Herpes Simplex Virus Type 1 Capsids Transit by the trans-Golgi Network, Where Viral Glycoproteins Accumulate Independently of Capsid Egress

ABSTRACT Egress of herpes capsids from the nucleus to the plasma membrane is a complex multistep transport event that is poorly understood. The current model proposes an initial envelopment at the inner nuclear membrane of capsids newly assembled in the nucleus. The capsids are then released in cytosol by fusion with the outer nuclear membrane. They are finally reenveloped at a downstream organelle before traveling to the plasma membrane for their extracellular release. Although the trans-Golgi network (TGN) is often cited as a potential site of reenvelopment, other organelles have also been proposed, including the Golgi, endoplasmic reticulum-Golgi intermediate compartment, aggresomes, tegusomes, and early or late endosomes. To clarify this important issue, we followed herpes simplex virus type 1 egress by immunofluorescence under conditions that slowed intracellular transport and promoted the accumulation of the otherwise transient reenvelopment intermediate. The data show that the capsids transit by the TGN and point to this compartment as the main reenvelopment site, although a contribution by endosomes cannot formally be excluded. Given that viral glycoproteins are expected to accumulate where capsids acquire their envelope, we examined this prediction and found that all tested could indeed be detected at the TGN. Moreover, this accumulation occurred independently of capsid egress. Surprisingly, capsids were often found immediately adjacent to the viral glycoproteins at the TGN.

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Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication

Cells ◽

10.3390/cells8020120 ◽

2019 ◽

Vol 8 (2) ◽

pp. 120 ◽

Cited By ~ 1

Author(s):

Natalia Saiz-Ros ◽

Rafal Czapiewski ◽

Ilaria Epifano ◽

Andrew Stevenson ◽

Selene Swanson ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Nuclear Membrane ◽

Herpes Simplex Virus Type ◽

Vesicle Fusion ◽

Nuclear Egress ◽

Simplex Virus ◽

Hsv 1

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

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UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

Journal of Virology ◽

10.1128/jvi.75.18.8803-8817.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8803-8817 ◽

Cited By ~ 211

Author(s):

Ashley E. Reynolds ◽

Brent J. Ryckman ◽

Joel D. Baines ◽

Yuping Zhou ◽

Li Liang ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Nuclear Envelope ◽

Gene Product ◽

Virus Type ◽

Nuclear Membrane ◽

Herpes Simplex Virus Type ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.

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Early, Active, and Specific Localization of Herpes Simplex Virus Type 1 gM to Nuclear Membranes

Journal of Virology ◽

10.1128/jvi.01180-09 ◽

2009 ◽

Vol 83 (24) ◽

pp. 12984-12997 ◽

Cited By ~ 16

Author(s):

Jie Zhang ◽

Claus-Henning Nagel ◽

Beate Sodeik ◽

Roger Lippé

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Dependent Manner ◽

Nuclear Membranes ◽

Simplex Virus ◽

Golgi Network ◽

Hsv 1

ABSTRACT Thirteen different glycoproteins are incorporated into mature herpes simplex virus type 1 (HSV-1) virions. Five of them play important roles during entry, while others intervene during egress of the virus. Although HSV-1 gM is not essential in cell culture, its deletion reduces viral yields and promotes syncytium formation. Furthermore, gM is conserved among herpesviruses, is essential for several of them, and can redirect the gD and gH/gL viral glycoproteins from the cell surface to the trans-Golgi network, where gM presumably modulates final capsid envelopment. Late in infection, gM reaches the nuclear envelope and decorates perinuclear virions. This process seemingly requires UL31 and UL34 and occurs when several markers of the trans-Golgi network have relocalized to the nucleus. However, the precise mechanism of gM nuclear targeting is unclear. We now report that gM is quickly and specifically targeted to nuclear membranes in a virus-dependent manner. This occurs prior to the HSV-1-induced reorganization of the trans-Golgi network and before gM enters the secretory pathway. The presence of a high-mannose glycosylation pattern on gM further corroborated these findings. While gM was targeted to the inner nuclear membrane early in infection, its partners gD, gH, gN, VP22, UL31, and UL34 did not colocalize with gM. These data suggest that nuclear gM fulfills an early nuclear function that is independent of its known interaction partners and its function in viral egress.

