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Thomas C Mettenleiter

Publications and source records attributed to Thomas C Mettenleiter.

At least 19 recordsLinked to original sources
PubMed2006

Efficient incorporation of tegument proteins pUL46, pUL49, and pUS3 into pseudorabies virus particles depends on the presence of pUL21.

The mature virion of the alphaherpesvirus pseudorabies virus (PrV) contains a minimum of 31 structural proteins which are recruited into the virus particle by a network of protein-protein interactions which is only incompletely understood. We show here that deletion of the tegument protein pUL21 resulted in a drastic decrease in the incorporation of the pUL46, pUL49, and pUS3 tegument components into mature virions. Moreover, the attenuated PrV strain Bartha (PrV-Ba), which, among other defects, carries mutations in pUL21, also fails to package pUL46, pUL49, and pUS3 efficiently. By the reconstitution of wild-type pUL21 expression to PrV-Ba and the transfer of mutated PrV-Ba pUL21 into wild-type PrV, we demonstrate that this phenotype is due to the mutated pUL21.

Animals↗
PubMed2006

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Coronaviridae↗
PubMed2006

Alpha-herpesvirus glycoprotein D interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites.

Alpha-herpesviruses constitute closely related neurotropic viruses, including herpes simplex virus in man and pseudorabies virus (PRV) in pigs. Peripheral sensory neurons, such as trigeminal ganglion (TG) neurons, are predominant target cells for virus spread and lifelong latent infections. We report that in vitro infection of swine TG neurons with the homologous swine alpha-herpesvirus PRV results in the appearance of numerous synaptophysin-positive synaptic boutons (varicosities) along the axons. Nonneuronal cells that were juxtaposed to these varicosities became preferentially infected with PRV, suggesting that varicosities serve as axonal exit sites for the virus. Viral envelope glycoprotein D (gD) was found to be necessary and sufficient for the induction of varicosities. Inhibition of Cdc42 Rho GTPase and p38 mitogen-activated protein kinase signaling pathways strongly suppressed gD-induced varicosity formation. These data represent a novel aspect of the cell biology of alpha-herpesvirus infections of sensory neurons, demonstrating that virus attachment/entry is associated with signaling events and neuronal changes that may prepare efficient egress of progeny virus.

Animals↗
PubMed2006

Herpesvirus assembly: a tale of two membranes.

Herpes virions are amongst the most complex virus particles: they comprise in excess of thirty virally encoded proteins, and also contain cellular components. Capsid formation and the cleavage and encapsidation of replicated viral DNA occur in the nucleus and resemble similar processes in tailed dsDNA (double-stranded DNA) bacteriophages, which indicates they might have common ancestry. In contrast, final virion maturation takes place in the cytoplasm. Nucleocapsids gain access to this compartment by envelopment at the inner nuclear membrane, which involves the interaction between viral and cellular proteins in order to locally alter nuclear architecture. Fusion of the primary viral envelope with the outer nuclear membrane results in translocation of the nucleocapsid to the cytoplasm. Here, the majority of the tegument - a structure, composed of a multitude of different proteins, that links the capsid and the envelope - is added to nucleocapsids, which obtain their final envelope by budding into glycoprotein-containing Golgi-derived vesicles. Thus, herpesvirus morphogenesis proceeds in two different cellular compartments, involving different viral and cellular proteins.

Animals↗
PubMed2006

Chemosensory properties of murine nasal and cutaneous trigeminal neurons identified by viral tracing.

