Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions

Guirakhoo, Farshad; Heinz, Franz X.; Mandl, Christian W.; Holzmann, Heidemarie; Künz, Christian

doi:10.1099/0022-1317-72-6-1323

Cited by 170 publications

(168 citation statements)

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“…However, a number of other studies have reported the secretion of DENV RSPs from mammalian cells expressing the DENV prM and E genes, either transiently (Fonseca et al, 1994;Raviprakash et al, 2000) or stably (Konishi & Fujii, 2002;Konishi et al, 2003). Previous studies have determined that furin-mediated cleavage of prM was necessary to activate the full fusogenic potential of flaviviruses (Guirakhoo et al, 1991(Guirakhoo et al, , 1992Stadler et al, 1997). In our experiments, the levels of secreted prM were high relative to intracellular prM levels, both from virally infected C6/36 cells and from pSVprM-E-transfected COS cells, suggesting that prM processing was impaired.…”

Section: Discussionmentioning

confidence: 99%

“…The structure of immature DENV and YFV particles obtained by cryo-EM reconstruction revealed pronounced spikes, each comprising three prM/E heterodimers projecting from the virion surface (Zhang et al, 2003b). The prM proteins associate to cap each spike, covering a fusion peptide located in the E protein, a mechanism that is believed to prevent premature fusion during virion transport through the low-pH environment of the secretory pathway (Guirakhoo et al, 1991(Guirakhoo et al, , 1992Heinz et al, 1994). Prior to or during release of the infectious virus from cells, the host enzyme furin cleaves the prM protein (Stadler et al, 1997), a process that is believed to dissociate the prM/E heterodimer, resulting in rearrangement of the E proteins into tightly packed dimers on the surface of the mature virus particle .…”

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confidence: 99%

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Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly

Pryor

Azzola

Wright

et al. 2004

Journal of General Virology

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The mature flavivirus particle comprises a nucleocapsid core surrounded by a lipid bilayer containing the membrane (M) (derived from the precursor prM) and envelope (E) proteins. The formation of intracellular prM/E heterodimers occurs rapidly after translation and is believed to be important for the assembly and secretion of immature virus particles. In this study, the role of the His residue at position 39 in the M protein (M39) of dengue virus type 2 (DENV-2) in the virus life cycle was investigated. Mutations encoding basic (Arg), non-polar (Leu and Pro) and uncharged polar (Asn, Gln and Tyr) amino acids at M39 were introduced into a DENV-2 genomic-length cDNA clone and their effects on virus replication were examined. Substitution of the His residue with non-polar amino acids abolished virus replication, whereas substitution with basic or uncharged polar amino acids decreased virus replication moderately (~2 log 10 p.f.u. ml "1 decrease in viral titre for Arg and Asn) or severely (>3?5 log 10 p.f.u. ml "1 decrease in viral titre for Gln and Tyr). Selected mutations were introduced into a prM-E gene cassette and expressed transiently in COS cells to investigate whether the mutations impaired prM/E association or secretion. None of the mutations was found to disrupt the formation of intracellular prM/E heterodimers. However, the mutations that abolished virus replication prevented secretion of prM/E complexes. The results of this study pinpoint a critical residue in the M protein that potentially plays a role in viral morphogenesis, secretion and entry. INTRODUCTIONDengue virus (DENV) is transmitted by mosquitoes and causes the most important arthropod-borne viral disease of humans, with 50-100 million individuals infected annually worldwide (Gubler, 2002). The four serotypes of DENV (1-4) are members of the genus Flavivirus within the family Flaviviridae, along with a number of other medically important viruses that include Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV) and yellow fever virus (YFV) (Burke & Monath, 2001).The mature flavivirus particle contains three structural proteins, the capsid (C), envelope (E) and membrane (M) (derived from the precursor prM) proteins, plus a lipid bilayer and a single strand of infectious RNA. The structure of the mature DENV-2 particle has been determined by fitting the X-ray crystallographic structure of the TBEV E protein (Rey et al., 1995) into a 24 Å resolution cryoelectron microscopic (cryo-EM) reconstruction of the DENV-2 particle (Kuhn et al., 2002). The structure revealed that 90 E protein dimers lying parallel to the membrane in a 'herringbone' conformation and 180 M proteins form a tightly packed outer icosahedral protein shell surrounding a lipid bilayer, which encloses a disordered nucleocapsid consisting of multiple copies of the C protein surrounding the RNA genome (Kuhn et al., 2002).The flavivirus RNA genome is approximately 11 kb in size and encodes a large polyprotein with the gene order NH 2 -C-prM-E-N...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly

Pryor

Azzola

Wright

et al. 2004

Journal of General Virology

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“…Recent studies on selected flaviviruses suggest that the processing of preM to M is a pH-dependent event, which takes place in post-golgi acid vesicles. This proteolytic event may be necessary for fusion of virus with susceptible cells (43,94). Some flaviviruses may, however, be released from infected cells predominantly in the immature form (43).…”

Section: P R O C E S S I N G O F T H E F L a V I V I R U S P O L Y P mentioning

confidence: 99%

“…This proteolytic event may be necessary for fusion of virus with susceptible cells (43,94). Some flaviviruses may, however, be released from infected cells predominantly in the immature form (43). Additional factors may be involved in the cellular release of the immature flavivirions.…”