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Inner tegument protein pUL37 of herpes simplex virus type 1 is involved in directing capsids to the trans-Golgi network for envelopment

Journal of General Virology ◽

10.1099/vir.0.022053-0 ◽

2010 ◽

Vol 91 (9) ◽

pp. 2145-2151 ◽

Cited By ~ 28

Author(s):

D. Pasdeloup ◽

F. Beilstein ◽

A. P. E. Roberts ◽

M. McElwee ◽

D. McNab ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Tegument Protein ◽

Trans Golgi Network ◽

Simplex Virus ◽

Golgi Network

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Comprehensive Characterization of Extracellular Herpes Simplex Virus Type 1 Virions

Journal of Virology ◽

10.1128/jvi.00904-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8605-8618 ◽

Cited By ~ 241

Author(s):

Sandra Loret ◽

Ginette Guay ◽

Roger Lippé

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Viral Envelope ◽

Host Proteins ◽

Viral Glycoproteins ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) genome is contained in a capsid wrapped by a complex tegument layer and an external envelope. The poorly defined tegument plays a critical role throughout the viral life cycle, including delivery of capsids to the nucleus, viral gene expression, capsid egress, and acquisition of the viral envelope. Current data suggest tegumentation is a dynamic and sequential process that starts in the nucleus and continues in the cytoplasm. Over two dozen proteins are assumed to be or are known to ultimately be added to virions as tegument, but its precise composition is currently unknown. Moreover, a comprehensive analysis of all proteins found in HSV-1 virions is still lacking. To better understand the implication of the tegument and host proteins incorporated into the virions, highly purified mature extracellular viruses were analyzed by mass spectrometry. The method proved accurate (95%) and sensitive and hinted at 8 different viral capsid proteins, 13 viral glycoproteins, and 23 potential viral teguments. Interestingly, four novel virion components were identified (UL7, UL23, UL50, and UL55), and two teguments were confirmed (ICP0 and ICP4). In contrast, UL4, UL24, the UL31/UL34 complex, and the viral UL15/UL28/UL33 terminase were undetected, as was most of the viral replication machinery, with the notable exception of UL23. Surprisingly, the viral glycoproteins gJ, gK, gN, and UL43 were absent. Analyses of virions produced by two unrelated cell lines suggest their protein compositions are largely cell type independent. Finally, but not least, up to 49 distinct host proteins were identified in the virions.

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Protein Kinase D-Dependent Trafficking of the Large Herpes simplex Virus Type 1 Capsids from the TGN to Plasma Membrane

Traffic ◽

10.1111/j.1600-0854.2009.00939.x ◽

2009 ◽

Vol 10 (8) ◽

pp. 1074-1083 ◽

Cited By ~ 30

Author(s):

Gaudeline Rémillard-Labrosse ◽

Constantina Mihai ◽

Johanne Duron ◽

Ginette Guay ◽

Roger Lippé

Keyword(s):

Plasma Membrane ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Protein Kinase ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Protein Kinase D ◽

Simplex Virus

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Effects of Targeting Herpes Simplex Virus Type 1 gD to the Endoplasmic Reticulum and trans-Golgi Network

Journal of Virology ◽

10.1128/jvi.73.11.9515-9520.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9515-9520 ◽

Cited By ~ 68

Author(s):

Alison Whiteley ◽

Birgitte Bruun ◽

Tony Minson ◽

Helena Browne

Keyword(s):

Endoplasmic Reticulum ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Wild Type ◽

Simplex Virus ◽

Golgi Network ◽

Hsv 1

ABSTRACT Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) was modified to encode targeting signals known to localize proteins to either the endoplasmic reticulum (ER) or the trans-Golgi network. These motifs conferred the predicted targeting properties on gD in transfected cells as judged by immunofluorescence staining, and the exclusion of targeted gD from the cell surface was confirmed by the fact that these molecules exhibited substantially reduced activity in cell-cell fusion assays. Recombinant viruses expressing Golgi-targeted forms of gD grew to wild-type levels in noncomplementing cells, exhibited unaltered particle/infectivity ratios, and were found to contain wild-type levels of gD, whereas a recombinant expressing ER-retained gD was helper cell dependent and, when grown on noncomplementing cells, produced virions of low specific infectivity with greatly reduced levels of gD. These data imply that HSV-1 acquires its final membrane from a post-ER compartment and lend support to the view that the virus undergoes de-envelopment and reenvelopment steps during virus egress.

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