BACKGROUND: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that provides innervation of diverse tissues like the face skin, the conjunctiva of the eyes, blood vessels and the mucouse membranes of the oral and nasal cavities. Trigeminal perception encompasses thermosensation, touch, and pain. Trigeminal chemosensation from the nasal epithelia mainly evokes stinging, burning, or pungent sensations. In vitro characterization of trigeminal primary sensory neurons derives largely from analysis of complete neuronal populations prepared from sensory ganglia. Thus, functional properties of primary trigeminal afferents depending on the area of innervation remain largely unclear. RESULTS: We established a PrV based tracing technique to identify nasal and cutaneous trigeminal neurons in vitro. This approach allowed analysis and comparison of identified primary afferents by means of electrophysiological and imaging measurement techniques. Neurons were challenged with several agonists that were reported to exhibit specificity for known receptors, including TRP channels and purinergic receptors. In addition, TTX sensitivity of sodium currents and IB4 binding was investigated. Compared with cutaneous neurons, a larger fraction of nasal trigeminal neurons showed sensitivity for menthol and capsaicin. These findings pointed to TRPM8 and TRPV1 receptor protein expression largely in nasal neurons whereas for cutaneous neurons these receptors are present only in a smaller fraction. The majority of nasal neurons lacked P2X3 receptor-mediated currents but showed P2X2-mediated responses when stimulated with ATP. Interestingly, cutaneous neurons revealed largely TTX resistant sodium currents. A significantly higher fraction of nasal and cutaneous afferents showed IB4 binding when compared to randomly chosen trigeminal neurons. CONCLUSION: In conclusion, the usability of PrV mediated tracing of primary afferents was demonstrated. Using this technique it could be shown that compared with neurons innervating the skin nasal trigeminal neurons reveal pronounced chemosensitivity for TRPM8 and TRPV1 channel agonists and only partially meet properties typical for nociceptors. In contrast to P2X3 receptors, TRPM8 and TRPV1 receptors seem to be of pronounced physiological relevance for intranasal trigeminal sensation.

Afferent Pathways↗
PubMed2006

Newcastle disease virus expressing H5 hemagglutinin gene protects chickens against Newcastle disease and avian influenza.

Newcastle disease virus (NDV)-expressing avian influenza virus (AIV) hemagglutinin (HA) of subtype H5 was constructed by reverse genetics. A cloned full-length copy of the genome of the lentogenic NDV strain Clone 30 was used for insertion of the ORF encoding the HA of the highly pathogenic AIV isolate A/chicken/Italy/8/98 (H5N2) in the intergenic region between the NDV fusion and hemagglutinin-neuraminidase (HN) genes. Remarkably, two species of HA transcripts were detected in cells infected with the resultant NDVH5. In a second recombinant (NDVH5m), a NDV transcription termination signal-like sequence located within the HA ORF was eliminated by silent mutations. Consequently, NDVH5m produced 2.7-fold more full-length HA transcripts, expressed higher levels of HA, and also incorporated more HA protein into its envelope than NDVH5. NDVH5m stably expressed the modified HA gene for 10 egg passages and both recombinants were found innocuous after intracerebral inoculation of 1-day-old chickens. Immunization of chickens with NDVH5m induced NDV- and AIVH5-specific antibodies and protected chickens against clinical disease after challenge with a lethal dose of velogenic NDV or highly pathogenic AIV, respectively. Remarkably, shedding of influenza virus was not observed. Furthermore, immunization with NDVH5m permitted serological discrimination of vaccinated and AIV field virus-infected animals based on antibodies against the nucleoprotein of AIV. Therefore, recombinant NDVH5m is suitable as a bivalent vaccine against NDV and AIV and may be used as marker vaccine for the control of avian influenza.

Animals↗
PubMed2006

Neuronal pathways of viral invasion in mice after intranasal inoculation of pseudorabies virus PrV-9112C2 expressing bovine herpesvirus 1 glycoprotein B.

In contrast to wild-type Pseudorabies virus (PrV), which infects the central nervous system mainly via fibres of the trigeminal and autonomous nerves, the PrV mutant PrV-9112C2, deleted in glycoprotein B but expressing its bovine herpesvirus 1 (BHV-1) homologue, was shown to infect the swine central nervous system (CNS) via the olfactory route. In this study application of PrV-9112C2 into the nose of mice resulted in CNS infection as described for wild-type PrV. These findings indicate that gB((BHV-1))-dependent changes in PrV's capability to infect swine olfactory sensory neurons (OSNs) are not prominent in mice and give evidence for viral entry receptors present in swine but not mice OSNs.

Administration, Intranasal↗
PubMed2006

The UL4 gene of pseudorabies virus encodes a minor infected-cell protein that is dispensable for virus replication.

