Molecular Biology of Pseudorabies Virus (PrV) – causative agent of Aujeszkys Disease
Pseudorabies Virus (PrV; also designated as Suid Alphaherpesvirus 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 pigs 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 1 and 2, the causative agents of Herpes labialis (cold sores) and genitalis, and varicella-zoster virus (Chickenpox/Shingles), 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 herpesvirus infection in vitro and in vivo.
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. A proteinaceous tegument, corresponding to the matrix in RNA viruses, surrounds the nucleocapsid. The envelope is derived from intracytoplasmic membranes and contains virally encoded, mostly glycosylated proteins (= glycoproteins).
PrV is amongst the functionally and structurally best-characterized herpesviruses. Figure 2 schematically depicts the herpesvirus infection cycle. We are especially interested in stages of virus entry (1-2), nuclear egress of mature nucleocapsids (9-11), as well as secondary envelopment in the cytoplasm (11-13). As neurotropic herpesvirus, PrV is also able to replicate and spread in neurons. The molecular mechanisms of this process are also subject of intensive investigations. In addition, function of specific viral proteins is analyzed in the animal, mainly in mouse and pig.
- 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 sulfate-containing cellular surface proteins. This weak contact is strengthened by interaction of viral gD with more specific cellular receptors. In a yet not completely understood interplay between gD, gB, and the gH/gL complex 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, only gB, gH and gL, but not gD are required for PrV membrane fusion. Although gB constitutes the bona fide fusion protein it ultimately depends on the presence of the gH/gL complex, whose function is yet unclear. While gH is absolutely essential, function of gL or the gL-binding domain in gH can be compensated by mutations in the other glycoproteins involved in fusion. Based on the recently solved crystal structures for gB and gH, the molecular mechanism of membrane fusion is now further analyzed by site-specific mutagenesis.
- Backovic M, DuBois RM, Cockburn JJ, Sharff AJ, Vaney MC, Granzow H, Klupp BG, Bricogne G, Mettenleiter TC, Rey FA. 2010. Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody. Proc Natl Acad Sci U S A. 107:22635-40.
- Schröter C, Vallbracht M, Altenschmidt J, Kargoll S, Fuchs W, Klupp BG, Mettenleiter TC. 2015. Mutations in Pseudorabies Virus Glycoproteins gB, gD, and gH Functionally Compensate for the Absence of gL. J. Virol. 90:2264-2272
- Vallbracht M, Schröter, C., Klupp, B. G. and Mettenleiter, T. C. 2017. Transient Transfection-based Fusion Assay for Viral Proteins. Bio-protocol 7.
- Vallbracht M, Rehwaldt S, Klupp BG, Mettenleiter TC, Fuchs W. 2017. Functional Relevance of the N-Terminal Domain of Pseudorabies Virus Envelope Glycoprotein H and Its Interaction with Glycoprotein L. J Virol. 91:. pii: e00061-17. doi: 10.1128/JVI.00061-17.
- Vallbracht M, Brun D, Tassinari M, Vaney MC, Pehau-Arnaudet G, Guardado-Calvo P, Haouz A, Klupp BG, Mettenleiter TC, Rey FA, Backovic M. 2017. Structure-function dissection of the Pseudorabies virus glycoprotein B fusion loops. J Virol. pii: JVI.01203-17.
- Vallbracht M, Fuchs W, Klupp BG, Mettenleiter TC. 2018. Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion. J Virol. 92. pii: e00376-18.
Nuclear egress of mature nucleocapsids
Herpesvirus capsids are assembled in the nucleus of infected cells. In this cellular compartment also replication, cleavage and packaging of viral DNA 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, membrane fission resulting in primary enveloped virions located in the perinuclear space, 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 We are now interested in uncovering the underlying molecular mechanism the viral proteins involved, pUL34 and pUL31, have been identified and characterized to some extent. pUL34 constitutes a membrane protein which is efficiently targeted to the nuclear membrane and forms, together with its interaction partner pUL31, the nuclear egress complex (NEC) at the inner nuclear membrane. Simultaneous expression of both proteins in the absence of virus infection results in formation of membranous vesicles from the inner nuclear membrane (Klupp et al., 2007) or even synthetic membranes (GUVs = giant unilamellar vesicles) (Lorenz et al., 2015) resembling primary envelopes indicating that these two viral proteins are sufficient for their formation. Cryo-electron microscopy (Hagen et al., 2016) as well crystallography (Zeev-Ben Mordehai et al., 2015) showed that oligomerization of the heterodimeric NEC results in membrane deformation and finally scission of vesicles.
We are now interested in uncovering the underlying molecular mechanism of fusion between the primary virion envelope and the outer nuclear membrane resulting in the release of the nucleocapsid into the cytosol. We already showed that viral glycoproteins necessary for fusion during entry are not required for this process (Klupp et al., 2008). Involvement of a putative cellular fusion mechanism is the focus of current research (Hellberg et al., 2016)
- 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.
- Mettenleiter TC, Müller F, Granzow H, Klupp BG. 2013. The way out: what we know and do not know about herpesvirus nuclear egress. Cell Microbiol. 15:170-178.
- Lorenz M, Vollmer B, Unsay JD, Klupp BG, García-Sáez AJ, Mettenleiter TC, Antonin W. 2015. A single herpesvirus protein can mediate vesicle formation in the nuclear envelope. J Biol Chem. 290:6962-6974.
- Zeev-Ben-Mordehai T, Weberruß M, Lorenz M, Cheleski J, Hellberg T, Whittle C, El Omari K, Vasishtan D, Dent KC, Harlos K, Franzke K, Hagen C, Klupp BG, Antonin W, Mettenleiter TC, Grünewald K. 2015. Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling. Cell Rep. 13:2645-2652.
- Hagen C, Dent KC, Zeev-Ben-Mordehai T, Grange M, Bosse JB, Whittle C, Klupp BG, Siebert CA, Vasishtan D, Bäuerlein FJ, Cheleski J, Werner S, Guttmann P, Rehbein S, Henzler K, Demmerle J, Adler B, Koszinowski U, Schermelleh L, Schneider G, Enquist LW, Plitzko JM, Mettenleiter TC, Grünewald K. 2015. Structural Basis of Vesicle Formation at the Inner Nuclear Membrane. Cell 163:1692-1701.
- Hellberg T, Paßvogel L, Schulz KS, Klupp BG, Mettenleiter TC. 2016. Nuclear Egress of Herpesviruses: The Prototypic Vesicular Nucleocytoplasmic Transport. Adv Virus Res.94:81-140.
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.
- 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 capsid protein with an autofluorescent tag 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.
Pathogenesis in the mouse model
Characterization of different deletion mutants showed that some proteins are nonessential for replication in cell culture but are important for spread in the animal. The mutants are characterized in-depth first in the mouse to understand the complex interactions between virus and host.
- Klopfleisch R, Klupp BG, Fuchs W, Kopp M, Teifke JP, Mettenleiter TC. 2006. Influence of pseudorabies virus proteins on neuroinvasion and neurovirulence in mice. J Virol. 80:5571-5576
- Mettenleiter TC, Ehlers B, Müller T, Yoon KJ, Teifke JP. 2012. Herpesviruses. In: Diseases of Swine (10th Edition), Edited by Zimmerman JJ, Karriker LA; Ramirez, A., Schwarz KJ, Stevenson GW. John Wiley & Sons.