Section: P R O C E S S I N G O F T H E F L a V I V I R U S P O L Y P mentioning

confidence: 99%

Insect-transmitted vertebrate viruses: Flaviviridae

Ludwig

Iacono-Connors

1993

In Vitro Cell Dev Biol - Animal

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SUMMARYThe Flaviviridae include almost 70 viruses, nearly half of which have been associated with human disease. These viruses are among the most important arthropod-borne viruses worldwide and include dengue, yellow fever, and Japanese encephalitis viruses. Morbidity and mortality caused by these viruses vary, but collectively they account for millions of encephalitis, hemorrhagic fever, arthralgia, rash, and fever cases per year. Most of the members of this family are transmitted between vertebrate hosts by arthropod vectors, most commonly mosquitoes or ticks. Transmission cycles can be simple or complex depending on the hosts, vectors, the virus, and the environmental factors affecting both hosts and viruses. Replication of virus in invertebrate hosts does not seem to result in any significant pathology, which suggests a close evolutionary relationship between virus and vector. Another example of this relationship is the ability of these viruses to grow in invertebrate ceil culture, where replication usually results in a steady state, persistent infection, often without cytopathic effect. Yields of virus from insect cell culture vary but are generally similar to yields in vertebrate cells. Replication kinetics are comparable between insect and vertebrate ceil lines, despite differences in incubation temperature. Both vertebrate and insect cell culture systems continue to play a significant role in flavivirus isolation and the diagnosis of disease caused by these agents. Additionally, these culture systems permit the study of flavivirus attachment, penetration, replication, and release from cells and have been instrumental in the production and characterization of live-attenuated vaccines. Both vertebrate and insect cell culture systems will continue to play a significant role in basic and applied flavivirus research in the future.

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“…For a long time the role of prM antibodies during flavivirus infection was not understood, as numerous functional studies revealed that immature prM-containing flavivirus particles are non-infectious (Elshuber & Mandl, 2005;Stadler et al, 1997). Indeed, studies on TBEV, DENV and WNV have shown that cleavage of prM to M is required to activate the membrane-fusion machinery of the virus (Guirakhoo et al, 1991;Moesker et al, 2010;Stadler et al, 1997;Zybert et al, 2008). Interestingly, however, we recently observed that prM antibodies bind to fully immature DENV particles and facilitate cellular entry through Fc receptor and fusion in a furin-dependent manner (Rodenhuis- Zybert et al, 2010).…”

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confidence: 99%

prM-antibody renders immature West Nile virus infectious in vivo

Colpitts

Rodenhuis-Zybert

Moesker

et al. 2011

Journal of General Virology

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West Nile virus (WNV) is a member of the family Flaviviridae and is a neurotropic pathogen responsible for severe human disease. Flavivirus-infected cells release virus particles that contain variable numbers of precursor membrane (prM) protein molecules at the viral surface. Consequently, antibodies are produced against the prM protein. These antibodies have been shown to activate the infectious potential of fully immature flavivirus particles in vitro. Here, we provide in vivo proof that prM antibodies render immature WNV infectious. Infection with antibody-opsonized immature WNV particles caused disease and death of mice, and infectious WNV was found in the brains and sera.West Nile virus (WNV) is a mosquito-borne flavivirus and an emerging pathogen responsible for encephalitis and neurological disease in humans (Mackenzie et al., 2004). Members of the family Flaviviridae, which also include dengue virus (DENV) and tick-borne encephalitis (TBEV) virus, are enveloped, ssRNA viruses and contain three structural proteins: capsid (C), envelope (E) and membrane (M). In mature virions, E is organized as 90 homodimers that lie flat against the viral surface forming a 'smooth' protein shell (Kuhn et al., 2002).Flaviviruses infect cells via receptor-mediated endocytosis, which is mediated by the E glycoprotein (Lindenbach, 2001;Mukhopadhyay et al., 2005;van der Schaar et al., 2007). Following RNA replication, immature virions are formed containing precursor membrane protein (prM) in a heterodimeric configuration with E extending from the viral surface as 60 trimeric spikes (Kuhn et al., 2002). These newly formed particles mature during virus egress through the secretory pathway, whereafter cleavage of prM by a furin-like protease generates infectious particles (Stadler et al., 1997). This cleavage is known to be fairly inefficient as the prM content of virus particles released from DENV-and WNV-infected cells is approximately 30 % (Moesker et al., 2010;Zybert et al., 2008). Furthermore, the prM content is variable on a per-particle basis, as recent studies have shown that both fully immature as well as partially immature, or nearly mature, particles exist in wild-type ( , 1997). Indeed, studies on TBEV, DENV and WNV have shown that cleavage of prM to M is required to activate the membrane-fusion machinery of the virus (Guirakhoo et al., 1991; Moesker et al., 2010;Stadler et al., 1997;Zybert et al., 2008). Interestingly, however, we recently observed that prM antibodies bind to fully immature DENV particles and facilitate cellular entry through Fc receptor and fusion in a furin-dependent manner (Rodenhuis-Zybert et al., 2010). Furthermore, antibodies against prM have been shown to enhance wt DENV infection (Huang et al., 2006) and the levels of prM antibodies were found to be higher in patients with secondary infections compared with sera from primary DENV infections (Lai et al., 2008). Moreover, a recent study showed that human anti-prM antibodies fail to efficiently neutralize immature DENV infection in primary m...

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Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions

Cited by 170 publications

References 33 publications

Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly

Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly

Insect-transmitted vertebrate viruses: Flaviviridae

prM-antibody renders immature West Nile virus infectious in vivo

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