Fluorescent Protein Approaches in Alpha Herpesvirus Research

Hogue, Ian B.; Bosse, Jens B.; Engel, Esteban A.; Scherer, Julian; Hu, Jiun-Ruey; Rio, Tony del; Enquist, Lynn W.

doi:10.3390/v7112915

Cited by 36 publications

(39 citation statements)

References 185 publications

(165 reference statements)

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“…Before infection, mNG-VP26 fluorescence was distributed throughout the cell. During infection, the signal changed its localization predominantly to the nucleus, where it formed characteristic intranuclear aggregate structures ("assemblons") (22). This pattern was consistent with that of wild-type and tagged VP26 proteins in infected cells (23).…”

Section: Resultssupporting

confidence: 74%

Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics

Scherer

Yaffe

Vershinin

et al. 2016

J Virol

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Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses.Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.A lphaherpesviruses (e.g., human herpes simplex virus [HSV] and swine pseudorabies virus [PRV]) are neuroinvasive double-stranded DNA (dsDNA) viruses with virions consisting of a nucleocapsid surrounded by a proteinaceous tegu...

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Section: Resultssupporting

confidence: 74%

Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics

Scherer

Yaffe

Vershinin

et al. 2016

J Virol

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“…To quantify the data and avoid subjective biases, we implemented an automated tracking script. However as capsid motility is rapid and improved capsid-tagged mutants 16 produced many more capsids in the nucleus, we needed a system that acquired images at high frame rates in the range of 30 and more frames per second (fps). Unfortunately, this speed of image capture would reduce the available signal dramatically so that commercial laser scanning confocal microscopy or spinning disk microscopy could not be used.…”

Section: Nuclear Capsid Motility Is Not Dependent On F-actinmentioning

confidence: 99%

The diffusive way out: Herpesviruses remodel the host nucleus, enabling capsids to access the inner nuclear membrane

Bosse

Enquist

2016

Nucleus

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Herpesviruses are large DNA viruses that utilize the host nucleus for genome replication as well as capsid assembly. After maturation, these 125 nm large capsid assemblies must cross the nucleoplasm to engage the nuclear envelope and bud into the cytoplasm. Here we summarize our recent findings how this motility is facilitated. We suggest that herpesvirus induced nuclear remodeling allows capsids to move by diffusion in the nucleus and not by motor-dependent transport.

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“…Then, the antiviral activity of His-Au NCs and MES-Au NCs on PRV was investigated by confocal microscopic analysis using the recombinant PRV expressing fluorescent protein (GFP-PRV). [27] In Figure 3a, obvious green fluorescence signals could be observed in GFP-PRV infected cells at 12 h postinfection.…”

Section: Effects Of His-au Ncs and Mes-au Ncs On Prv Proliferationmentioning

confidence: 93%

Different Effects of His‐Au NCs and MES‐Au NCs on the Propagation of Pseudorabies Virus

et al. 2018

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In a previous work, gold nanoclusters (Au NCs) are found to inactivate RNA virus, but the effect of surface modification of Au NCs on its proliferation is still largely unknown. Here, the effect of surface modification of Au NCs on the proliferation of pseudorabies virus (PRV) by synthesizing two types of gold clusters with different surface modification, histidine stabilized Au NCs (His‐Au NCs) and mercaptoethane sulfonate and histidine stabilized Au NCs (MES‐Au NCs), is investigated. His‐Au NCs rather than MES‐Au NCs could strongly inhibit the proliferation of PRV, as indicated by the results of plaque assay, confocal microscopic analysis, Western blot assay, and quantitative real‐time polymerase chain reaction (PCR) assay. Further study reveals that His‐Au NCs perform the function via blockage of the viral replication process rather than the processes of attachment, penetration, or release. Additionally, His‐Au NCs are found to be mainly localized to nucleus, while MES‐Au NCs are strictly distributed in cytoplasm, which may explain why His‐Au NCs can suppress the proliferation of PRV, but not MES‐Au NCs. These results demonstrate that surface modification plays a key role in the antiviral effects of Au NCs and a potential antiviral agent can be developed by changing the Au NC surface modification.

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Fluorescent Protein Approaches in Alpha Herpesvirus Research

Cited by 36 publications

References 185 publications

Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics

Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics

The diffusive way out: Herpesviruses remodel the host nucleus, enabling capsids to access the inner nuclear membrane

Different Effects of His‐Au NCs and MES‐Au NCs on the Propagation of Pseudorabies Virus

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