Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.

Section: Discussionsupporting

confidence: 92%

The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge

Peng

Ponce-de-León

Jiang³

et al. 1998

“…These studies suggested that VZV gL acts as chaperone for gH and is not transported to the cell surface or released into the medium (9,10). For HSV-1 and BHV-1, coprocessing and colocalization of gH and gL on the cell surface have been reported (27,32,75). When expressed in the absence of gH, HSV-1 gL was released into the medium (8).…”

mentioning

confidence: 99%

“…42-kDa protein and a 125-kDa protein, respectively (39,40,77). The importance of complex formation with gL for intracellular transport and processing of gH has been analyzed mostly after transient expression (65), expression from recombinant vaccinia viruses in mammalian cells (3,9,10,15,27,32,40,45,77), or expression from recombinant baculovirus in insect cells (22,23,75). These studies suggested that VZV gL acts as chaperone for gH and is not transported to the cell surface or released into the medium (9,10).…”

mentioning

confidence: 99%

Pseudorabies virus glycoprotein L is necessary for virus infectivity but dispensable for virion localization of glycoprotein H

Klupp

Fuchs

Weiland

et al. 1997

Herpesviruses contain a number of envelope glycoproteins which play important roles in the interaction between virions and target cells. Although several glycoproteins are not present in all herpesviruses, others, including glycoproteins H and L (gH and gL), are conserved throughout the Herpesviridae. To elucidate common properties and differences in herpesvirus glycoprotein function, corresponding virus mutants must be constructed and analyzed in different herpesvirus backgrounds. Analysis of gH ؊ mutants of herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV) showed that in both viruses gH is essential for penetration and cell-to-cell spread and that its presence is required for virion localization of gL. Since gH homologs are found complexed with gL, it was of interest to assess the phenotype of gL ؊ mutant viruses. By using this approach, HSV-1 gL has been shown to be required for entry and for virion localization of gH (C. Roop, L. Hutchinson, and D. Johnson, J. Virol. 67:2285-2297, 1993). To examine whether a similar phenotype is associated with lack of gL in another alphaherpesvirus, PrV, we constructed two independent gL ؊ PrV mutants by insertion and deletion-insertion mutagenesis. The salient findings are as follows: (i) PrV gL is required for penetration of virions and cell-to-cell spread; (ii) unlike HSV-1, PrV gH is incorporated into the virion in the absence of gL; (iii) virion localization of gH in the absence of gL is not sufficient for infectivity; (iv) in the absence of gL, N-glycans on PrV gH are processed to a greater extent than in the presence of gL, indicating masking of N-glycans by association with gL; and (v) an anti-gL polyclonal antiserum is able to neutralize virion infectivity but did not inhibit cell-to-cell spread. Thus, whereas PrV gL is essential for virus replication, as is HSV-1 gL, gL ؊ PrV mutants exhibit properties strikingly different from those of HSV-1. In conclusion, our data show an important functional role for PrV gL in the viral entry process, which is not explained by a chaperone-type mechanism in gH maturation and processing.

“…The gH-gL complex is conserved throughout the herpesviruses, although the nature of the linkage between the two components may differ. In various systems it has been shown that gH requires gL for proper folding and intracellular transport (2,6,32,36,37). In HSV-1, virion localization of gH requires the presence of gL and vice versa (25).…”

mentioning

confidence: 99%

Glycoprotein gL-Independent Infectivity of Pseudorabies Virus Is Mediated by a gD-gH Fusion Protein

Klupp

Mettenleiter

1999

Envelope glycoproteins gH and gL, which form a complex, are conserved throughout the family Herpesviridae. The gH-gL complex is essential for the fusion between the virion envelope and the cellular cytoplasmic membrane during penetration and is also required for direct viral cell-to-cell spread from infected to adjacent noninfected cells. It has been proposed for several herpesviruses that gL is required for proper folding, intracellular transport, and virion localization of gH. In pseudorabies virus (PrV), glycoprotein gL is necessary for infectivity but is dispensable for virion localization of gH. A virus mutant lacking gL, PrV-ΔgLβ, is defective in entry into target cells, and direct cell-to-cell spread is drastically reduced, resulting in only single or small foci of infected cells (B. G. Klupp, W. Fuchs, E. Weiland, and T. C. Mettenleiter, J. Virol. 71:7687–7695, 1997). We used this limited cell-to-cell spreading ability of PrV-ΔgLβ for serial passaging of cells infected with transcomplemented virus by coseeding with noninfected cells. After repeated passaging, plaque formation was restored and infectivity in the supernatant was observed. One single-plaque isolate, designated PrV-ΔgLPass, was further characterized. To identify the mutation leading to this gL-independent infectious phenotype, Southern and Western blot analyses, radioimmunoprecipitations, and DNA sequencing were performed. The results showed that rearrangement of a genomic region comprising part of the gH gene into a duplicated copy of part of the unique short region resulted in a fusion fragment predicted to encode a protein consisting of the N-terminal 271 amino acids of gD fused to the C-terminal 590 residues of gH. Western blotting and radioimmunoprecipitation with gD- and gH-specific antibodies verified the presence of a gDH fusion protein. To prove that this fusion protein mediates infectivity of PrV-ΔgLPass, cotransfection of PrV-ΔgLβ DNA with the cloned fusion fragment was performed, and a cell line, Nde-67, carrying the fusion gene was established. After cotransfection, infectious gL-negative PrV was recovered, and propagation of PrV-ΔgLβ on Nde-67 cells produced infectious virions. Thus, a gDH fusion polypeptide can compensate for function of the essential gL in entry and cell-to-cell spread of PrV.