Axel Karger

Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.

The Pseudorabies Virus UL11 Protein Is a Virion Component Involved in Secondary Envelopment in the Cytoplasm

ABSTRACT Homologs of the small tegument protein encoded by the UL11 gene of herpes simplex virus type 1 are conserved throughout all herpesvirus subfamilies. However, their function during viral replication has not yet been conclusively shown. Using a monospecific antiserum and an appropriate viral deletion and rescue mutant, we identified and functionally characterized the UL11 protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL11 encodes a protein with an apparent molecular mass of 10 to 13 kDa that is primarily detected at cytoplasmic membranes during viral replication. In the absence of the UL11 protein, viral titers were decreased approximately 10-fold and plaque sizes were reduced by 60% compared to wild-type virus. Intranuclear capsid maturation and nuclear egress resulting in translocation of DNA-containing capsids into the cytoplasm were not detectably affected. However, in the absence of the UL11 protein, intracytoplasmic membranes were distorted. Moreover, in PrV-ΔUL11-infected cells, capsids accumulated in the cytoplasm and were often found associated with tegument in aggregated structures such as had previously been demonstrated in cells infected with a PrV triple-mutant virus lacking glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Thus, the PrV UL11 protein, like glycoproteins E, I, and M, appears to be involved in secondary envelopment.

Composition of Pseudorabies Virus Particles Lacking Tegument Protein US3, UL47, or UL49 or Envelope Glycoprotein E

ABSTRACT Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.

Effects of Truncation of the Carboxy Terminus of Pseudorabies Virus Glycoprotein B on Infectivity

ABSTRACT Glycoproteins homologous to the type I membrane glycoprotein B (gB) of herpes simplex virus 1 (HSV-1) are the most highly conserved glycoproteins within the family Herpesviridae and are present in members of each herpesvirus subfamily. In the alphaherpesvirus pseudorabies virus (PrV), gB is required for entry into target cells and for direct viral cell-to-cell spread. These processes, though related, appear to be distinct, and thus it was interesting to analyze whether they require different functions of gB. To this end, we established cell lines stably expressing different carboxy-terminally truncated versions of PrV gB by deleting either (i) one predicted intracytoplasmic α-helical domain encompassing putative YQRL and dileucine internalization signals, (ii) two predicted intracytoplasmic α-helical domains, (iii) the complete intracytoplasmic domain, or (iv) the intracytoplasmic domain and the transmembrane anchor region. Confocal laser scanning microscopy showed that gB derivatives lacking at least the last 29 amino acids (aa) localize close to the plasma membrane, while the full-length protein accumulates in intracellular aggregations. Trans-complementation studies with a gB-deleted PrV (PrV-gB−) demonstrated that the 29-aa truncated form lacking the putative internalization signals and the C-terminal α-helical domain (gB-008) was efficiently incorporated into PrV-gB− virions and efficiently complemented infectivity and cell-to-cell spread. Moreover, gB-008 exhibited an enhanced fusogenic activity. In contrast, gB proteins lacking both α-helical domains (gB-007), the complete intracytoplasmic domain, or the intracytoplasmic domain and transmembrane anchor were only inefficiently or not at all incorporated into PrV-gB− virions and did not complement infectivity. However, gB-007 was able to mediate cell-to-cell spread of PrV-gB−. Similar phenotypes were observed when virus recombinants expressing gB-008 or gB-007, respectively, instead of wild-type gB were isolated and analyzed. Thus, our data show that internalization of gB is not required for gB incorporation into virions nor for its function in either entry or cell-to-cell spread. Moreover, they indicate different requirements for gB in these membrane fusion processes.

Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes: G and F proteins bind heparin.

Bovine respiratory syncytial virus (BRSV) encodes three transmembrane envelope glycoproteins, namely the small hydrophobic (SH) protein, the attachment glycoprotein (G) and the fusion glycoprotein (F). The BRSV reverse genetics system has been used to generate viable recombinant BRSV lacking either the G gene or the SH gene or both genes. The deletion mutants were fully competent for multicycle growth in cell culture, proving that, of the BRSV glycoprotein genes, the SH and G genes are non-essential. Virus morphogenesis was not impaired by either of the deletions. The deletion mutants were used to study the role of the F glycoprotein and the contributions of SH and G with respect to virus attachment. Attachment mediated by the F protein alone could be blocked by soluble heparin, but not by chondroitin sulphate. Heparin affinity chromatography revealed that both the BRSV G and F glycoproteins have heparin-binding activity, with the affinity of the F glycoprotein being significantly lower than that of G. Therefore, the roles of the BRSV glycoproteins in virus attachment and receptor binding have to be reconsidered.

