skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Attention:

The NSF Public Access Repository (PAR) system and access will be unavailable from 11:00 PM ET on Friday, May 16 until 2:00 AM ET on Saturday, May 17 due to maintenance. We apologize for the inconvenience.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering
Title: Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering
ABSTRACT During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro , providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world’s population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.  more » « less
Award ID(s):
1808459
PAR ID:
10422614
Author(s) / Creator(s):
; ; ; ; ; ;
Editor(s):
Shenk, Thomas
Date Published:
Journal Name:
mBio
Volume:
12
Issue:
4
ISSN:
2150-7511
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; (, Journal of Virology)
    Longnecker, Richard M. (Ed.)
    ABSTRACT Nuclear envelope budding in herpesvirus nuclear egress may be negatively regulated, since the pUL31/pUL34 nuclear egress complex heterodimer can induce membrane budding without capsids when expressed ectopically or on artificial membranes in vitro , but not in the infected cell. We have previously described a pUL34 mutant that contained alanine substitutions at R158 and R161 and that showed impaired growth, impaired pUL31/pUL34 interaction, and unregulated budding. Here, we determine the phenotypic contributions of the individual substitutions to these phenotypes. Neither substitution alone was able to reproduce the impaired growth or nuclear egress complex (NEC) interaction phenotypes. Either substitution, however, could fully reproduce the unregulated budding phenotype, suggesting that misregulated budding may not substantially impair virus replication. In addition, the R158A substitution caused relocalization of the NEC to intranuclear punctate structures and recruited lamin A/C to these structures, suggesting that this residue might be important for recruitment of kinases for dispersal of nuclear lamins. IMPORTANCE Herpesvirus nuclear egress is a complex, regulated process coordinated by two virus proteins that are conserved among the herpesviruses that form a heterodimeric nuclear egress complex (NEC). The NEC drives budding of capsids at the inner nuclear membrane and recruits other viral and host cell proteins for disruption of the nuclear lamina, membrane scission, and fusion. The structural basis of individual activities of the NEC, apart from membrane budding, are not clear, nor is the basis of the regulation of membrane budding. Here, we explore the properties of NEC mutants that have an unregulated budding phenotype, determine the significance of that regulation for virus replication, and also characterize a structural requirement for nuclear lamina disruption. 
    more » « less
  2. ; (, Viruses)
    The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spurred unprecedented and concerted worldwide research to curtail and eradicate this pathogen. SARS-CoV-2 has four structural proteins: Envelope (E), Membrane (M), Nucleocapsid (N), and Spike (S), which self-assemble along with its RNA into the infectious virus by budding from intracellular lipid membranes. In this paper, we develop a model to explore the mechanisms of RNA condensation by structural proteins, protein oligomerization and cellular membrane–protein interactions that control the budding process and the ultimate virus structure. Using molecular dynamics simulations, we have deciphered how the positively charged N proteins interact and condense the very long genomic RNA resulting in its packaging by a lipid envelope decorated with structural proteins inside a host cell. Furthermore, considering the length of RNA and the size of the virus, we find that the intrinsic curvature of M proteins is essential for virus budding. While most current research has focused on the S protein, which is responsible for viral entry, and it has been motivated by the need to develop efficacious vaccines, the development of resistance through mutations in this crucial protein makes it essential to elucidate the details of the viral life cycle to identify other drug targets for future therapy. Our simulations will provide insight into the viral life cycle through the assembly of viral particles de novo and potentially identify therapeutic targets for future drug development. 
    more » « less
  3. ; ; ; ; ; ; ; ; (, Journal of Virology)
    ABSTRACT In eukaryotic cells, the s oluble N -ethylmaleimide- s ensitive f actor (NSF) a ttachment protein re ceptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV. IMPORTANCE Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses. 
    more » « less
  4. ; ; ; ; ; ; ; ; ; ; et al (, Science Advances)
    Nuclear import of the hepatitis B virus (HBV) nucleocapsid is essential for replication that occurs in the nucleus. The ~360-angstrom HBV capsid translocates to the nuclear pore complex (NPC) as an intact particle, hijacking human importins in a reaction stimulated by host kinases. This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end of the HBV coat protein Cp183 carboxyl-terminal domain (CTD). This NLS is exposed to the capsid surface through a pore at the icosahedral quasi-sixfold vertex. Phosphorylation at serine-155, serine-162, and serine-170 promotes CTD compaction but does not affect the affinity for importin α1. The binding of 30 importin α1/β1 augments HBV capsid diameter to ~620 angstroms, close to the maximum size trafficable through the NPC. We propose that phosphorylation favors CTD externalization and prompts its compaction at the capsid surface, exposing the NLS to importins. 
    more » « less
  5. ; ; ; ; ; ; ; (, Viruses)
    null (Ed.)
    Positive-strand RNA viruses universally remodel host intracellular membranes to form membrane-bound viral replication complexes, where viral offspring RNAs are synthesized. In the majority of cases, viral replication proteins are targeted to and play critical roles in the modulation of the designated organelle membranes. Many viral replication proteins do not have transmembrane domains, but contain single or multiple amphipathic alpha-helices. It has been conventionally recognized that these helices serve as an anchor for viral replication protein to be associated with membranes. We report here that a peptide representing the amphipathic α-helix at the N-terminus of the poliovirus 2C protein not only binds to liposomes, but also remodels spherical liposomes into tubules. The membrane remodeling ability of this amphipathic alpha-helix is similar to that recognized in other amphipathic alpha-helices from cellular proteins involved in membrane remodeling, such as BAR domain proteins. Mutations affecting the hydrophobic face of the amphipathic alpha-helix severely compromised membrane remodeling of vesicles with physiologically relevant phospholipid composition. These mutations also affected the ability of poliovirus to form plaques indicative of reduced viral replication, further underscoring the importance of membrane remodeling by the amphipathic alpha-helix in possible relation to the formation of viral replication complexes. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email