Incorporation of green fluorescent protein into the essential envelope glycoprotein B of herpes simplex virus type 1

134

180

We report here the construction of a triply fluorescent-tagged herpes simplex virus 1 (HSV-1) expressing capsid protein VP26, tegument protein VP22, and envelope protein gB as fusion proteins with monomeric yellow, red, and cyan fluorescent proteins, respectively. The recombinant virus enabled us to monitor the dynamics of these capsid, tegument, and envelope proteins simultaneously in the same live HSV-1-infected cells and to visualize single extracellular virions with three different fluorescent emissions. In Vero cells infected by the triply fluorescent virus, multiple cytoplasmic compartments were found to be induced close to the basal surfaces of the infected cells (the adhesion surfaces of the infected cells on the solid growth substrate). Major capsid, tegument, and envelope proteins accumulated and colocalized in the compartments, as did marker proteins for the trans-Golgi network (TGN) which has been implicated to be the site of HSV-1 secondary envelopment. Moreover, formation of the compartments was correlated with the dynamic redistribution of the TGN proteins induced by HSV-1 infection. These results suggest that HSV-1 infection causes redistribution of TGN membranes to form multiple cytoplasmic compartments, possibly for optimal secondary envelopment. This is the first real evidence for the assembly of all three types of herpesvirus proteins-capsid, tegument, and envelope membrane proteins-in TGN.Herpes simplex virus 1 (HSV-1) consists of three morphologically distinct structures: nucleocapsid, tegument, and envelope (58). The nucleocapsid contains the linear doublestranded DNA viral genome in an icosahedral capsid (58). The nucleocapsid is surrounded by a proteinaceous layer designated the tegument, which is enclosed by an envelope that is a host cell-derived lipid bilayer containing virus-encoded glycoproteins (58). After virus entry into the host cell, the deenveloped nucleocapsids reach the nucleopores, and the viral genome enters the nucleus (58). Subsequently, viral DNA replication and packaging of nascent progeny virus genomes into assembling capsids take place within nuclear compartments, called replication compartments (12,56,58). The progeny nucleocapsids then acquire primary envelopes by budding through the inner nuclear membrane into the space between the inner and the outer nuclear envelopes, called the perinuclear space (39, 58). While assembly of progeny nucleocapsids within the nucleus and primary envelopment of nucleocapsids at the inner nuclear membrane have been well documented, the route of the nascent virions from the perinuclear space to the extracellular space is controversial (7,34,41,42,58,66). It is now generally accepted that perinuclear virions lose their envelopes by fusion with the outer nuclear membrane, thereby releasing naked nucleocapsids in the cytoplasm (40,41,60). These nucleocapsids must acquire tegument proteins in the cytoplasm and/or upon budding through the nuclear membrane and must be enveloped again at cytoplasmic membranes (secondary envelopment), probabl...

Section: Construction Of Triply Fluorescent Virusessupporting

confidence: 93%

Simultaneous Tracking of Capsid, Tegument, and Envelope Protein Localization in Living Cells Infected with Triply Fluorescent Herpes Simplex Virus 1

Sugimoto

Uema²,

Sagara

et al. 2008

134

180

“…The gB-null K082 virus (10) was propagated on the gBexpressing D6 cell line as described previously (44). Both the K082 virus and the D6 cell line were kindly provided by P. Desai and S. Person (Johns Hopkins University, Baltimore, Md.).…”

Section: Cells and Virusesmentioning

confidence: 99%

“…To define their role, we used a deletion and point mutation approach to delineate domains involved in the intracellular transport of the protein and in the production of infectious particles. Members of our laboratory previously showed that a chimeric GFP-gB protein, containing the enhanced green fluorescent protein (EGFP) fused to the ectodomain of HSV-1 gB, is processed and localized in infected cells like native gB (44,45). We constructed GFP-gB mutant forms and studied their processing, cellular localization, and capacity to complement a defective gB-null HSV-1.…”

mentioning

confidence: 99%

Effects of Mutations in the Cytoplasmic Domain of Herpes Simplex Virus Type 1 Glycoprotein B on Intracellular Transport and Infectivity

