Parvovirus-Host Cell Interactions

The Parvoviridae is a large family of small, nonenveloped, single-stranded DNA viruses that infect a large range of hosts from insects to man, causing infections going from asymptomatic to lethal. In addition to species pathogenic for humans and animals, the parvovirus family includes agents that have promising applications in gene and cancer therapy.

The entire parvovirus biology is influenced by their small size and the limited coding capacity of their genome. Lacking the accessory proteins for their own replication, parvoviruses follow a complex route from the plasma membrane to the nucleus where they can access the host replication machinery. This implies that the incoming particle has to overcome a series of obstacles before they can deliver the DNA into the nucleus. To do so, parvoviruses follow a sequence of complex structural rearrangements mediated by intracellular factors. These structural changes, which are tightly regulated in a spatial and temporal manner, promote interactions with cellular proteins at specific sites to assist the intracellular transport and nuclear targeting of the incoming particles.

Moreover, because they lack a transcriptionally competent duplex template and cannot induce the resting host cell to enter the S-phase, parvovirus infections are commonly restricted to actively dividing cells. But being small is not just about disadvantages. They can mimic cargo molecules and associate to organelles and transport proteins without disturbing cell functions and can enter the nucleus through the nuclear pore without the need to disassemble.

Simultaneous detection of viral genomes (red) and capsids (green) in infected UT7Epo cells by FISH.

Like all viruses, the first step of the parvovirus infection is the recognition of one or more specific receptor molecules in the plasma membrane of the cell. Parvoviruses use a wide variety of receptors, which explain their differences in tissue tropism and pathogenesis. The viral particles are internalized mostly via clathrin-dependent, receptor-mediated endocytosis and become sequestered in endocytic vesicles where they are structurally modified by the low endosomal pH. Following endosomal escape, parvoviruses exploit the cytoskeleton and motor proteins to move to the nuclear vicinity. Considering their small size, parvoviruses can potentially enter the nucleus through the nuclear pore without disassembly.

The understanding of how viruses enter the cell is a complex task, because viruses can use various pathways when infecting a cell, each pathway consists of a number of steps and each step consists of a series of specific and sequential interactions. Increasing the level of complexity is the fact that virus-host interactions are dynamic and that actually many of those interactions are irrelevant and not productive. Although the task is extensive, it will also be the benefits. A better knowledge of the complex process of parvovirus entry will help to better understand the tropism of the virus, the pathogenesis, and the disease resulting from the infection. It will also be beneficial to develop novel antiviral approaches or to improve and expand the application of parvovirus vectors in cancer and gene therapy.

Pathogen Safety

We collaborate with CSL Behring, a global leader in the protein biotherapeutics industry, to gain a better understanding of the mechanisms of virus inactivation and removal.

Virus inactivation

Viruses have developed structural solutions to combine a high stability in the extracellular milieu with an unstable structure inside the target cells. The final goal of such strategy is to ensure transmission without inactivation while allowing an efficient viral genome delivery in the appropriate cell compartment. We are particularly interested in understanding the mechanisms by which the highly stable parvovirus particles are destabilized and uncoated inside the cell. Besides a better understanding of the uncoating process, the information obtained is useful to understand the molecular mechanisms underlying virus inactivation, which is crucial to the safety of plasma-derived medicinal products.

Transmission electron microscopy of B19 virus. In the absence of divalent cations capsids are destablized and release their DNA (arrows)

Virus removal by filtration

Virus filtration uses a membrane barrier to selectively retain virus particles and represents an effective virus clearance technology commonly employed to improve virus safety in the manufacture of biotech- and plasma-derived medicinal products. We use confocal scanning microscopy to detect virus particles within filters. The method is used to examine the influence of factors such as virus size, capsid structure, electrostatic potential or operating conditions in virus retention.

Visualization of SV4 Pegasus membrane with MVM-542 (red), CPV-633 (blue) and recombinant MS2-488 (green)

Hepatitis E surrogate

Hepatitis E virus (HEV) belongs to the genus Hepevirus in the Hepeviridae family. HEV is a small (27-34 nm) non-enveloped virus with a positive-sense, single-stranded RNA genome (7,2 kb). HEV is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality in pregnant women is particularly high. Chronic infections can occur in transplant and other immune-compromised patients. Our understanding of HEV has changed enormously over the last years, from a waterborne infection causing outbreaks of acute hepatitis in developing countries to an infection of global distribution causing a wide range of syndromes. Therefore, HEV infection is an emerging concern in industrialized countries and in particular HEV has raised concern of transmission through blood donation. Transmission of HEV through blood donation must happen, but is currently unrecognized because blood samples are not tested for HEV and most infections are subclinical.

Efficient culture systems are essential to study HEV. Unfortunately, HEV has proven difficult to cultivate, which hampers its study. An alternative approach is the use of a surrogate virus. A HEV-related virus was isolated from trout and named cutthroat trout virus (CTV). The CTV genome has a comparable size to the HEV genome and is organized in a similar way. CTV is not pathogenic to humans and animals, thus avoiding safety concerns associated with HEV and other closely related agents such as rat and avian HEV.

Confocal immunofluorescent analysis of CTV-infected RTGill-W1 cells. Three-dimensional reconstruction.

We have successfully replicated CTV in cell culture from fish and developed and infectivity assay based on the detection and quantification of viral RNA. The study of CTV infection in cell culture will be a valuable instrument for the understanding of fundamental questions regarding hepevirus in general and HEV in particular. Different aspects of virus-host interactions, such as entry and uncoating, are currently under investigation. The physicochemical properties of CTV capsids and their inactivation profile is also an important aspect of our research. The fish HEV has substantial similarities to human HEV. Accordingly, the fast-replicating, non-pathogenic CTV represents a practical virus model for virus clearance studies.