Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus

ABSTRACT Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38°C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.

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Obtention of Porcine Aminopeptidase-N Transgenic Mice and Analysis of Their Susceptibility to Transmissible Gastroenteritis Virus

Advances in Experimental Medicine and Biology - Coronaviruses and Arteriviruses ◽

10.1007/978-1-4615-5331-1_7 ◽

1998 ◽

pp. 53-59 ◽

Cited By ~ 2

Author(s):

L. Benbacer ◽

M.-G. Stinackre ◽

H. Laude ◽

B. Delmas

Keyword(s):

Transgenic Mice ◽

Aminopeptidase N ◽

Transmissible Gastroenteritis Virus ◽

Gastroenteritis Virus ◽

Transmissible Gastroenteritis

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Papain-Like Protease 1 from Transmissible Gastroenteritis Virus: Crystal Structure and Enzymatic Activity toward Viral and Cellular Substrates

Journal of Virology ◽

10.1128/jvi.00898-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 10063-10073 ◽

Cited By ~ 36

Author(s):

Justyna A. Wojdyla ◽

Ioannis Manolaridis ◽

Puck B. van Kasteren ◽

Marjolein Kikkert ◽

Eric J. Snijder ◽

...

Keyword(s):

Host Cell ◽

Tertiary Structure ◽

Nonstructural Protein ◽

Cysteine Proteases ◽

Proteolytic Processing ◽

Transmissible Gastroenteritis Virus ◽

Cell Functions ◽

Gastroenteritis Virus ◽

Transmissible Gastroenteritis

ABSTRACT Coronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two precursor polyproteins of the viral replicase and are also thought to modulate host cell functions to facilitate infection. The papain-like protease 1 (PL1pro) domain is present in nonstructural protein 3 (nsp3) of alphacoronaviruses and subgroup 2a betacoronaviruses. It participates in the proteolytic processing of the N-terminal region of the replicase polyproteins in a manner that varies among different coronaviruses and remains poorly understood. Here we report the first structural and biochemical characterization of a purified coronavirus PL1pro domain, that of transmissible gastroenteritis virus (TGEV). Its tertiary structure is compared with that of severe acute respiratory syndrome (SARS) coronavirus PL2pro, a downstream paralog that is conserved in the nsp3's of all coronaviruses. We identify both conserved and unique structural features likely controlling the interaction of PL1pro with cofactors and substrates, including the tentative mapping of substrate pocket residues. The purified recombinant TGEV PL1pro was shown to cleave a peptide mimicking the cognate nsp2|nsp3 cleavage site. Like its PL2pro paralogs from several coronaviruses, TGEV PL1pro was also found to have deubiquitinating activity in an in vitro cleavage assay, implicating it in counteracting ubiquitin-regulated host cell pathways, likely including innate immune responses. In combination with the prior characterization of PL2pro from other alphacoronaviruses, e.g., human coronaviruses 229E and NL63, our results unequivocally establish that these viruses employ two PLpros with overlapping specificities toward both viral and cellular substrates.

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Molecular Characterization of Attenuated Vaccine Strains of Transmissible Gastroenteritis Virus

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879400600104 ◽

1994 ◽

Vol 6 (1) ◽

pp. 16-22 ◽

Cited By ~ 5

Author(s):

Karen B. Register ◽

Ronald D. Wesley

Keyword(s):

Transmissible Gastroenteritis Virus ◽

Coding Region ◽

S1 Nuclease ◽

Live Virus ◽

Gastroenteritis Virus ◽

Sensitive Tool ◽

Transmissible Gastroenteritis ◽

Genomic Cdna ◽

Cdna Fragments

Previous studies in our laboratory demonstrated that 2 attenuated strains of transmissible gastroenteritis virus (TGEV) contain deletions affecting messenger (m) RNAs 2, 3, or 4. In this report, we have compared mRNAs of four modified-live virus vaccines for TGEV with the virulent Miller PP3 isolate to determine whether any transcriptional patterns are shared among attenuated strains. Using northern blot analysis, all vaccine viruses expressed mRNAs indistinguishable in size from those of Miller PP3. However, using S1 nuclease protection experiments, alterations in the regions of the genome from which mRNAs 2 and 3 are transcribed were detected in 2 of the vaccine strains. When genomic cDNA fragments derived from the coding region for mRNA 2 were sequenced, a 6-nucleotide deletion, also found in the attenuated strain Purdue- 115, was discovered. The product of mRNA 2, a spike glycoprotein, was visualized by western blotting for each vaccine strain, and no profound differences in mobility were detected relative to Miller PP3. Alterations in the region of the genome from which mRNA 3 is transcribed appear to be identical or very similar to sequence alterations already described in this region for Purdue- 115, one of which is likely to alter the polypeptide product of mRNA 3. Insertions or deletions in mRNAs 2 or 3 may contribute to attenuation but are not a prerequisite for this phenotype. The S1 nuclease protection analysis is a sensitive tool for differentiating particular strains of TGEV.

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