scholarly journals Mutation of the YXXL Endocytosis Motif in the Cytoplasmic Tail of Pseudorabies Virus gE

1999 ◽  
Vol 73 (4) ◽  
pp. 2717-2728 ◽  
Author(s):  
R. S. Tirabassi ◽  
L. W. Enquist
Keyword(s):  
Central Nervous System ◽  
Pseudorabies Virus ◽  
Cultured Cells ◽  
Cytoplasmic Tail ◽  
Viral Envelope ◽  
Small Plaque ◽  
Viral Mutant ◽  
Plaque Phenotype ◽  
Gi Proteins

ABSTRACT The role of alphaherpesvirus membrane protein internalization during the course of viral infection remains a matter of speculation. To determine the role of internalization of the pseudorabies virus (PRV) gE and gI proteins, we constructed viral mutants encoding specific mutations in the cytoplasmic tail of the gE gene that inhibited internalization of the gE-gI complex. We used these mutants to assess the role of gE-gI endocytosis in incorporation of the proteins into the viral envelope and in gE-mediated spread or gE-promoted virulence. In addition, we report that another viral mutant, PRV 25, which encodes a gE protein defective in endocytosis, contains an additional, previously uncharacterized mutation in the gE gene. We compared PRV 25 to another viral mutant, PRV 107, that does not express the cytoplasmic tail of the gE protein. The gE protein encoded by PRV 107 is also defective in endocytosis. We conclude that efficient endocytosis of gE is not required for gE incorporation into virions, gE-mediated virulence, or spread of virus in the rat central nervous system. However, we do correlate the defect in endocytosis to a small-plaque phenotype in cultured cells.

scholarly journals Role of the Cytoplasmic Tail of Pseudorabies Virus Glycoprotein E in Virion Formation

2000 ◽  
Vol 74 (9) ◽  
pp. 4004-4016 ◽  
Author(s):  
Alexandra R. Brack ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Rebecca Tirabassi ◽  
Lynn W. Enquist ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Triple Mutant ◽  
Small Plaque ◽  
Gene Encoding ◽  
One Step ◽  
Plaque Phenotype ◽  
Virus Mutant ◽  
Virion Formation

ABSTRACT Glycoproteins M (gM), E (gE), and I (gI) of pseudorabies virus (PrV) are required for efficient formation of mature virions. The simultaneous absence of gM and the gE/gI complex results in severe deficiencies in virion morphogenesis and cell-to-cell spread, leading to drastically decreased virus titers and a small-plaque phenotype (A. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364–5372, 1999). Serial passaging in noncomplementing cells of a virus mutant unable to express gM, gE, and gI resulted in a reversion of the small-plaque phenotype and restoration of infectious virus formation to the level of a gM− mutant. Genetic analyses showed that reversion of the phenotype was accompanied by a genomic rearrangement which led to the fusion of a portion of the gE gene encoding the cytoplasmic domain to the 3′ end of the glycoprotein D gene, resulting in expression of a chimeric gD-gE protein. Since this indicated that the intracytoplasmic domain of gE was responsible for the observed phenotypic alterations, the UL10 (gM) gene was deleted in a PrV mutant, PrV-107, which specifically lacked the cytoplasmic tail of gE. Regarding one-step growth, plaque size, and virion formation as observed under the electron microscope, the mutant lacking gM and the gE cytoplasmic tail proved to be very similar to the gE/I/M triple mutant. Thus, our data indicate that it is the cytoplasmic tail of gE which is responsible for the observed phenotypic effects in conjunction with deletion of gM. We hypothesize that the cytoplasmic domain of gE specifically interacts with components of the capsid and/or tegument, leading to efficient secondary envelopment of intracytoplasmic capsids.

scholarly journals Functional Role of the Cytoplasmic Tail Domain of the Major Envelope Fusion Protein of Group II Baculoviruses