Zarate

Kaelin

Rozenberg

2004

Self Cite

Herpes simplex virus type 1 (HSV-1) is a human pathogen of the alphaherpesvirus family which infects and spreads in the nervous system. Glycoproteins play a key role in the process of assembly and maturation of herpesviruses, which is essential for neuroinvasion and transneuronal spread. Glycoprotein B (gB) is a main component of the HSV-1 envelope and is necessary for the production of infectious particles. The cytoplasmic domain of gB, the longest one among HSV-1 glycoproteins, contains several highly conserved peptide sequences homologous to motifs involved in intracellular sorting. To determine the specific roles of these motifs in processing, subcellular localization, and the capacity of HSV-1 gB to complement a gB-null virus, we generated truncated or point mutated forms of a green fluorescent protein (GFP)-tagged gB. GFP-gB with a deletion in the acidic cluster DGDADEDDL (amino acids [aa] 896 to 904) behaved the same as the parental form. Deletion or disruption of the YTQV motif (aa 889 to 892) abolished internalization and reduced complementation by 60%. Disruption of the LL motif (aa 871 to 872) impaired the return of the protein to the trans-Golgi network (TGN) while enhancing its recycling to the plasma membrane. Truncations from residue E 857 abolished transport and processing of the truncated proteins, which had null complementation activity, through the Golgi complex. Altogether, our results favor a model in which HSV-1 gets its final envelope in the TGN, and they suggest that endocytosis, albeit not necessary, might play a role in infectivity.Herpes simplex virus type 1 (HSV-1) is a human alphaherpesvirus which generally causes benign infections of the skin and mucosa. The virus also infects neurons of the sensory ganglia, where it establishes latency. HSV-1 periodically reactivates to cause peripheral recurrences and occasionally spreads transneuronally to cause severe central nervous system infections. Although the mechanisms of neuroinvasion and transneuronal spread of alphaherpesviruses are not fully understood, they seem to be linked to the process of assembly and maturation of virions in infected cells, in which glycoproteins play a major role (33, 56).How HSV-1 particles are assembled and enveloped in infected cells is not completely known. The current view is that after primary envelopment at the inner nuclear membrane, capsids are de-enveloped through their exit from the nucleus and acquire their final envelope at the trans-Golgi network (TGN), where mature glycoproteins are incorporated (reviewed in reference 32). In support of this model, it has been shown that retention of glycoprotein D (gD) or gH in the endoplasmic reticulum (ER) of infected cells prevents their insertion into virions (6, 52, 61). Thus, proper intracellular transport of glycoproteins is necessary for successful incorporation into virions.gB, one of the most conserved envelope glycoproteins among herpesviruses, is essential at many steps of the viral replication cycle. In vitro, gB is essential for attachment and entr...

“…The addition of GFP to VP26 interferes neither with cell entry and dynein-mediated microtubule transport to the nucleus (30) nor with dynein and dynactin binding to isolated capsids or capsid transport along microtubules in vitro (96). The addition of GFP to the envelope glycoprotein gB of HSV1 reduces plaque sizes by three-to fivefold and virus titers by 100-fold (70), whereas the addition of GFP or yellow fluorescent protein (YFP) to glycoprotein D (gD) seems to be better tolerated (66,80).…”

mentioning

confidence: 99%

Nuclear Egress and Envelopment of Herpes Simplex Virus Capsids Analyzed with Dual-Color Fluorescence HSV1(17 ⁺ )

Nagel

Döhner

Fathollahy

et al. 2008

122

To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as ␤-galactosidase expression cassettes. When the BAC pHSV1(17 ؉ )blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17 ؉ )blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17 ؉ )blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.Herpes simplex virus type 1 (HSV1) causes severe human diseases such as herpes encephalitis or herpes keratoconjunctivitis (18). Its double-stranded DNA genome of 152 kb that codes for more than 80 open reading frames is enclosed in an icosahedral capsid with a diameter of 125 nm. HSV1 is a spherical, enveloped virus with a diameter of about 250 nm. Between the capsid and the viral membrane, there is an amorphous, asymmetric layer, the tegument, which consists of about 20 different proteins (45,74). HSV1 enters cells by fusion at the plasma membrane or after endocytosis by fusion with an endosomal membrane (19,42,66,67,82). After dynein-mediated transport along microtubules (32,56,59,81), capsids reach the nuclear pore where the viral genome is released into the nucleoplasm (68) for viral transcription and DNA replication (74). Progeny viral genomes are packaged into preassembled nuclear capsids, which translocate to the inner nuclear membrane. The subsequent steps of herpesvirus morphogenesis are controversial (12, 13, 65).HSV1 capsids can leave the nucleus by primary envelopment at the inner nuclear membrane (6, 64). According to the luminal or single-envelopment hypothesis, these enveloped virions present in the lumen of the nuclear envelope or the endoplasmic reticulu...