Fluorescence photobleaching recovery measurements reveal differences in envelopment of sindbis and vesicular stomatitis viruses

Johnson, David C.; Schlesinger, Milton J.; Elson, Elliot L.

doi:10.1016/0092-8674(81)90137-9

“…Previous FRAP studies with labeled antibodies showed that SINV envelope proteins on the PM are immobilized early in infection and suggested that this effect is mediated primarily by interactions between the envelope proteins, rather than E2-Cp interactions (54). Our FRAP experiments confirmed that the GFP-E2 protein was strikingly immobile on the PM of WT virusinfected cells.…”

Section: Discussionsupporting

confidence: 77%

Imaging the Alphavirus Exit Pathway

Martínez¹,

Snapp²,

Perumal

³

et al. 2015

Microsc Microanal

1

0

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Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ϳ2 to 4 m in length and excluded the PM marker, and tubulin-positive extensions that were >10 m long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites. IMPORTANCEAlphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.

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“…Previous morphological studies of alphavirus assembly and budding were based primarily on electron microscopy methods, such as transmission, scanning, freeze-etch, and freeze fracture electron microscopy (26)(27)(28), and/or on surface labeling of cells with antibodies to the envelope proteins (54,55). These early studies demonstrated local enrichment of envelope proteins in patches and extensions and exclusion of host membrane proteins from nascent virus particles but by necessity were based on static measurements.…”

Section: Discussionmentioning

confidence: 99%

“…Although earlier studies showed that E1/E2 heterodimers become relatively immobile at late times of SINV infection, the meaning of this finding remained unclear (54). We used fluorescence recovery after photobleaching (FRAP) to study the mobility of PM-GFP, the E2 protein from WT-mCherry, and the E2 protein from Y400K-mCherry.…”

Section: Construction and Characterization Of E2-tagged Sinvmentioning

confidence: 99%

Imaging the Alphavirus Exit Pathway

Martínez

¹

,

Snapp

²

,

Perumal

³

et al. 2014

J Virol

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Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ϳ2 to 4 m in length and excluded the PM marker, and tubulin-positive extensions that were >10 m long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites. IMPORTANCEAlphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.

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“…Oligomerization of pE2 and E1 is also a requirement for the transport of the glycoprotein to the cell surface, but the presence of the CP is not required. SINV glycoproteins can first be detected at the cell surface at 1.5 h and virus budding is evident by 3 h post-infection [182]. …”

Section: Alphavirus Lifecyclementioning

confidence: 99%

A structural and functional perspective of alphavirus replication and assembly

Jose

¹

,

Snyder

²

,

Kühn

³

2009

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Alphaviruses are small, spherical, enveloped, positive-sense ssRNA viruses responsible for a considerable number of human and animal diseases. Alphavirus members include Chikungunya virus, Sindbis virus, Semliki Forest virus, the western, eastern and Venezuelan equine encephalitis viruses, and the Ross River virus. Alphaviruses can cause arthritic diseases and encephalitis in humans and animals and continue to be a worldwide threat. The viruses are transmitted by blood-sucking arthropods, and replicate in both arthropod and vertebrate hosts. Alphaviruses form spherical particles (65–70 nm in diameter) with icosahedral symmetry and a triangulation number of four. The icosahedral structures of alphaviruses have been defined to very high resolutions by cryo-electron microscopy and crystallographic studies. In this review, we summarize the major events in alphavirus infection: entry, replication, assembly and budding. We focus on data acquired from structural and functional studies of the alphaviruses. These structural and functional data provide a broader perspective of the virus lifecycle and structure, and allow additional insight into these important viruses.

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Fluorescence photobleaching recovery measurements reveal differences in envelopment of sindbis and vesicular stomatitis viruses

Cited by 40 publications

References 31 publications

Imaging the Alphavirus Exit Pathway

Imaging the Alphavirus Exit Pathway

Imaging the Alphavirus Exit Pathway

A structural and functional perspective of alphavirus replication and assembly

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