Glycoprotein gH of pseudorabies virus is essential for penetration and propagation in cell culture and in the nervous system of mice.

Glycoprotein H (gH) of pseudorabies virus (PrV) is a structural component of the virion and forms a complex with another glycoprotein, gL. For a detailed analysis of the function of PrV gH, we isolated a gH-deficient mutant on trans-complementing gH-expressing cells after insertion of a beta-galactosidase expression cassette into a partially deleted gH gene. The absence of gH did not affect primary or secondary attachment of PrV but the mutant was not infectious. The defect in infectivity could partially be overcome by experimentally induced membrane fusion using PEG, which suggests that gH was necessary for fusion between virion and cellular membranes. After intranasal inoculation into mice, the LD50 of complemented gH- PrV was more than four orders of magnitude higher than that of wild-type PrV. Infection of the respiratory epithelium was much less efficient with complemented gH- PrV as compared with rescued PrV, reflecting the lack of direct cell-to-cell spread. Complemented gH- PrV was able to penetrate into a few trigeminal and sympathetic first order neurons accessible from the nasal cavity, whereas transneuronal transfer in the second order neurons was not observed. In summary, gH is essential for entry and cell-to-cell spread in cell culture, and for propagation in the nervous system of mice. This substantiates the hypothesis that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms.

Simple and rapid purification of alphaherpesviruses by chromatography on a cation exchange membrane

A simple and rapid method is described for the purification of two alphaherpesviruses, pseudorabies virus (PrV) and bovine herpesvirus 1, by chromatography on a cation exchange membrane. Cell culture supernatants were passed over a sulfonic-acid modified filter membrane and virions were eluted with a potassium chloride-containing buffer. Over 85% of the virus was eluted within a single fraction and specific infectivity of the resulting virus preparation was over 10-fold higher than that of sucrose gradient-purified virions. Cation exchange was also used for purification of PrV mutants deleted in several glycoproteins which grow in cell culture to titers 10- to 100-fold lower than those obtained by wildtype PrV. For PrV, the presence of non-essential glycoprotein gC, which mediates interaction of virions with cell surface heparin sulfate during attachment, was crucial for the successful purification by cation exchange.

Species determination and characterization of developmental stages of ticks by whole-animal matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of crude bacterial samples has been introduced as a very cost-efficient and rapid, yet highly informative tool to identify and classify bacteria. The potential of this approach to characterize whole animals, so far preferentially insects, is only evolving. Here, a simple protocol was developed to perform MALDI-MS analysis on extracts from whole ticks of 7 species and 4 developmental stages. Using commercially available software designed for the identification of bacteria, a reference database of spectra was constructed that allowed the species determination of ticks using larvae, nymphs, or adult individuals as starting material. Cluster analysis on the basis of MALDI mass spectra indicated that the primary determinant for the mass spectra was the species, followed by the developmental stages, which formed distinct clusters within the given species. With certain limitations, species identification was also possible using body parts and engorged animals. Spectra of developing Ixodes ricinus eggs showed dramatic changes with time, suggesting that, beyond its usefulness for species determination, MALDI-typing may have applications in developmental biology.

Post-Endoplasmic Reticulum Rescue of Unstable MHC Class I Requires Proprotein Convertase PC7

The function of the peptide-loading complex (PLC) is to facilitate loading of MHC class I (MHC I) molecules with antigenic peptides in the endoplasmic reticulum and to drive the selection of these ligands toward a set of high-affinity binders. When the PLC fails to perform properly, as frequently observed in virus-infected or tumor cells, structurally unstable MHC I peptide complexes are generated, which are prone to disintegrate instead of presenting Ags to cytotoxic T cells. In this study we show that a second quality control checkpoint dependent on the serine protease proprotein convertase 7 (PC7) can rescue unstable MHC I, whereas the related convertase furin is completely dispensable. Cells with a malfunctioning PLC and silenced for PC7 have substantially reduced MHC I surface levels caused by high instability and significantly delayed surface accumulation of these molecules. Instead of acquiring stability along the secretory route, MHC I appears to get largely routed to lysosomes for degradation in these cells. Moreover, mass spectrometry analysis provides evidence that lack of PLC quality control and/or loss of PC7 expression alters the MHC I-presented peptide profile. Finally, using exogenously applied peptide precursors, we show that liberation of MHC I epitopes may directly require PC7. We demonstrate for the first time an important function for PC7 in MHC I-mediated Ag presentation.

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

BackgroundBurkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms.ResultsA collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain.ConclusionsHandling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.