The hepatitis B virus X protein (HBx) is thought to be implicated in the development of hepatocellular carcinoma, but its exact function remains controversial. Transgenic mice from PEX7 and AX16 lineages that express HBx in the liver under control of di erent viral regulatory elements develop no liver pathology (Billet et al., 1995). We have crossed these two mouse lineages with WHV/c-myc oncomice in which liver-speci®c expression of c-myc driven by woodchuck hepatitis virus (WHV) regulatory sequences causes liver cancer in all animals. The average tumor latency was shortened by 2 to 3 months in bitransgenic animals from all populations compared with simple c-myc transgenic littermates. At preneoplastic stages, adult bitransgenic mice showed four to ®vefold enhanced expression of the c-myc transgene, increased hepatocyte proliferation and more extensive liver lesions compared with simple WHV/c-myc transgenics. Thus in this model, HBx alone has no direct pathological e ect but it is shown to accelerate tumor development induced by c-myc. The data presented here ®rmly establish the oncogenic potential of HBx, apparently acting as a tumor promoter. This model o ers unique opportunities to investigate the mechanisms by which HBx trans-activates the expression of target genes and deregulates the hepatocyte growth control in vivo.

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.

Adenovirus fibers from most serotypes bind the D1 domain of coxsackie and adenovirus receptor (CAR), although the binding residues are not strictly conserved. To understand this further, we determined the crystal structures of canine adenovirus serotype 2 (CAV-2) and the human adenovirus serotype 37 (HAd37) in complex with human CAR D1 at 2.3 and 1.5 Å resolution, respectively. Structure comparison with the HAd12 fiber head-CAR D1 complex showed that the overall topology of the interaction is conserved but that the interfaces differ in number and identity of interacting residues, shape complementarity, and degree of conformational adaptation. Using surface plasmon resonance, we characterized the binding affinity to CAR D1 of wild type and mutant CAV-2 and HAd37 fiber heads. We found that CAV-2 has the highest affinity but fewest direct interactions, with the reverse being true for HAd37. Moreover, we found that conserved interactions can have a minor contribution, whereas serotype-specific interactions can be essential. These results are discussed in the light of virus evolution and design of adenovirus vectors for gene transfer. Adenoviruses (Ads)5 are nonenveloped icosahedral particles (70 -90 nm in diameter) containing double-stranded DNA genomes of 28 -42 kbp. They have been isolated from a wide variety of vertebrates, including mammals, birds, reptiles, amphibians, and fish (1). The ϳ50 human Ad serotypes (HAd) (divided into species A-F) lead to serotype-specific respiratory, ocular, and enteric infections that are usually self-limiting diseases in immunocompetent individuals. However, Ad infections are a significant cause of morbidity and mortality in newborns and immunosuppressed individuals (2). Notably, bone marrow and solid organ transplant patients who have severe lymphocytopenia are at the highest risk for Ad-induced disease (3). In addition, there are no drugs currently available that efficiently prevent or treat infections from all HAd serotypes.The major adenovirus capsid protein is the trimeric hexon, 240 of which form the 20 facets of the icosahedron. The 12 vertices are composed of the penton complex, which comprises the pentameric penton base and the externally projecting trimeric fiber. The fiber has a shaft of variable length with a terminal globular knob or head domain. In most cell types in vitro, the fiber head and penton base mediate attachment and entry, respectively. Crystal structures are available of the HAd2 and HAd5 hexons (4, 5); the HAd2 penton base (6); the fiber head domains of HAd2, HAd3, HAd5, HAd12, HAd37, HAd41 (short); and a construct of HAd2 fiber head plus part of its shaft (7-13) (reviewed in Ref. 14). In addition, there are recent high resolution cryoelectron microscopy reconstructions of the entire virus (15, 16). Based on similarities in overall capsid architecture and capsid protein structure, Adenoviridae probably share an ancient common ancestor with bacteriophages of the Tectiviridae family (17-19).The initial stages of adenoviral infection, cellular attachment, ...

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The contribution of the hepatitis B virus enhancers I and II in the regulation of the activity of the core and the X promoters was assessed in transgenic mice. Surprisingly, despite the presence of heterologous promoters linked 5 of the X gene, the transgene expression is mostly due to core promoter (Cp) activity present in the X coding sequence. Moreover, the restriction of Cp activity to hepatic tissue required the combined action of both enhancers I and II, whereas the proximity of these two enhancers was insufficient to confer tissue specificity on Xp activity. Furthermore, the liver-specific activity of the Cp was developmentally regulated in an enhancer I-independent manner.

The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P213. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA) domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion) and S (shell) domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination.

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