Glycoprotein D-Independent Infectivity of Pseudorabies Virus Results in an Alteration of In Vivo Host Range and Correlates with Mutations in Glycoproteins B and H

Schmidt, Jerg; Gerdts, Volker; Beyer, Jörg; Klupp, Barbara G.; Mettenleiter, Thomas C.

doi:10.1128/jvi.75.21.10054-10064.2001

Cited by 27 publications

(21 citation statements)

References 44 publications

(66 reference statements)

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“…6), nor were any syncytia seen when transfected C10 cells were stained with Giemsa stain (data not shown). These results provide a clear distinction between the HSV gDgH chimeras and the PrV gDgH protein, as the latter protein replaced gL in several functional assays (11,24,25,49). It was disappointing that chimera 22 was not adequately presented on the cell surface without gL, because we are precluded from assigning a clear function to HSV-1 gH or gL.…”

Section: Resultsmentioning

confidence: 95%

See 1 more Smart Citation

Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras

Cairns

Milne

Ponce-de-León

et al. 2003

J Virol

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In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replicationcompetent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.For many alphaherpesviruses, four glycoproteins are required for virus entry into mammalian cells (32,52,53). In the case of herpes simplex virus (HSV) and bovine herpesvirus type 1, gB, gD, and the gH-gL heterodimer function independently or in concert to effect fusion of the virion envelope with the plasma membrane. All four glycoproteins are essential for the spread of these viruses from cell to cell and for cell fusion (2,35,46,57). However, little is known about the mechanism of these processes. Interestingly, although the same set of four glycoproteins are required for pseudorabies virus (PrV) entry, gD and gL are not essential for cell-cell spread (24, 25). Thus, HSV and PrV glycoproteins have evolved to have somewhat different functions.Klupp and Mettenleiter passaged a gL-null virus in cell culture and obtained a virus, PrV-⌬gLpass, that was able to enter cells, replicate, and spread from cell to cell (24). PrV-⌬gLpass had undergone gene rearrangements such that it lacked wildtype (WT) gH and instead contained a gDgH chimera resulting from a fusion between the first 271 amino acids of gD and the C-terminal 590 am...

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Section: Resultsmentioning

confidence: 95%

“…Work done with PrV demonstrated that the requirement for gL during virus entry could be superseded by a gDgH chimeric protein (24,25,49). We questioned whether a similar protein could be constructed for HSV and if the minimal protein domains needed for entry would mimic those of PrV.…”

Section: Resultsmentioning

confidence: 99%

Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras

Cairns

Milne

Ponce-de-León

et al. 2003

J Virol

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“…Our results raise the possibility that for other herpesviruses lacking gD, gH might have dual function in entry, including receptor binding. Also of interest are the findings that for some gD-containing ␣-herpesviruses, gD-deleted variants have been selected that have a restored ability to spread by cell-cell fusion; mutations in gH are associated with this (45,46). Perhaps the modified gH has acquired receptor binding activity, although this has not yet been demonstrated.…”

Section: Discussionmentioning

confidence: 99%

Interaction of Glycoprotein H of Human Herpesvirus 6 with the Cellular Receptor CD46

Santoro

Greenstone

Insinga³

et al. 2003

Journal of Biological Chemistry

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Human herpesvirus 6 (HHV-6) employs the complement regulator CD46 (membrane cofactor protein) as a receptor for fusion and entry into target cells. Like other known herpesviruses, HHV-6 encodes multiple glycoproteins, several of which have been implicated in the entry process. In this report, we present evidence that glycoprotein H (gH) is the viral component responsible for binding to CD46. Antibodies to CD46 co-immunoprecipitated an ϳ110-kDa protein band specifically associated with HHV-6-infected cells. This protein was identified as gH by selective depletion with an anti-gH monoclonal antibody, as well as by immunoblot analysis with a rabbit hyperimmune serum directed against a gH synthetic peptide. In reciprocal experiments, a monoclonal antibody against HHV-6 gH was found to co-immunoprecipitate CD46. Studies using monoclonal antibodies directed against specific CD46 domains, as well as engineered constructs lacking defined CD46 regions, demonstrated a close correspondence between the CD46 domains involved in the interaction with gH and those previously shown to be critical for HHV-6 fusion (i.e. short consensus repeats 2 and 3).Human herpesvirus 6 (HHV-6) 1 is a member of the ␤-herpesvirus subfamily (reviewed in Ref. 1). Two major HHV-6 variants, A and B, have been defined that form two segregated clusters with unique genetic, biologic, and immunologic characteristics. Primary infection with HHV-6 B occurs almost universally in early childhood and has been etiologically linked to exanthema subitum. In adult life, HHV-6 infection and/or reactivation have been associated with a wide range of diseases, including multiple sclerosis, but most of these associations have yet to be substantiated by rigorous epidemiological studies. In immunocompromised people, including those infected with human immunodeficiency virus, HHV-6 B has been implicated as an opportunistic agent that may cause life-threatening respiratory tract and central nervous system infections, as well as bone marrow or organ graft failure. Moreover, HHV-6 may act as a cofactor to accelerate progression of human immunodeficiency virus disease.The best studied members of the herpesvirus family enter cells by direct fusion with the plasma membrane in a process involving binding of viral glycoproteins to specific protein receptors on the target cell (reviewed in Ref.2). Fusion is also facilitated by virus interactions with cell surface glycosaminoglycans. Recently, we identified CD46 (membrane cofactor protein) as a cellular receptor mediating fusion and entry for both HHV-6 variants (3). This glycoprotein also serves as a cellular receptor mediating critical events in the infectious process of other human pathogens, including binding/fusion/ entry of measles virus, pilus binding of different pathogenic Neisseriae, and adhesion of group A Streptococci (reviewed in Ref. 4). CD46 is a member of the regulators of complement activation (RCA) protein family. It is a type I transmembrane glycoprotein of 45-67 kDa, expressed on most or all human nucleated ce...

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“…For HSV-1, it has been further shown that a defined domain of gD triggers membrane fusion, presumably via interaction with gB or gH (9). Nevertheless, replication-competent PrV and bovine herpesvirus 1 could be isolated after serial passage of gD-deleted viruses, which had acquired compensatory mutations within gB and gH (55,56).…”

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confidence: 99%

Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus

Fuchs

Backović

Klupp

et al. 2012

J Virol

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To investigate the functional relevance of these structures, nonconservative amino acid substitutions were introduced by site-directed mutagenesis. The mutated proteins were tested for correct expression, fusion activity, and functional complementation of gH-deleted PrV. Several single amino acid changes within the flap and the hydrophobic patch were tolerated, and deletion of the glycosylation site had only minor effects. However, multiple alanine substitutions within the flap or the hydrophobic patch led to significant defects. gH function was also severely affected by disruption of the disulfide bond at the C terminus of the flap and after introduction of cysteine pairs designed to bridge the central part of the flap with the hydrophobic patch. Interestingly, all mutated gH proteins were able to complement gH-deleted PrV, but fusion-deficient gH mutants resulted in a pronounced delay in virus entry.

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Glycoprotein D-Independent Infectivity of Pseudorabies Virus Results in an Alteration of In Vivo Host Range and Correlates with Mutations in Glycoproteins B and H

Cited by 27 publications

References 44 publications

Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras

Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras

Interaction of Glycoprotein H of Human Herpesvirus 6 with the Cellular Receptor CD46

Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus

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