Intracellular processing, glycosylation, and cell-surface expression of the measles virus fusion protein (F) encoded by a recombinant adenovirus

doi:10.1016/0042-6822(90)90207-8

Virology

Volume 175, Issue 1, March 1990, Pages 262-270

https://doi.org/10.1016/0042-6822(90)90207-8 Get rights and content

Abstract

The membrane fusion protein of measles virus (MVF) is a surface glycoprotein which is essential for initiation of viral infection. The F protein mediates penetration of the host cell through a process of membrane fusion between the viral envelope and the host cell plasma membrane. To study the structure-function relationship of the MVF protein, a recombinant adenovirus, Ad5MVF, was constructed which expressed the F protein in mammalian cells. The MVF gene was inserted into the Ad5 genome by homologous recombination, which resulted in replacement of most of the E1 region. This recombinant virus was stable and replicated efficiently in the 293 cell line which complemented the deleted E1 functions. Human 293 cells infected with Ad5MVF synthesized an authentic MVF protein precursor (F₀) which appeared to be cleaved efficiently to the F₁ and F₂ polypeptides. This recombinant F protein was glycosylated, transported to the cell surface, and found to be capable of inducing syncytia formation and hemolysis of monkey erythrocytes. The hemagglutinin protein (HA), provided by a coinfecting adenovirus, was not able to increase the biological activity of the F protein. Treatment of MV or Ad5MVF-infected cells with tunicamycin, an inhibitor of Winked glycosylation, abolished processing of the F protein. This observation suggests that glycosylation might play an important role in cleavage-dependent activation of the precursor F₀ protein or in its transport to the subcellular region where proteolytic cleavage occurs.

References (32)

G. Alkhatib et al.
The predicted primary structure of the measles virus hemagglutinin
Virology
(1986)
S. Gibson et al.
Fusion of a Sendai mutant deficient in HN protein (ts271) with cardiolipin liposomes
Virology
(1988)
F.L. Graham et al.
A new technique for the assay of infectivity of human adenovirus 5 DNA
Virology
(1973)
M.C. Graves et al.
Measles virus polypeptide synthesis in infected cells
Virology
(1978)
B. Hirt
Selective extraction of polyoma DNA from infected mouse cell cultures
J. Mol. Biol.
(1967)
D.C. Johnson et al.
Abundant expression of herpes simplex virus glycoprotein gB using an adenovirus vector
Virology
(1988)
W.J. Mcgrory et al.
A simple technique for the rescue of early region 1 mutations into infectious human adenovirus type 5
Virology
(1988)
R.G. Paterson et al.
Ability of the hydrophobic fusion-related external domain of a paramyxovirus F protein to act as a membrane anchor
Cell
(1987)
C.D. Richardson et al.
The nucleotide sequence of the mRNA encoding the fusion protein of measles virus (Edmonston strain): A comparison of fusion proteins from several different paramyxoviruses
Virology
(1986)
A. Scheid et al.
Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus
Virology
(1974)

A. Scheid et al.

Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses

Virology

(1977)

K.C. Stallcup et al.

The replication of measles virus in the presence of tunicamycin

Virology

(1981)

T.M. Varsanyi et al.

Isolation and characterization of the measles virus F1 polypeptide: Comparison with other paramyxovirus fusion proteins

Virology

(1985)

G. Alkhatib et al.

High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system

J. Virol.

(1988)

G. Alkhatib et al.

Expression of bicistronic measles virus P/C mRNA by using hybrid adenoviruses: Levels of C protein synthesized in vivo are unaffected by the presence or absence of the upstream P initiator codon

J. Virol.

(1988)

K.L. Berkner et al.

Abundant expression of polyomavirus T antigen and dihydrofolate reductase in an adenovirus recombinant

J. Virol.

(1987)

Cited by (56)

