Intracellular trafficking and maturation of herpes simplex virus type 1 gB and virus egress require functional biogenesis of multivesicular bodies (original) (raw)
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Journal of Virology, 2012
Herpes simplex virus 1 (HSV-1) viral glycoproteins gD (carboxyl terminus), gE, gK, and gM, the membrane protein UL20, and membrane-associated protein UL11 play important roles in cytoplasmic virion envelopment and egress from infected cells. We showed previously that a recombinant virus carrying a deletion of the carboxyl-terminal 29 amino acids of gD (gDΔct) and the entire gE gene (ΔgE) did not exhibit substantial defects in cytoplasmic virion envelopment and egress (H. C. Lee et al., J. Virol. 83:6115–6124, 2009). The recombinant virus ΔgM2, engineered not to express gM, produced a 3- to 4-fold decrease in viral titers and a 50% reduction in average plaque sizes in comparison to the HSV-1(F) parental virus. The recombinant virus containing all three mutations, gDΔct-ΔgM2-ΔgE, replicated approximately 1 log unit less efficiently than the HSV-1(F) parental virus and produced viral plaques which were on average one-third the size of those of HSV-1(F). The recombinant virus ΔUL11-ΔgM2...
Journal of Virology, 2001
Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, 1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74: . Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.
Journal of Virology, 2006
Assembly of herpes simplex viruses (HSV) is a poorly understood process involving multiple redundant interactions between large number of tegument and envelope proteins. We have previously shown (G. E. Lee, G. A. Church, and D. W. Wilson, J. Virol. 77:2038-2045, 2003) that the virion host shutoff (Vhs) tegument protein is largely insoluble in HSV-infected cells and is also stably associated with membranes. Here we demonstrate that both insolubility and stable membrane binding are stimulated during the course of an HSV infection. Furthermore, we have found that the amino-terminal 42 residues of Vhs are sufficient to mediate membrane association and tegument incorporation when fused to a green fluorescent protein (GFP) reporter. Particle incorporation correlates with sorting to cytoplasmic punctate structures that may correspond to sites of HSV assembly. We conclude that the amino terminus of Vhs mediates targeting to sites of HSV assembly and to the viral tegument.
HSV-1 Cytoplasmic Envelopment and Egress
International Journal of Molecular Sciences
Herpes simplex virus type 1 (HSV-1) is a structurally complex enveloped dsDNA virus that has evolved to replicate in human neurons and epithelia. Viral gene expression, DNA replication, capsid assembly, and genome packaging take place in the infected cell nucleus, which mature nucleocapsids exit by envelopment at the inner nuclear membrane then de-envelopment into the cytoplasm. Once in the cytoplasm, capsids travel along microtubules to reach, dock, and envelope at cytoplasmic organelles. This generates mature infectious HSV-1 particles that must then be sorted to the termini of sensory neurons, or to epithelial cell junctions, for spread to uninfected cells. The focus of this review is upon our current understanding of the viral and cellular molecular machinery that enables HSV-1 to travel within infected cells during egress and to manipulate cellular organelles to construct its envelope.
Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2017
Herpes simplex virus (HSV)-encoded glycoprotein B (gB) is the most abundant protein in the viral envelope and promotes fusion of the virus with the cellular membrane. In the present study, we found that gB impacts on the major histocompatibility complex (MHC)-II pathway of antigen presentation by fostering homotypic fusion of early endosomes and trapping MHC-II molecules in these altered endosomes. By using an overexpression approach, we demonstrated that transient expression of gB induces giant vesicles of early endosomal origin, which contained Rab5, early endosomal antigen 1 (EEA1), large amounts of MHC-II molecules [human leukocyte antigen (HLA)-DR, and HLA-DM] but no CD63. In HSV-1-infected and stably transfected cell lines that expressed lower amounts of gB, giant endosomes were not observed, but strongly increased amounts of HLA-DR and HLA-DM were found in EEA1(+) early endosomes. We used these giant vesicles as a model system and revealed that gB interacts with Rab5 and EEA1...
