Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

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Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an in situ protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.

mentioning

confidence: 99%

Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity

González-Magaldi

Postigo

Torre

et al. 2012

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“…For VSV protein detection by dot blot, infection medium samples were loaded onto a nitrocellulose membrane, which was blocked, incubated with a guinea pig hyperimmune serum against VSV Indiana and then with horseradishperoxidase (HRP)-labeled goat anti-guinea pig IgG, and subsequently developed using an ECL kit (Amersham). These antibodies were also used for Western blot detection (38).…”

mentioning

confidence: 99%

Inhibition of Enveloped Virus Infection of Cultured Cells by Valproic Acid

Vázquez-Calvo

Saiz

Sánchez‐Madrid

et al. 2011

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Valproic acid (VPA) is a short-chain fatty acid commonly used for treatment of neurological disorders. As VPA can interfere with cellular lipid metabolism, its effect on the infection of cultured cells by viruses of seven viral families relevant to human and animal health, including eight enveloped and four nonenveloped viruses, was analyzed. VPA drastically inhibited multiplication of all the enveloped viruses tested, including the zoonotic lymphocytic choriomeningitis virus and West Nile virus (WNV), while it did not affect infection by the nonenveloped viruses assayed. VPA reduced vesicular stomatitis virus infection yield without causing a major blockage of either viral RNA or protein synthesis. In contrast, VPA drastically abolished WNV RNA and protein synthesis, indicating that this drug can interfere the viral cycle at different steps of enveloped virus infection. Thus, VPA can contribute to an understanding of the crucial steps of viral maturation and to the development of future strategies against infections associated with enveloped viruses.

“…NTPDase6 belongs to a family of enzymes known to modulate a variety of important cellular responses by controlling extracellular nucleotide concentrations; these include blood clotting and neural signaling transmission (29,55,56; for a review, see reference 27). However, NTPDase6, unlike other members of the NTPDase family, exists as both a soluble form and a membrane-bound form, raising the possibility that this enzyme may be implicated in membrane rearrangement events that occur during normal FMDV replication (32,37,39). The cell clone containing an antisense NTPDase6 EST was further characterized.…”

Section: Resultsmentioning

confidence: 99%

“…In the case of FMDV, the replication complex recently has been described, and the involvement of the Golgi apparatus or the endoplasmic reticulum has been reported (32,37,39,41). Preliminary electromicroscopy analysis of NTPDase6-expressing cell clone 30 infected with FMDV at an MOI of 10 showed a reduced number of replication complexes at 4 hpi compared to infected LF-BK tTA cells.…”

Section: Discussionmentioning

confidence: 98%

Identification of Cellular Genes Affecting the Infectivity of Foot-and-Mouth Disease Virus

Piccone

Yin²,

Chang³

et al. 2009

Foot-and-mouth disease virus (FMDV) produces one of the most infectious of all livestock diseases, causing extensive economic loss in areas of breakout. Like other viral pathogens, FMDV recruits proteins encoded by host cell genes to accomplish the entry, replication, and release of infectious viral particles. To identify such host-encoded proteins, we employed an antisense RNA strategy and a lentivirus-based library containing approximately 40,000 human expressed sequence tags (ESTs) to randomly inactivate chromosomal genes in a bovine kidney cell line (LF-BK) that is highly susceptible to FMDV infection and then isolated clones that survived multiple rounds of exposure to the virus. Here, we report the identification of ESTs whose expression in antisense orientation limited host cell killing by FMDV and restricted viral propagation. The role of one such EST, that of ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase6; also known as CD39L2), a membrane-associated ectonucleoside triphosphate diphosphohydrolase that previously was not suspected of involvement in the propagation of viral pathogens and which we now show is required for normal synthesis of FMDV RNA and proteins, is described in this report.