Strategies for enhancing intratumoral spread of oncolytic adenoviruses
2020, Pharmacology and Therapeutics
Citation Excerpt :
Alkhatib et al. investigated the heterologous expression of the F protein of MeV in a non-replicating Ad. They reported that the expression of the F protein induces syncytium formation in HeLa and 293 cell lines (Alkhatib, Richardson, & Shen, 1990). Galanis et al. constructed two Ad vectors expressing H and F proteins, AdH and AdF, and tested their efficacy in a U87 xenograft glioma nude mice.
Oncolytic viruses, effectively replicate viruses within malignant cells to lyse them without affecting normal ones, have recently shown great promise in developing therapeutic options for cancer. Adenoviruses (Ads) are one of the candidates in oncolytic virotheraoy due to its easily manipulated genomic DNA and expression of wide rane of its receptors on the various cancers. Although systematic delivery of oncolytic adenoviruses can target both primary and metastatic tumors, there are some drawbacks in the effective systematic delivery of oncolytic adenoviruses, including pre-existing antibodies and liver tropism. To overcome these limitations, intratumural (IT) administration of oncolytic viruses have been proposed. However, IT injection of Ads leaves much of the tumor mass unaffected and Ads are not able to disperse more in the tumor microenvironment (TME). To this end, various strategies have been developed to enhance the IT spread of oncolytic adenoviruses, such as using extracellular matrix degradation enzymes, junction opening peptides, and fusogenic proteins. In the present paper, we reviewed different oncolytic adenoviruses, their application in the clinical trials, and strategies for enhancing their IT spread.
RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs
2016, Journal of Virological Methods
During 2007–2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0–90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4–66.7% identity). In contrast, this region was highly conserved among the local strains (94.1–100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks.
Mucosal adenovirus-vectored vaccine for measles
2010, Vaccine
Citation Excerpt :
In this study, we evaluated recombinant adenovirus as a vaccine candidate against measles. In line with the previously published data [15–17], we demonstrated that our recombinant adenoviruses were capable to express the biologically active recombinant F and H proteins that were similar to the authentic MV F and H proteins and were present at the surface of the infected cells. In contrast to the previously published data [17], our study showed that the mice vaccinated with the recombinant adenoviruses either intramuscularly or intranasally elicited robust MV-specific antibody immune responses even after a single immunization.
Several problems associated with the available anti-measles vaccine emphasize the need for a single shot anti-measles vaccine which is efficacious by mucosal route of administration and functional in the presence of anti-measles neutralizing antibodies. To achieve these goals, we constructed two recombinant human adenoviruses (collectively designated Ad-F/H) carrying genes for measles virus (MV) fusion (F) and haemagglutinin (H) proteins. Single intranasal or intramuscular vaccination of mice and cotton rats with Ad-F/H elicited high MV-specific serum neutralizing-antibody titers. Furthermore, bronchoalveolar lavage samples from mice vaccinated intranasally with Ad-F/H showed a 100-fold increase in MV-specific IgA titers compared with intramuscularly vaccinated mice. Moreover, Ad-F/H vaccine administered intranasally, but not intramuscularly, completely protected challenged cotton rats from MV replication in the lungs.
Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein
2008, Virology
Citation Excerpt :
Whereas the buried surface area for each monomer in the PIV5 HN dimer is 1810 Å2 the HN dimer-of-dimers interface only involves 10 residues and buries only 657 Å2. For some paramyxoviruses, such as the W3A strain of PIV 5 and perhaps in some strains of measles virus, there is a limited or even lack of requirement for HN/H or G for cell–cell fusion to occur (Alkhatib et al., 1990; Bagai and Lamb, 1995; Horvath and Lamb, 1992; Horvath et al., 1992; Paterson et al., 1985; Vialard et al., 1990). However, it has been shown that even for the relatively HN-independent W3A strain of PIV 5, fusion is still significantly enhanced by the presence of HN (Russell et al., 2001).
The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high α-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.
Effect of the alterations in the fusion protein of measles virus isolated from brains of patients with subacute sclerosing panencephalitis on syncytium formation
2007, Virus Research
Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell–cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell–cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV.
Inhibitory potential of Crotalus durissus terrificus venom on measles virus growth
2003, Toxicon
This paper presents the antiviral activity found in a snake with Crotalus durissus terrificus venom (Cdt), studied by use of microplate inhibition assay, using measles virus (MV). Cdt at concentrations below 100 μg/ml showed no cytotoxicity for Vero cells. This study shows the optimal conditions for cell treatment and infection. Two factors that affect virus binding and infection efficiency were studied: the use of an adsorption step, where infection volume was varied; and the concentration of fetal bovine serum (FBS). The adsorption step, with or without FBS, increased the bound virus percentage, whereas it increased bound virus at equilibrium only in FBS-free until 2.5% FBS. In contrast, the addition of 10% FBS decreased the bound virus percentage.
The inhibition of MV replication in Vero cells was observed when Cdt was added either before or during cell infection with virus. Its inhibitory concentration against MV replication was 0.1 until 100 μg/ml, respectively. The anti-MV effect of the Cdt was gradually decreased when it was added before or during infection, and little inhibition was observed when Cdt was added 1 h after infection, suggesting that the MV infection was inhibited at the time of the initial events such as at the moment of adsorption and penetration of the viral cycle. In conclusion, Cdt contains anti-MV effects that may be of potential clinical interest.

View all citing articles on Scopus

View full text

Intracellular processing, glycosylation, and cell-surface expression of the measles virus fusion protein (F) encoded by a recombinant adenovirus

Abstract

Virology

Virology

Virology

Virology

J. Mol. Biol.

Virology

Virology

Cell

Virology

Virology

Virology

Virology

Virology

High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system

J. Virol.

Expression of bicistronic measles virus P/C mRNA by using hybrid adenoviruses: Levels of C protein synthesized in vivo are unaffected by the presence or absence of the upstream P initiator codon

J. Virol.

Abundant expression of polyomavirus T antigen and dihydrofolate reductase in an adenovirus recombinant

J. Virol.