Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes simplex virus

Alphaherpesviruses, including pseudorabies virus (PRV), spread directionally within the nervous systems of their mammalian hosts. Three viral membrane proteins are required for efficient anterograde-directed spread of infection in neurons, including Us9 and a heterodimer composed of the glycoproteins gE and gI. We previously demonstrated that the kinesin-3 motor KIF1A mediates anterograde-directed transport of viral particles in axons of cultured peripheral nervous system (PNS) neurons. The PRV Us9 protein copurifies with KIF1A, recruiting the motor to transport vesicles, but at least one unidentified additional viral protein is necessary for this interaction. Here we show that gE/gI are required for efficient anterograde transport of viral particles in axons by mediating the interaction between Us9 and KIF1A. In the absence of gE/gI, viral particles containing green fluorescent protein (GFP)-tagged Us9 are assembled in the cell body but are not sorted efficiently into axons. Importantly, we found that gE/gI are necessary for efficient copurification of KIF1A with Us9, especially at early times after infection. We also constructed a PRV recombinant that expresses a functional gE-GFP fusion protein and used affinity purification coupled with mass spectrometry to identify gE-interacting proteins. Several viral and host proteins were found to associate with gE-GFP. Importantly, both gI and Us9, but not KIF1A, copurified with gE-GFP. We propose that gE/gI are required for efficient KIF1A-mediated anterograde transport of viral particles because they indirectly facilitate or stabilize the interaction between Us9 and KIF1A.

Section: Discussionmentioning

confidence: 99%

Glycoproteins gE and gI Are Required for Efficient KIF1A-Dependent Anterograde Axonal Transport of Alphaherpesvirus Particles in Neurons

Kratchmarov

Kramer

Greco

et al. 2013

“…The following day, the cells were transfected with 50 pmol Smartpool siRNA using Lipofectamine 2000; 6 h after transfection, the cells were infected at a multiplicity of infection (MOI) of 0.2 in DMEM containing 5% fetal bovine serum. As previously described (16), after 120 h, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS (lacking Ca 2ϩ and Mg 2ϩ ) at pH 7.4, and then autofluorescence was quenched by incubation for 15 min in 50 mM ammonium chloride. The cells were subsequently permeabilized and then successively incubated with the primary antibodies (described in Table 1) and fluorescence-tagged secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG; both from Molecular Probes, Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Exactly how virions mature in the cVAC remains a mystery, and it has not been demonstrated whether a fully formed cVAC is necessary for efficient virion production. Although structures morphologically similar to the HCMV cVAC have not been described for other herpesviruses, a recent study found that herpes simplex virus 1 virions acquire their envelope at tubular structures that share many properties with recycling endosomes (16).…”

mentioning

confidence: 99%

Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex

Das

Ortiz

Gurczynski

et al. 2014

Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis. IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development.

“…It is generally accepted that capsids then bud into the inner nuclear membrane to generate primary enveloped perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing mature nucleocapsids ("naked" capsids) into the cytoplasm (1-3). These capsids subsequently undergo secondary envelopment at a cytoplasmic organelle to assemble the mature, infectious virion (4)(5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Herpes Simplex Virus Capsid Localization to ESCRT-VPS4 Complexes in the Presence and Absence of the Large Tegument Protein UL36p

Kharkwal

Smith

Wilson

2016

UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of tegument, the complex protein layer between the capsid and envelope. UL36p performs multiple functions in the HSV life cycle, including a critical but unknown role in capsid cytoplasmic envelopment. We tested whether UL36p is essential for envelopment because it is required to engage capsids with the cellular ESCRT/Vps4 apparatus. A green fluorescent protein (GFP)-fused form of the dominant negative ATPase Vps4-EQ was used to irreversibly tag ESCRT envelopment sites during infection by UL36p-expressing and UL36-null HSV strains. Using fluorescence microscopy and scanning electron microscopy, we quantitated capsid/Vps4-EQ colocalization and examined the ultrastructure of the corresponding viral assembly intermediates. We found that loss of UL36p resulted in a two-thirds reduction in the efficiency of capsid/Vps4-EQ association but that the remaining UL36p-null capsids were still able to engage the ESCRT envelopment apparatus. It appears that although UL36p helps to couple HSV capsids to the ESCRT pathway, this is likely not the sole reason for its absolute requirement for envelopment. IMPORTANCEEnvelopment of the HSV capsid is essential for the assembly of an infectious virion and requires the complex interplay of a large number of viral and cellular proteins. Critical to envelope assembly is the virally encoded protein UL36p, whose function is unknown. Here we test the hypothesis that UL36p is essential for the recruitment of cellular ESCRT complexes, which are also known to be required for envelopment. Herpesviruses replicate their genomes and assemble DNApackaged capsids in the cell nucleus. It is generally accepted that capsids then bud into the inner nuclear membrane to generate primary enveloped perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing mature nucleocapsids ("naked" capsids) into the cytoplasm (1-3). These capsids subsequently undergo secondary envelopment at a cytoplasmic organelle to assemble the mature, infectious virion (4-10).Herpesvirus cytoplasmic envelopment is extremely complex, requiring the coordinated interaction of multiple viral and cellular polypeptides (8,9,(11)(12)(13)(14)(15)(16)). An essential component of the envelopment apparatus is the conserved multifunctional protein UL36p (VP1/2) (17-21). UL36p is the largest structural polypeptide encoded by the members of the Herpesviridae (22, 23), and it forms the innermost layer (18, 24-32) of tegument, the complex protein scaffold between the capsid and envelope (8,16,33). UL36p attaches the capsid (18, 31, 32, 34) to multiple outer tegument components (24-30, 35, 36) that in turn bind integral membrane envelope proteins (1, 9, 37-40) and the lipid envelope (41-43). One important function of UL36p is to recruit UL37p (19,20,25,29,44,45), a putative mimic of cellular multisubunit tethering complexes (28) that mediates capsid docking to organelles, including the trans-Golgi netwo...