Journal of Virology, 2014
The herpes simplex virus type 1 (HSV-1) UL20 gene encodes a 222-amino-acid nonglycosylated envelope protein which forms a complex with viral glycoprotein K (gK) that functions in virion envelopment, egress, and virus-induced cell fusion. To investigate the role of the carboxyl terminus of the UL20 protein (UL20p) in cytoplasmic virion envelopment, a cadre of mutant viruses was constructed and characterized. The deletion of six amino acids from the carboxyl terminus of UL20p caused an approximately 1-log reduction in infectious virus production compared to that of the wild-type virus. Surprisingly, a phenylalanine-toalanine replacement at amino acid position 210 caused a gain-of-function phenotype, increasing infectious virus production up to 1 log more than in the wild-type virus. In contrast, the replacement of two membrane-proximal phenylalanines with alanines caused drastic inhibition of infectious virion production and cytoplasmic virion envelopment. Prediction of the membrane topology of UL20p revealed that these two amino acid changes cause retraction of the carboxyl terminus of UL20p from the intracellular space. Confocal microscopy revealed that none of the engineered UL20 mutations affected intracellular transport of UL20p to trans-Golgi network membranes. In addition, a proximity ligation assay showed that none of the UL20 mutations affected UL20p colocalization and potential interactions with the UL37 protein recently found to interact with the gK/UL20 protein complex. Collectively, these studies show that phenylalanine residues within the carboxyl terminus of UL20p are involved in the regulation of cytoplasmic virion envelopment and infectious virus production. IMPORTANCE We have shown previously that the UL20/gK protein complex serves crucial roles in cytoplasmic virion envelopment and that it interacts with the UL37 tegument protein to facilitate cytoplasmic virion envelopment. In this study, we investigated the role of phenylalanine residues within the carboxyl terminus of UL20p, since aromatic and hydrophobic amino acids are known to be involved in protein-protein interactions through stacking of their aromatic structures. Characterization of mutant viruses carrying phenylalanine (Phe)-to-alanine (Ala) mutations revealed that the two membrane-proximal Phe residues were critical for the proper UL20p membrane topology and efficient virion envelopment and infectious virus production. Surprisingly, a Phe-to-Ala change located approximately in the middle of the UL20p carboxyl terminus substantially enhanced cytoplasmic envelopment and overall production of infectious virions. This work revealed that Phe residues within the UL20p carboxyl terminus are involved in the regulation of cytoplasmic virion envelopment and infectious virus production.
Journal of Virology, 2007
Egress of herpes simplex virus type 1 (HSV-1) from the nucleus of the infected cell to extracellular spaces involves a number of distinct steps, including primary envelopment by budding into the perinuclear space, de-envelopment into the cytoplasm, cytoplasmic reenvelopment, and translocation of enveloped virions to extracellular spaces. UL20/gK-null viruses are blocked in cytoplasmic virion envelopment and egress, as indicated by an accumulation of unenveloped or partially enveloped capsids in the cytoplasm. Similarly, UL11-null mutants accumulate unenveloped capsids in the cytoplasm. To assess whether UL11 and UL20/gK function independently or synergistically in cytoplasmic envelopment, recombinant viruses having either the UL20 or UL11 gene deleted were generated. In addition, a recombinant virus containing a deletion of both UL20 and UL11 genes was constructed using the HSV-1(F) genome cloned into a bacterial artificial chromosome. Ultrastructural examination of virus-infected cells showed that both UL20-and UL11-null viruses accumulated unenveloped capsids in the cytoplasm. However, the morphology and distribution of the accumulated capsids appeared to be distinct, with the UL11-null virions forming aggregates of capsids having diffuse tegument-derived material and the UL20-null virus producing individual capsids in close juxtaposition to cytoplasmic membranes. The UL20/UL11 double-null virions appeared morphologically similar to the UL20-null viruses. Experiments on the kinetics of viral replication revealed that the UL20/UL11 double-null virus replicated in a manner similar to the UL20-null virus. Additional experiments revealed that transiently expressed UL11 localized to the trans-Golgi network (TGN) independently of either gK or UL20. Furthermore, virus infection with the UL11/UL20 double-null virus did not alter the TGN localization of transiently expressed UL11 or UL20 proteins, indicating that these proteins did not interact. Taken together, these results show that the intracellular transport and TGN localization of UL11 is independent of UL20/gK functions, and that UL20/gK are required and function prior to UL11 protein in virion cytoplasmic envelopment.
Virology Journal, 2007
The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20null virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.
Journal of Virology, 2009
Herpes simplex virus type 1 (HSV-1) acquires its final envelope by budding into cytoplasmic vesicles thought to be derived from trans -Golgi network membranes. This process is facilitated by interactions among the carboxyl termini of viral glycoproteins and tegument proteins. To directly investigate the relative importance of the carboxyl terminus of glycoprotein D (gD) in the presence or absence of gE, a recombinant virus (gDΔct) was constructed to specify a truncated gD lacking the carboxy-terminal 29 amino acids. Furthermore, two additional recombinant viruses were constructed by mutating from ATG to CTG the initiation codons of gE (gEctg) or both gE and gM (gEctg+gMctg), causing lack of expression of gE or both gE and gM, respectively. A fourth mutant virus was constructed to specify the gEctg+gDΔct mutations. The replication properties of these viruses were compared to those of a newly constructed recombinant virus unable to express UL20 due to alteration of the two initiation ...
Virology, 2014
The herpes simplex virus type 1 (HSV-1) tegument proteins pUL36 (VP1/2) and pUL37 are essential for viral egress. We previously defined a minimal domain in HSV-1 pUL36, residues 548-572, as important for binding pUL37. Here, we investigated the role of this region in binding to pUL37 and facilitating viral replication. We deleted residues 548-572 in frame in a virus containing a mRFP tag at the N-terminus of the capsid protein VP26 and an eGFP tag at the C-terminus of pUL37 (HSV-1pUL36∆548-572). This mutant virus was unable to generate plaques in Vero cells, indicating that deletion of this region of pUL36 blocks viral replication. Imaging of HSV-1pUL36∆548-572-infected Vero cells, in comparison to parental and resucant, revealed a block in secondary envelopment of cytoplasmic capsids. In addition, immunoblot analysis suggested that failure to bind pUL37 affected the stability of pUL36. This study provides further insight into the role of this essential interaction.