Although homologues of the open reading frame (ORF) UL4 of herpes simplex virus 1 (Human herpesvirus 1) have been found in the genomes of all hitherto-analysed alphaherpesviruses, little is known about their function. In a project to analyse systematically, in an isogenic and standardized assay system, the gene products of the alphaherpesvirus pseudorabies virus (PrV; Suid herpesvirus 1), the PrV UL4 gene product was identified using a monospecific rabbit antiserum prepared against a bacterial fusion protein. Western blot and immunofluorescence analyses revealed that the 146 codon UL4 ORF of PrV was translated into a nuclear 15 kDa protein which was detectable from 6 h after infection of rabbit kidney cells, but was not found in purified virus particles. For functional analysis, a UL4-negative virus recombinant (PrV-DeltaUL4F) was generated by mutagenesis of an infectious full-length clone of the PrV genome in E. coli. PrV-DeltaUL4F was replication-competent in rabbit kidney cells, and plaque formation was not affected by the mutation. However, maximum virus titres of PrV-DeltaUL4F were decreased about fivefold compared with wild-type PrV, and electron microscopy of infected cells demonstrated an impairment of release of mature virions. This growth defect of PrV-DeltaUL4F could be corrected completely by propagation in UL4-expressing cells. Correlating with the inconspicuous in vitro phenotype, neurovirulence of PrV-DeltaUL4F was also not affected significantly. Thus, UL4 encodes a non-structural protein of PrV that enhances virion formation but is not essential for PrV replication in vitro or in vivo.

Animals↗
PubMed2006

Pseudorabies virus particles lacking tegument proteins pUL11 or pUL16 incorporate less full-length pUL36 than wild-type virus, but specifically accumulate a pUL36 N-terminal fragment.

Proteins of the virion tegument of alphaherpesviruses are involved in protein-protein interactions, which play important roles in virus morphogenesis. Seven single-gene deletion mutants of Pseudorabies virus were analysed for alterations in the overall composition of the virion beyond the loss of the targeted protein. The UL36 protein (pUL36) was present in equal amounts in wild-type virions and mutants lacking pUL21, pUL49, pUL51, pUS3 or pUS8. Virions lacking pUL11 or pUL16 incorporated less full-length pUL36 than wild-type particles, but contained increased amounts of an N-terminal fragment of pUL36 that is present only in traces in wild-type virus and the other mutants.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

Identification of a 709-amino-acid internal nonessential region within the essential conserved tegument protein (p)UL36 of pseudorabies virus.

Tegument proteins homologous to the essential herpes simplex virus type 1 UL36 gene product (p)UL36 are conserved throughout the Herpesviridae and constitute the largest herpesvirus-encoded proteins. So far, only limited information is available on their functions, which include complex formation with the (p)UL37 homologs via an N-terminal domain and a deubiquitinating activity in the extreme N terminus. For further analysis we constructed deletion mutants lacking 437, 784, 926, 1,046, 1,217, or 1,557 amino acids (aa) from the C terminus. While none of them supported replication of a pseudorabies virus (PrV) UL36 deletion mutant, a mutant polypeptide with an internal deletion from aa 2087 to 2795, which comprises a proline/alanine-rich region, fully complemented the lethal replication defect. Thus, our data indicate that the extreme C terminus of (p)UL36 fulfills an essential role in PrV replication, while a large internal portion of the C-terminal half of the protein is dispensable for replication in cell culture.

Amino Acids↗
PubMed2006

Influence of pseudorabies virus proteins on neuroinvasion and neurovirulence in mice.

Neurotropism is a distinctive feature of members of the Alphaherpesvirinae. However, its molecular basis remains enigmatic. In the past, research has been focused mainly on the role of viral envelope proteins in modulating herpesvirus neuroinvasion and neurovirulence (T. C. Mettenleiter, Virus Res. 92:192-206, 2003). To further analyze the molecular requirements for neuroinvasion of the alphaherpesvirus pseudorabies virus (PrV), adult mice were infected intranasally with a set of single- or multiple-deletion mutants lacking the UL3, UL4, UL7, UL11, UL13, UL16, UL17, UL21, UL31, UL34, UL37, UL41, UL43, UL46, UL47, UL48, UL51, US3, US9, glycoprotein E (gE), gM, UL11/US9, UL11/UL16, UL16/UL21, UL11/UL16/UL21, UL11/gE, UL11/gM, UL43/gK, UL43/gM, or UL43/gK/gM genes. Neurovirulence was evaluated by measuring mean survival times compared to that after wild-type virus infection. Furthermore, by immunohistochemical detection of infected neurons, the kinetics of viral spread in the murine central nervous system was investigated.