2006 ◽  
Vol 80 (22) ◽  
pp. 11226-11234 ◽  
Author(s):  
Gang Long ◽  
Xiaoyu Pan ◽  
Marcel Westenberg ◽  
Just M. Vlak
Keyword(s):  
Fusion Proteins ◽  
Functional Role ◽  
Cytoplasmic Tail ◽  
Viral Envelope ◽  
Repair System ◽  
Dependent Manner ◽  
Tail Domain ◽  
F Protein ◽  
Group Ii

ABSTRACT F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD), ranging from 48 (Spodoptera litura multicapsid NPV [MNPV]) to 78 (Adoxophyes honmai NPV) amino acid (aa) residues, with a nonassigned function. This CTD is much longer than the CTD of GP64-like envelope fusion proteins (7 aa), which appear to be nonessential for BV infectivity. Here we have investigated the functional role of the CTD of Helicoverpa armigera single-capsid NPV (HearNPV), a group II NPV. We combined a newly constructed HearNPV f-null bacmid knockout-repair system and an Autographa californica MNPV (AcMNPV) gp64-null bacmid knockout-pseudotype system with mutation and rescue experiments to study the functional role of the baculovirus F protein CTD. We show that except for the 16 C-terminal aa, the HearNPV F CTD is essential for virus spread from cell to cell. In addition, the CTD of HearNPV F is involved in BV production in a length-dependent manner and is essential for BV infectivity. The tyrosine residue Y658, located 16 aa from the C terminus, seems to be critical. However, HearNPV F without a CTD still rescues the infectivity of gp64-null AcMNPV BV, indicating that the CTD is not involved in processing and fusogenicity. Altogether, our results indicate that the F protein is essential for baculovirus BV infectivity and that the CTD is important for F protein incorporation into BV.

scholarly journals Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion

2018 ◽  
Vol 92 (12) ◽  
Author(s):  
Melina Vallbracht ◽  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Membrane Fusion ◽  
Fusion Proteins ◽  
Essential Role ◽  
Pseudorabies Virus ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Class Iii ◽  
Complex Needs ◽  
Hsv 1

ABSTRACTHerpesvirus membrane fusion depends on the core fusion machinery, comprised of glycoproteins B (gB) and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays, arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy, we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH and replaced the TMD with a glycosylphosphatidylinositol (gpi) anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid linker, or comprising the PrV gD TMD were nonfunctional, highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD, pointing to functional conservation.IMPORTANCEEnveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins, herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona fide fusion proteins, it depends on gH and its complex partner, gL, for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear, and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH, the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.

scholarly journals Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH

2018 ◽  
Vol 92 (9) ◽  
pp. e00084-18 ◽  
Author(s):  
Melina Vallbracht ◽  
Sascha Rehwaldt ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
Walter Fuchs
Keyword(s):  
Pseudorabies Virus ◽  
Viral Envelope ◽  
Fusion Activity ◽  
Virus Particles ◽  
Glycosylation Sites ◽  
Functional Relevance ◽  
And Function ◽  
Cell Spread

ABSTRACTMany viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reducedin vitrofusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in theVaricellovirusgenus, resulted in enhancedin vitrofusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles.IMPORTANCEHerpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport,in vitrofusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

A putative latency promoter/enhancer (PLAT2) region of pseudorabies virus contains a virulence determinant

Microbiology ◽  
2000 ◽  
Vol 81 (2) ◽  
pp. 415-420 ◽  
Author(s):  
Zsolt Boldogköi ◽  
Ferenc Erdélyi ◽  
István Fodor
Keyword(s):  
Pseudorabies Virus ◽  
Cultured Cells ◽  
Outer Region ◽  
Wild Type Virus ◽  
Virulence Determinant ◽  
Internal Repeat ◽  
The Difference ◽  
Repeat Junction ◽  
Intraperitoneal Inoculation

Contradictory data have recently been reported on the role of the unique long–internal repeat junction area of pseudorabies (Aujeszky’s disease) virus (PrV) genome in the virulence of the virus. To investigate the basis of the difference, four recombinant PrVs mutated at the outer region of inverted repeats that involved a putative latency promoter (PLAT2) were constructed in this study. Propagation characteristics of mutant viruses in cultured cells were similar to those of the wild-type virus. However, a 757 bp deletion at this location caused significant reduction in the virulence of PrV after intraperitoneal inoculation of mice and a moderate decrease in the virulence after intracranial inoculation. These results indicate that the PLAT2 region is an important virulence determinant that may be implicated in the neuroinvasive capability of the virus.

