Flavivirus internalization is regulated by a size-dependent endocytic pathway

Hackett, Brent A.; Cherry, Sara

doi:10.1073/pnas.1720032115

Cited by 99 publications

(98 citation statements)

References 36 publications

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“…We validated that YFVΔSK/Nluc gene expression was neutralized by YFV-specific antibodies and was dependent on several known pathways of flavivirus entry, including clathrin-and dynamin-mediated endocytosis (10,30,(65)(66)(67), endosomal acidification (45,(66)(67)(68)(69), E protein-dependent fusion (31), and dependence on LY6E (32,33) and RPLP1 (34).…”

Section: Discussionmentioning

confidence: 77%

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A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

Ramanathan

Zhang

Douam

et al. 2020

mBio

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While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replicationdefective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin-and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

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Section: Discussionmentioning

confidence: 77%

“…Furthermore, YFV-mediated Nluc expression was reduced by genetic ablation of LY6E ( Fig. 2H), a host factor that facilitates internalization of flaviviruses and other viruses (32,33), or by RNAi-mediated knockdown of RPLP1 ( Fig. 2I), a ribosomal protein required for efficient flavivirus translation (34).…”

Section: Downloaded Frommentioning

confidence: 99%

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

Ramanathan

Zhang

Douam

et al. 2020

mBio

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“…Once attached to cells, flaviviruses are taken up by clathrindependent endocytic vesicles. While this same host machinery is involved in the internalization of multiple types of cellular cargo, recent studies identified host molecules required by flaviviruses to exploit the endocytic pathway for infectious entry including RNASEK, lymphocyte antigen locus 6 (LY6E) and microtubules [43][44][45] . Flavivirus membrane fusion occurs in the low pH compartments of the endosome and is catalysed by conformational changes in the E protein that involve the formation of E protein trimers, penetration of the highly conserved E-DII fusion loop into the adjacent host membranes and the folding of the E protein helical stem against the exterior surface of the newly formed E protein trimer 46 .…”

Section: E-diismentioning

confidence: 99%

The continued threat of emerging flaviviruses

Pierson

Diamond²

2020

Nat Microbiol

668

627

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F laviviruses are single-stranded RNA viruses vectored principally by arthropods that cause severe illnesses in humans. The extensive global spread and epidemic transmission of flaviviruses during the last seven decades has been remarkable. The mosquito-borne dengue viruses (DENV) infect an estimated 400 million humans each year; more than a quarter of the world's population lives in areas where DENV is now endemic 1 . By comparison, only sporadic DENV epidemics were documented before the Second World War 2 . The introductions of West Nile (WNV) and Zika (ZIKV) viruses into the Western Hemisphere was followed by rapid geographical spread, large numbers of human infections and considerable morbidity 3,4 . Ongoing yellow fever virus (YFV) transmission and its encroachment on urban environments, despite the existence of an effective vaccine, poses a serious public health challenge 5-7 . Other flaviviruses present ongoing health risks or are beginning to emerge in different parts of the world, including Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and Usutu virus (USUV).The epidemic potential of flaviviruses reflects many factors related to the unique characteristics of their insect vectors, the consequences of poorly planned urbanization that creates ideal arthropod breeding habitats, the geographical expansion of vectors, changing environmental conditions and extensive global travel 8,9 . Beyond arthropods and humans, flaviviruses are also known to infect a wide array of animal species and can be important veterinary pathogens that threaten economically important domesticated animals 10-14 . These vertebrate animal hosts may constitute important stable reservoirs and contribute to defining conditions that support the introduction of new viral species and transmission among humans 15 . The continued threat of flavivirus emergence and re-emergence highlights a need for a detailed fundamental understanding of the biology of these viruses, the immune responses that can contain them and the possible countermeasures that can blunt their impact on public health should new outbreaks occur. Flavivirus structure and replicationFlaviviruses are small (~50 nm) spherical virus particles that incorporate a single genomic RNA of positive-sense polarity encoding three structural and seven non-structural proteins (Fig. 1a). Our knowledge of the biology of flaviviruses has advanced considerably with the availability of high-resolution structures of viral structural proteins and of virions at different stages of the replication cycle or in complex with antibodies or host factors 16 . Crystal structures of the enzymatic non-structural proteins also have been solved, accelerating advances in an understanding of virus replication and pathogenesis [17][18][19] and enabling structure-guided drug discovery, as reviewed elsewhere 20 .Virion structure and morphogenesis. Flaviviruses are assembled using three viral structural proteins (C, prM and E), a host lipid envelope and the viral genomic RNA. The structure of ...

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“…The initial events of ZIKV entry have been identified [10][11][12]; but data regarding its fate thereafter are limited. Upon attachment to a permissive cell, ZIKV crosses the plasma membrane via clathrin-and mucolipin-2-dependent endocytosis, accompanied by formation of LY6E tubules [13,14]. The dependence of ZIKV on endocytosis has been confirmed in a variety of cell models [15].…”

mentioning

confidence: 89%

Zika virus: mapping and reprogramming the entry

Owczarek

Chykunova

Jassoy³

et al. 2019

Cell Commun Signal

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Background The flaviviridae family comprises single-stranded RNA viruses that enter cells via clathrin-mediated pH-dependent endocytosis. Although the initial events of the virus entry have been already identified, data regarding intracellular virus trafficking and delivery to the replication site are limited. The purpose of this study was to map the transport route of Zika virus and to identify the fusion site within the endosomal compartment. Methods Tracking of viral particles in the cell was carried out with confocal microscopy. Immunostaining of two structural proteins of Zika virus enabled precise mapping of the route of the ribonucleocapsid and the envelope and, consequently, mapping the fusion site in the endosomal compartment. The results were verified using RNAi silencing and chemical inhibitors. Results After endocytic internalization, Zika virus is trafficked through the endosomal compartment to fuse in late endosomes. Inhibition of endosome acidification using bafilomycin A1 hampers the infection, as the fusion is inhibited; instead, the virus is transported to late compartments where it undergoes proteolytic degradation. The degradation products are ejected from the cell via slow recycling vesicles. Surprisingly, NH 4 Cl, which is also believed to block endosome acidification, shows a very different mode of action. In the presence of this basic compound, the endocytic hub is reprogrammed. Zika virus-containing vesicles never reach the late stage, but are rapidly trafficked to the plasma membrane via a fast recycling pathway after the clathrin-mediated endocytosis. Further, we also noted that, similarly as other members of the flaviviridae family, Zika virus undergoes furin- or furin-like-dependent activation during late steps of infection, while serine or cysteine proteases are not required for Zika virus maturation or entry. Conclusions Zika virus fusion occurs in late endosomes and is pH-dependent. These results broaden our understanding of Zika virus intracellular trafficking and may in future allow for development of novel treatment strategies. Further, we identified a novel mode of action for agents commonly used in studies of virus entry. Schematic representation of differences in ZIKV trafficking in the presence of Baf A1 and NH 4 Cl Electronic supplementary material The online version of this article (10.1186/s12964-019-0349-z) contains supplementary material, which is available to authorized users.

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Flavivirus internalization is regulated by a size-dependent endocytic pathway

Cited by 99 publications

References 36 publications

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

The continued threat of emerging flaviviruses

Zika virus: mapping and reprogramming the entry

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