV2 or STIV1. The structure and mechanism of these virus-associated pyramids (VAPs) has been studied by cryo-electron tomography and complementary biochemical techniques, revealing that VAPs are formed by multiple copies of a virus-encoded 10-kDa protein (PVAP) that integrate into the cell membrane and assemble into hollow, sevenfold symmetric pyramids. In this process, growing VAPs puncture the protective surface layer and ultimately open to release newly replicated viral particles into the surrounding medium. PVAP has the striking capability to spontaneously integrate and self-assemble into VAPs in biological membranes of the archaea, bacteria and eukaryotes. This renders the VAP a universal membrane remodelling system. In this review, we provide an overview of the VAP structure and assembly mechanism and discuss the possible use of VAPs in nano-biotechnology.

Baculovirus Entry and Egress from Insect Cells.

Baculoviruses are large DNA viruses of insects that are highly pathogenic in many hosts. In the infection cycle, baculoviruses produce two types of virions. These virion phenotypes are physically and functionally distinct, and each serves a critical role in the biology of the virus. One phenotype, the occlusion-derived virus (ODV), is occluded within a crystallized protein that facilitates oral infection of the host. A large complex of at least nine ODV envelope proteins called per os infectivity factors are critically important for ODV infection of insect midgut epithelial cells. Viral egress from midgut cells is by budding to produce a second virus phenotype, the budded virus (BV). BV binds, enters, and replicates in most other tissues of the host insect. Cell recognition and entry by BV are mediated by a single major envelope glycoprotein: GP64 in some baculoviruses and F in others. Entry and egress by the two virion phenotypes occur by dramatically different mechanisms and reflect a life cycle in which ODV is specifically adapted for oral infection while BV mediates dissemination of the infection within the animal.

Archaeal viruses at the cell envelope: entry and egress.

The cell envelope represents the main line of host defense that viruses encounter on their way from one cell to another. The cytoplasmic membrane in general is a physical barrier that needs to be crossed both upon viral entry and exit. Therefore, viruses from the three domains of life employ a wide range of strategies for perforation of the cell membrane, each adapted to the cell surface environment of their host. Here, we review recent insights on entry and egress mechanisms of viruses infecting archaea. Due to the unique nature of the archaeal cell envelope, these particular viruses exhibit novel and unexpected mechanisms to traverse the cellular membrane.