Molecular Biology of Pseudorabies Virus (PrV) – causative agent of Aujeszkys Disease
Pseudorabies Virus (PrV; also designated as Suid Herpesvirus 1 (SuHV-1) or Aujeszky's Disease Virus (ADV)) is the causative agent of Aujeszky's disease of pigs. Due to its broad host range, PrV can infect numerous mammals with infection invariably leading to death. Only pigs can survive a productive PrV infection dependent on the age of the animal and the virulence of the virus and are therefore considered as the natural host of PrV. Higher primates including humans and equids are resistant against infection (Mettenleiter, 2008).
PrV is grouped into the subfamily Alphaherpesvirinae of the family Herpesviridae which comprises also the human pathogens Herpes simplex virus type 1 und 2, the causative agents of Herpes labialis (cold sores) and genitalis, and varicella-zoster virus as well as important animal pathogens including the virus causing bovine infectious rhinotracheitis and pustular vulvovaginitis (bovine herpesvirus 1, BoHV-1), infectious laryngotracheitis of poultry (ILTV) or Marek's disease of poultry (MDV) (Roizman & Pellet, 2001).
Due to its fast lytic spread and broad host range, absence of pathogenicity for humans and the availability of the natural host, the pig, as experimental animal PrV is an excellent model to study the molecular mechanisms of
All herpesvirus particles (= virions) are morphologically identical comprising four different substructures (Fig. 1). The inner core contains the linear double-stranded DNA genome, which in PrV encompasses ca. 143.000 base pairs. The genome is enclosed in an icosahedral capsid, which together form the nucleocapsid. This, in turn, is surrounded by a proteinaceous tegument, corresponding to the matrix in RNA viruses. The envelope is derived from intracytoplasmic membranes and contains virally encoded, mostly glycosylated proteins.
PrV is amongst the functionally and structurally best characterized herpesviruses. The herpesvirus infection cycle is schematically depicted in Figure 2. We are especially interested in stages of virus entry (1-2); transport to and docking at the nuclear pore (3-5), nuclear egress of mature nucleocapsids (9-11) and secondary envelopment in the cytoplasm (11-13). Another major focus is the functional characterization of all ca. 70 gene products of PrV. The gene annotations summarize the known facts on their function. As neutrotropic herpesvirus PrV is also able to replicate and spread in neurons. The molecular mechanisms of this process are also subject of intensive investigations.
- Mettenleiter, T.C., B.G. Klupp, and H. Granzow. 2009. Herpesvirus assembly: an update. Virus Res. 143:222-234.
- Mettenleiter T C. 2008. Pseudorabies Virus. Encyclopedia of Virology, 5 vols. (B.W.J. Mahy and M.H.V. Van Regenmortel, Editors), pp. 341-351 Oxford: Elsevier.
Function of viral proteins during virus entry
Infection of host cells starts with attachment of virions to the plasma membrane. The first contact is mediated by glycoprotein (g)C and heparan sulphate-containing cellular surface proteins. This weak contact is strengthened by interaction of viral gD with more specific cellular receptors, i.e. nectins. In a not completely understood interplay between gB, gH and gL fusion of viral envelope and plasma membrane occurs releasing the nucleocapsid and the tegument into the cytoplasm. Fusion of membranes can be mimicked in vitro by transfection of glycoprotein-expressing plasmids. In contrast to HSV-1 (Turner et al., 1998) only gB, gH and gL, but not gD are required for PrV membrane fusion. Mutagenesis of the fusion partners gB, gH and gL will help to elucidate the molecular function of the different components.
- Klupp B.G., R. Nixdorf, and T.C. Mettenleiter. 2000. Pseudorabies virus glycoprotein M inhibits membrane fusion. J. Virol. 74: 6760-6768.
- Turner, A, B. Bruun, T. Minson, and H. Browne. 1998. Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J. Virol. 72:873-875.
Transport the incoming nucleocapsid along microtubules to the nuclear pore
After penetration the tegument and the nucleocapsid are released into the cytoplasm. The nucleocapsid is transported along microtubules to the nuclear pore. However, it is still unclear which viral proteins are necessary for interaction with cellular motor proteins during this transport. In immune electron microscopic studies (Granzow et al., 2005) we could show that the tegument proteins pUL36 and pUL37 as well as the viral kinase pUS3 remain at the capsid during its transport through the cytosol and are therefore prime candidates, together with the capsid proteins, to mediate the interaction with the cellular motors and/or adaptors. Using virus mutants expressing a green fluorescent protein (GFP) tagged capsid protein we investigate this transport in live cell imaging confocal microscopy. In vitro interaction studies, like yeast-two-hybrid analyses are applied to further elucidate the virus-cell interactions.
- Granzow, H., B.G. Klupp, and T.C. Mettenleiter. 2005. Entry of pseudorabies virus: an immunogold-labeling study. J. Virol. 79:3200-3205.
- Krautwald, M., C. Maresch, B.G. Klupp, W. Fuchs, and T.C. Mettenleiter. 2008. Deletion or green fluorescent protein tagging of the pUL35 capsid component of pseudorabies virus impairs virus replication in cell culture and neuroinvasion in mice. J. Gen. Virol. 89, 89:1346-1351.