Animals↗
PubMed2006

The capsid-associated UL25 protein of the alphaherpesvirus pseudorabies virus is nonessential for cleavage and encapsidation of genomic DNA but is required for nuclear egress of capsids.

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.

Animals↗
PubMed2006

Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E.

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.

Animals↗
PubMed2006

Enhancement of pathogenicity of Newcastle disease virus by alteration of specific amino acid residues in the surface glycoproteins F and HN.

Recombinant viruses were rescued after site-specific mutagenesis of a full-length clone of the lentogenic Newcastle disease virus (NDV) strain Clone 30. To assess the contribution of different amino acids to virulence, specific alterations were introduced into the fusion (F) protein and in the hemagglutinin-neuraminidase (HN) protein based on sequence comparison between NDV strains of different virulence. Modification of the proteolytic cleavage site in the F protein to a polybasic motif increased the intracerebral pathogenicity index (ICPI) from 0.0 to 1.28. Moreover, the additional exchange of amino acid 123 of the HN protein from tryptophan to cysteine in combination with alteration of amino acid 27 of the F protein from cysteine to arginine increased the ICPI to 1.5. The HN mutation visibly altered conformation of the protein, resulting in the formation of disulfide-linked HN dimers that may indicate that this HN conformation is beneficial for the virulent phenotype.

Amino Acid Substitution↗
PubMed2005

Tracing from fat tissue, liver, and pancreas: a neuroanatomical framework for the role of the brain in type 2 diabetes.

The hypothalamus uses hormones and the autonomic nervous system to balance energy fluxes in the body. Here we show that the autonomic nervous system has a distinct organization in different body compartments. The same neurons control intraabdominal organs (intraabdominal fat, liver, and pancreas), whereas sc adipose tissue located outside the abdominal compartment receives input from another set of autonomic neurons. This differentiation persists up to preautonomic neurons in the hypothalamus, including the biological clock, that have a distinct organization depending on the body compartment they command. Moreover, we demonstrate a neuronal feedback from adipose tissue that reaches the brainstem. We propose that this compartment-specific organization offers a neuroanatomical perspective for the regional malfunction of organs in type 2 diabetes, where increased insulin secretion by the pancreas and disturbed glucose metabolism in the liver coincide with an augmented metabolic activity of visceral compared with sc adipose tissue.

Adipose Tissue↗
PubMed2005

Intriguing interplay between viral proteins during herpesvirus assembly or: the herpesvirus assembly puzzle.

Herpes virions are complex particles that consist of more than 30 different virally encoded proteins. The molecular basis of how this complicated structure is assembled is only recently beginning to emerge. After replication in the host cell nucleus viral DNA is incorporated into preformed capsids which leave the nucleus by budding at the inner nuclear membrane resulting in the formation of primary enveloped virions in the perinuclear space. The primary envelope then fuses with the outer leaflet of the nuclear membrane, thereby releasing nucleocapsids into the cytoplasm. Final envelopment including the acquisition of more than 15 tegument and more than 10 envelope (glyco)proteins occurs by budding into Golgi-derived vesicles. Mature virions are released after fusion of the vesicle membrane with the plasma membrane of the cell. Thus, herpesvirus morphogenesis requires a sequence of envelopment--de-envelopment--re-envelopment processes which are distinct not only in the subcellular compartments in which they occur but also in the viral proteins involved. This review summarizes recent advances in our understanding of the complex protein-protein interactions involved in herpesvirus assembly and egress.

Animals↗
PubMed2005

CNS neurons with links to both mood-related cortex and sympathetic nervous system.

Cardiovascular changes occur during mental stress and in certain types of mood disorders. The neural basis for this phenomenon is unknown but it may be dependent on CNS neurons that provide branched projections to affective processing regions of the brain, such as the medial prefrontal cortex, and to the sympathetic outflow system. Because these putative neurons may be connected to these two target sites by chains of neurons, we performed double virus transneuronal tracing experiments and show here that a select subset of neurons in the medial preoptic nucleus (MPN), lateral hypothalamic area (LHA), and nucleus tractus solitarius (NTS) are co-linked to these two sites. Neurotensin MPN, orexin-containing LHA, and catecholamine NTS neurons were the major phenotypes involved in these projections. This novel class of neurons may coordinate cardiovascular changes seen in different emotional states.

Affect↗