The sensitivity of cell-associated dengue virus proteins to trypsin and the detection of trypsin-resistant fragments of the nonstructural glycoprotein NS1

Cauchi, Mark R.; Henchal, Erik A.; Wright, P.J.

doi:10.1016/0042-6822(91)90079-q

Cited by 22 publications

(18 citation statements)

References 28 publications

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“…The protease sensitivity of the major flaviviral replicase proteins NS5 and NS3 had suggested a cytoplasmic orientation for the membrane bound RC (27,28). In contrast, electron microscopic analysis carried out on KUNV-infected cells as well as DENVinfected cells displayed a dominant association of the RF as well as replicase proteins with membranous vesicle packets that were in turn enclosed by an outer membrane (15,21).…”

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confidence: 94%

Architecture of the Flaviviral Replication Complex

Uchil¹,

Satchidanandam

2003

Journal of Biological Chemistry

154

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Flavivirus infection causes extensive proliferation and reorganization of host cell membranes to form specialized structures called convoluted membranes/ paracrystalline arrays and vesicle packets (VP), the latter of which is believed to harbor flaviviral replication complexes. Using detergents and trypsin and micrococcal nuclease, we provide for the first time biochemical evidence for a double membrane compartment that encloses the replicative form (RF) RNA of the three pathogenic flaviviruses West Nile, Japanese encephalitis, and dengue viruses. The bounding membrane enclosing the VP was readily solubilized with nonionic detergents, rendering the catalytic amounts of enzymatically active protein component(s) of the replicase machinery partially sensitive to trypsin but allowing limited access for nucleases only to the vRNA and single-stranded tails of the replicative intermediate RNA. The RF co-sedimented at high speed from nonionic detergent extracts of virus-induced heavy membrane fractions along with the released individual inner membrane vesicles whose size of 75-100 nm as well as association with viral NS3 was revealed by immunoelectron microscopy. Viral RF remained nuclease-resistant even after ionic detergents solubilized the more refractory inner VP membrane. All of the viral RNA species became nuclease-sensitive following membrane disruption only upon prior trypsin treatment, suggesting that proteins coat the viral genomic RNA as well as RF within these membranous sites of flaviviral replication. These results collectively demonstrated that the newly formed viral genomic RNA associated with the VP are oriented outwards, while the RF is located inside the nonionic detergent-resistant vesicles.Although replication of flaviviruses has been an extensively studied aspect, the precise mechanism adopted and intricate interactions among the factors involved are yet to be unraveled. The flavivirus genome is a single-stranded positive-sense RNA ϳ11-kb long, lacking a 3Ј-poly(A) tail but with a 5Ј-type I cap. This genomic RNA upon uncoating utilizes the host translational machinery to direct synthesis of an ϳ3,400 amino acid long polyprotein that is processed co-translationally and posttranslationally by the host signalase and a virus-encoded proteinase to give three structural (capsid, premembrane/membrane, and envelope) and seven nonstructural (NS) 1 proteins (NS1-NS5) (1). The replication of the viral genome is thought to take place using putative complexes composed of viral as well as hypothetical host protein(s) (2). This process is initiated by the synthesis of a negative strand RNA complementary to the viral genomic plus strand, resulting in a double-stranded (ds) replicative form (RF). Asymmetric and semi-conservative synthesis of RNA (3, 4) from the RF results in formation of replicative intermediates (RI) with nascent single-stranded RNA tails that resolve upon completion of strand synthesis to generate one molecule of single-stranded RNA and a RF.Two decades of scientific effort have revealed the puta...

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confidence: 94%

Architecture of the Flaviviral Replication Complex

Uchil¹,

Satchidanandam

2003

Journal of Biological Chemistry

154

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“…Mice inoculated with an equivalent volume of PBS were used as negative controls. At different times postinjection (1,3,6, or 24 h), the spleen, liver, kidney, and brain were recovered from mice that received sNS1 and from the control animals, and tissues were embedded in paraffin for histology and immunohistochemistry studies. No obvious histopathological alterations or inflammatory infiltrates could be observed by standard microscopy.…”

Section: Hepatocytes Are Target Cells For the Den Sns1 Protein In Vivomentioning

confidence: 99%

The Secreted Form of Dengue Virus Nonstructural Protein NS1 Is Endocytosed by Hepatocytes and Accumulates in Late Endosomes: Implications for Viral Infectivity

Alcon-LePoder

Drouet

Roux

et al. 2005

J Virol

107

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The flavivirus nonstructural protein NS1 is expressed as three discrete species in infected mammalian cells: an intracellular, membrane-associated form essential for viral replication, a cell surface-associated form that may be involved in signal transduction, and a secreted form (sNS1), the biological properties of which remain elusive. To determine the distribution of the dengue virus (DEN) sNS1 protein in vivo, we have analyzed by immunohistological means the tissue tropism of purified DEN sNS1 injected intravenously into adult mice. The sNS1 protein was found predominantly associated with the liver, where hepatocytes appeared to represent a major target cell. We further showed that sNS1 could be efficiently endocytosed by human Huh7 and HepG2 hepatocytes in vitro. After its internalization, the protein was detected intracellularly for at least 48 h without being substantially degraded. Colocalization studies of sNS1 with markers of the endolysosomal compartments revealed that the protein was specifically targeted to lysobisphosphatidic acid-rich structures reminiscent of late endosomes, as confirmed by electron microscopy. Intracellular accumulation of sNS1 in Huh7 cells enhanced the fluid phase uptake of rhodamine-labeled dextran. Furthermore, preincubation of Huh7 cells with sNS1 increased dengue virus production after infection with the homologous strain of DEN-1 virus. Our results demonstrate that the accumulation of DEN sNS1 in the late endosomal compartment of hepatocytes potentializes subsequent dengue virus infection in vitro, raising the possibility that sNS1 may contribute to viral propagation in vivo.

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“…The WNV NS1 protein also presents as an interesting target antigen. Although the precise function of the NS1 protein during replication is unknown, investigations of a variety of flaviviruses, including West Nile virus, indicate the presence of intracellular, cell-associated, and secreted forms of the NS1 protein that can exist as monomeric, dimeric, and multimeric species (10,17,22,52,61,78,79). The NS1 protein and antibodies reactive to NS1 have been detected in vivo during both induced (9,20,24,28,29,63,64) and natural (16,21,34,76) flaviviral infections, and recent antigen capture studies have revealed high levels of dengue virus NS1 protein circulating in the serum of dengue virusinfected patients (1,45,81).…”

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confidence: 99%

NS1 Protein Secretion during the Acute Phase of West Nile Virus Infection

Macdonald

Tonry

Hall

et al. 2005

J Virol

108

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The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

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The sensitivity of cell-associated dengue virus proteins to trypsin and the detection of trypsin-resistant fragments of the nonstructural glycoprotein NS1

Cited by 22 publications

References 28 publications

Architecture of the Flaviviral Replication Complex

Architecture of the Flaviviral Replication Complex

The Secreted Form of Dengue Virus Nonstructural Protein NS1 Is Endocytosed by Hepatocytes and Accumulates in Late Endosomes: Implications for Viral Infectivity

NS1 Protein Secretion during the Acute Phase of West Nile Virus Infection

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