Mechanism of action of a pestivirus antiviral compound

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The glucose-derived iminosugar derivatives N-butyl-and N-nonyl-deoxynojirimycin (DNJ) have an antiviral effect against a broad spectrum of viruses including Bovine viral diarrhea virus (BVDV). For BVDV, this effect has been attributed to the reduction of viral secretion due to an impairment of viral morphogenesis caused by the ability of DNJ-based iminosugar derivatives to inhibit ER ␣-glucosidases (N. Zitzmann, A. S. Mehta, S. Carrouée, T. D. Butters, F. M. Platt, J. McCauley, B. S. Blumberg, R. A. Dwek, and T. M. Block, Proc. Natl. Acad. Sci. USA 96:11878-11882, 1999). Here we present the antiviral features of newly designed DNJ derivatives and report for the first time the antiviral activity of long-alkyl-chain derivatives of deoxygalactonojirimycin (DGJ), a class of iminosugars derived from galactose which does not inhibit endoplasmic reticulum (ER) ␣-glucosidases. We demonstrate the lack of correlation between the ability of long-alkyl-chain DNJ derivatives to inhibit ER ␣-glucosidases and their antiviral effect, ruling out ER ␣-glucosidase inhibition as the sole mechanism responsible. Using short-and long-alkyl-chain DNJ and DGJ derivatives, we investigated the mechanisms of action of these drugs. First, we excluded their potential action at the level of the replication, protein synthesis, and protein processing. Second, we demonstrated that DNJ derivatives cause both a reduction in viral secretion and a reduction in the infectivity of newly released viral particles. Long-alkyl-chain DGJ derivatives exert their antiviral effect solely via the production of viral particles with reduced infectivity. We demonstrate that long-alkyl-chain DNJ and DGJ derivatives induce an increase in the quantity of E2-E2 dimers accumulated within the ER. The subsequent enrichment of these homodimers in secreted virus particles correlates with their reduced infectivity.Iminosugar derivatives containing the glucose analogue deoxynojirimycin (DNJ) exert antiviral effects against viruses of different families, including Human immunodeficiency virus (HIV) (9-12, 16), Hepatitis B virus (HBV) (3,18,19), Woodchuck hepatitis virus (4), Bovine viral diarrhea virus (BVDV) (29), and Dengue virus (8). The antiviral effects were either proven (10, 11) or assumed to be associated with the inhibitory action of DNJ-containing iminosugar derivatives on enzymes in the endoplasmic reticulum (ER), ␣-glucosidases I and II. DNJ, a ring-nitrogen-containing and unmetabolizable glucose analogue, competitively binds to these enzymes and prevents them from performing the stepwise removal of three glucose residues attached to the N-linked glycans carried by newly synthesized polypeptides. This in turn prevents these polypeptides from interacting with the ER chaperones calnexin and calreticulin, which bind to monoglucosylated glycoproteins (14). Interaction with these ER chaperones is crucial for the correct folding of some but not all glycoproteins (13,17,22). Potentially all viruses which encode glycoproteins that depend on calnexin interaction for prope...

mentioning

confidence: 99%

Study of the Mechanism of Antiviral Action of Iminosugar Derivatives against Bovine Viral Diarrhea Virus

Durantel

Branza‐Nichita

Carrouée-Durantel

et al. 2001

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130

“…In the absence of an efficient cell culture system able to support hepatitis C virus (HCV) replication, BVDV has been adopted as a model and surrogate for HCV (1), as both viruses share molecular and virological features. HCV and BVDV are small, enveloped viruses with positive single-stranded RNA genomes of approximately 9,600 and 12,600 nucleotides, respectively.…”

mentioning

confidence: 99%

Antiviral Effect of N -Butyldeoxynojirimycin against Bovine Viral Diarrhea Virus Correlates with Misfolding of E2 Envelope Proteins and Impairment of Their Association into E1-E2 Heterodimers

Branza‐Nichita

Durantel

Carrouée-Durantel

et al. 2001

The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum ␣-glucosidase inhibitor, has an antiviral effect against bovine viral diarrhea virus (BVDV). In this report, we investigate the molecular mechanism of this inhibition by studying the folding pathway of BVDV envelope glycoproteins in the presence and absence of NB-DNJ. Our results show that, while the disulfide-dependent folding of E2 glycoprotein occurs rapidly (2.5 min), the folding of E1 occurs slowly (30 min). Both BVDV envelope glycoproteins associate rapidly with calnexin and dissociate with different kinetics. The release of E1 from the interaction with calnexin coincides with the beginning of E1 and E2 association into disulfide-linked heterodimers. In the presence of NB-DNJ, the interaction of E1 and E2 with calnexin is prevented, leading to misfolding of the envelope glycoproteins and inefficient formation of E1-E2 heterodimers. The degree of misfolding and the lack of association of E1 and E2 into disulfide-linked complexes in the presence of NB-DNJ correlate with the dose-dependent antiviral effect observed for this iminosugar.Bovine viral diarrhea virus (BVDV) is a pestivirus member of the Flaviviridae family, which also comprises the genera Flavivirus and Hepacivirus (24). In the absence of an efficient cell culture system able to support hepatitis C virus (HCV) replication, BVDV has been adopted as a model and surrogate for HCV (1), as both viruses share molecular and virological features. HCV and BVDV are small, enveloped viruses with positive single-stranded RNA genomes of approximately 9,600 and 12,600 nucleotides, respectively. The polypeptide precursor is transcribed from a single large open reading frame and subsequently co-and posttranslationally processed into structural and nonstructural proteins. Most BVDV and HCV proteins are functionally homologous. The envelope glycoproteins E1 and E2 interact either noncovalently (HCV) (7) or through disulfide bonds (BVDV) (26, 28) to form a dimer, which has been proposed as the functional complex present on the surfaces of mature virions.We have previously shown that endoplasmic reticulum (ER) ␣-glucosidase inhibitors containing the glucose analogue deoxynojirimycin (DNJ) as the head group have a strong antiviral effect on BVDV (32). Castanospermine (CST), another ␣-glucosidase inhibitor, was shown to cause misfolding of recombinant HCV E1 and E2 glycoproteins expressed in BHK-21 cells and reduce their association into native dimers (5). Similarly, CST affected the morphogenesis and assembly of Dengue virus (DENV), another member of the Flaviviridae family (6).The ER ␣-glucosidases perform the stepwise removal of the three glucose residues on N-linked glycans attached to nascent polypeptides. This removal enables folding intermediates to associate with the lectin-like ER chaperones calnexin and calreticulin, which interact with monoglucosylated glycoproteins and which retain incompletely folded polypeptides and oligomers in the ER (2, 23). However, not all cellular proteins are...

“…The C-terminal NS3 domain encodes an RNA helicase that is required for an unknown step(s) in RNA replication (16,17,51). NS5B, the viral RNAdependent RNA polymerase, has been expressed, purified, and extensively compared to the corresponding enzyme of HCV (1,24,57). Essentially nothing is known about the functions of p7, NS2, NS4B, or NS5A, although NS5A has been shown to be a serine phosphoprotein that is tightly associated with one or more cellular kinases (37).…”

mentioning

confidence: 99%

Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity

McMullan

Rice

2001

Self Cite

Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production.