scholarly journals Influence of Pseudorabies Virus Proteins on Neuroinvasion and Neurovirulence in Mice

2006 ◽  
Vol 80 (11) ◽  
pp. 5571-5576 ◽  
Author(s):  
Robert Klopfleisch ◽  
Barbara G. Klupp ◽  
Walter Fuchs ◽  
Martina Kopp ◽  
Jens P. Teifke ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Past Research ◽  
Viral Envelope ◽  
Wild Type Virus ◽  
Survival Times ◽  
Glycoprotein E ◽  
Viral Spread ◽  
Adult Mice ◽  
Kinetics Of

ABSTRACT Neurotropism is a distinctive feature of members of the Alphaherpesvirinae. However, its molecular basis remains enigmatic. In the past, research has been focused mainly on the role of viral envelope proteins in modulating herpesvirus neuroinvasion and neurovirulence (T. C. Mettenleiter, Virus Res. 92:192-206, 2003). To further analyze the molecular requirements for neuroinvasion of the alphaherpesvirus pseudorabies virus (PrV), adult mice were infected intranasally with a set of single- or multiple-deletion mutants lacking the UL3, UL4, UL7, UL11, UL13, UL16, UL17, UL21, UL31, UL34, UL37, UL41, UL43, UL46, UL47, UL48, UL51, US3, US9, glycoprotein E (gE), gM, UL11/US9, UL11/UL16, UL16/UL21, UL11/UL16/UL21, UL11/gE, UL11/gM, UL43/gK, UL43/gM, or UL43/gK/gM genes. Neurovirulence was evaluated by measuring mean survival times compared to that after wild-type virus infection. Furthermore, by immunohistochemical detection of infected neurons, the kinetics of viral spread in the murine central nervous system was investigated.

scholarly journals Glycoproteins Required for Entry Are Not Necessary for Egress of Pseudorabies Virus

2008 ◽  
Vol 82 (13) ◽  
pp. 6299-6309 ◽  
Author(s):  
Barbara Klupp ◽  
Jan Altenschmidt ◽  
Harald Granzow ◽  
Walter Fuchs ◽  
Thomas C. Mettenleiter
Keyword(s):  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Virus Entry ◽  
Viral Envelope ◽  
Detectable Effect ◽  
Nuclear Egress ◽  
Viral Glycoproteins ◽  
Simplex Virus

ABSTRACT In the current perception of the herpesvirus replication cycle, two fusion processes are thought to occur during entry and nuclear egress. For penetration, glycoproteins gB and gH/gL have been shown to be essential, whereas a possible role of these glycoproteins in nuclear egress remains unclear. Viral envelope glycoproteins have been detected by immunolabeling in the nuclear membrane as well as in primary enveloped particles in several herpesviruses, indicating that they might be involved in the fusion process. Moreover, a herpes simplex virus type 1 mutant simultaneously lacking gB and gH was described to be deficient in nuclear egress (A. Farnsworth, T. W. Wisner, M. Webb, R. Roller, G. Cohen, R. Eisenberg, and D. C. Johnson, Proc. Natl. Acad. Sci. USA 104:10187-10192, 2007). To analyze the situation in the related alphaherpesvirus pseudorabies virus (PrV), mutants carrying single and double deletions of glycoproteins gB, gD, gH, and gL were constructed and characterized. We show here that the simultaneous deletion of gB and gD, gB and gH, gD and gH, or gH and gL has no detectable effect on PrV egress, implying that none of these glycoproteins either singly or in the tested combinations is required for nuclear egress. In addition, immunolabeling studies using different mono- or polyclonal sera raised against various PrV glycoproteins did not reveal the presence of viral glycoproteins in the inner nuclear membrane or in primary virions. Thus, our data strongly suggest that different fusion mechanisms are active during virus entry and egress.

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