- Krautwald, M., W. Fuchs, B.G. Klupp, and T.C. Mettenleiter. 2009. Translocation of incoming pseudorabies virus capsids to the cell nucleus is delayed in the absence of tegument protein pUL37. J. Virol. 83:3389-3396.
Nuclear egress of mature nucleocapsids
Herpesvirus capsids are assembled in the nucleus of infected cells. In this cellular compartment replication, cleavage and packaging of viral DNA also occurs. For further maturation the nucleocapsid has to leave the nucleus and gain access to the cytosol. This occurs by budding of nucleocapsids at the inner nuclear membrane, fission of the primary enveloped virion, and subsequent fusion of the primary envelope with the outer nuclear membrane thereby releasing the nucleocapsid into the cytosol. While the underlying cellular mechanism is still unclear the viral proteins involved, pUL34 and pUL31, have been identified and characterized. pUL34 constitutes a membrane protein which is targeted to the nuclear membrane. pUL31 is a soluble nuclear protein and is found diffusely distributed in the nucleus in absence of other viral proteins. Presence of its interaction partner pUL34 partially relocates pUL31 to the nuclear rim. Simultaneous expression of both proteins in the absence of virus infection results in formation of membranous vesicles from the inner nuclear membrane resembling primary envelopes indicating that these two viral proteins are sufficient for their formation. We are now interested in the underlying molecular mechanism of vesicle formation, fission and fusion. We already showed that viral glycoproteins necessary for fusion during entry are not required for this process (Klupp et al., 2008).
- Klupp B.G., H. Granzow, W. Fuchs, G.M. Keil, S. Finke, and T.C. Mettenleiter. 2007. Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins. Proc. Natl. Acad. Sci. U S A. 104: 7241-7246.
- Klupp, B.G., J. Altenschmidt, H. Granzow, W. Fuchs, and T.C. Mettenleiter. 2008. Glycoproteins required for entry are not necessary for egress of pseudorabies virus. J. Virol. 82:6299-309.
Virus assembly in the cytoplasm
Herpesvirus particles consist of more than 30 different proteins, which have to assemble into the four structural subunits of the virion (Fig. 1). While capsid assembly and genome packaging occur in the nucleus of the infected cell, the major portion of the tegument as well as the envelope are added in the cytosol. This process which had been designated as secondary envelopment, involves a complex pattern of protein-protein interactions (Fig. 6). Elucidation of these interactions and the role the different virion proteins play in this process are major topics of our studies.
- Mettenleiter, T.C., B.G. Klupp, and H. Granzow. 2009. Herpesvirus assembly: an update. Virus Res. 143:222-234. Review
Functional and structural characterization of PrV gene products
The PrV genome encodes at least 70 different proteins. While the function of several of them is well known, others have not yet been functionally characterized. To gain a complete picture of protein function in PrV, we sequentially mutate in an isogenic strain background viral genes and analyze their phenotype in a standardized cell culture system. Description of the identified genes and function of the corresponding gene products is provided in "PrV Gene Annotations".
- Klupp B.G., C.J. Hengartner, T.C. Mettenleiter, and L.W. Enquist. 2004. Complete, annotated sequence of the pseudorabies virus genome. J. Virol. 78: 424-440.
Molecular mechanism of neurotropism
As a neurotropic alphaherpesvirus PrV spreads transneuronally after infection of peripheral epithelial cells and nerve endings. The molecular details for this neuronal spread are largely unknown. Using virus mutants expressing a GFP-tagged capsid protein infection and transport in axons can by visualized and tracked by confocal microscopy. In combination with electron microscopy and targeted mutagenesis we are investigating the molecular principles of neuronal infection and spread.
PrV´s property to spread along neurons is increasingly being used for mapping neuronal networks. We genetically engineered an attenuated live vaccine strain of PrV (strain Bartha) to express marker proteins so that infected cells can easily be visualized by simple detection methods. Differentially labelled virus mutants allow the concomitant tracing of several neuronal circuits. Viral tracing is becoming a basic tool in neuroanatomy and neurobiology. Compared to HSV or rabies virus, PrV offers the advantage that it is not pathogenic for humans.
- Rothermel M., N. Schöbel, N. Damann, B.G. Klupp, T.C. Mettenleiter, H. Hatt, and C.H. Wetzel. 2007. Anterograde transsynaptic tracing in the murine somatosensory system using Pseudorabies virus (PrV): a "live-cell"-tracing tool for analysis of identified neurons in vitro. J. Neurovirol. 13: 579-585.
- Rothermel, M., D. Brunert, B.G. Klupp, M. Luebbert, T.C. Mettenleiter, and H. Hatt. 2009. Advanced tracing tools: functional neuronal expression of virally encoded fluorescent calcium indicator proteins. J. Neurovirol. 15:458-464.
- Maresch, C., H. Granzow, A. Negatsch, B.G. Klupp, W. Fuchs, J.P. Teifke and T.C. Mettenleiter. 2010. Ultrastructural analysis of virion formation and anterograde intraaxonal transport of the alphaherpesvirus pseudorabies virus in primary neurons. J. Virol., in press.