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The Underlying Mechanism of 3-Hydroxyphthalic Anhydride-Modified Bovine Beta-Lactoglobulin to Block Human Papillomavirus Entry Into the Host Cell.

ABSTRACT: We have previously demonstrated that 3-hydroxyphthalic anhydride (3HP)-modified bovine beta-lactoglobulin (3HP-?-LG) is highly effective in inhibiting entry of pseudovirus (PsV) of high- and low-risk human papillomavirus (HPV) into the target cell. Intravaginally applied 3HP-?-LG-containing vaginal gel could significantly inhibit HPV infection and reduce viral load in the cervical region. However, we still do not understand the underlying molecular mechanism by which 3HP-?-LG is able to inhibit HPV infection. Here, though, we showed that 3HP-?-LG did not inactivate HPV PsV, but rather blocked entry of HPV PsV into the target cell via its interaction with virus, not cell. It bound to the positively charged region in the HPV L1 protein, suggesting that 3HP-?-LG binds to HPV L1 protein through the interaction between the negatively charged region in 3HP-?-LG and the positively charged region in HPV L1 protein, thus competitively blocking the binding of HPV to the receptor on the basement membrane in vaginal mucosa. Although 3HP-modified chicken ovalbumin (3HP-OVA) also carries high net negative charges, it exhibited no anti-HPV activity, suggesting that the interaction between 3HP-modified protein and HPV L1 protein relies on both electrostatic and matchable conformation of the binding sites in both proteins. When topically applied, 3HP-?-LG did not enter the host cell or blood circulation. These findings suggest that 3HP-?-LG targets HPV L1 protein and blocks HPV entry into the host cell, thus being safe and effective for topical application in the treatment of HPV infection.

SUBMITTER: Hua C

PROVIDER: S-EPMC6775479 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The Underlying Mechanism of 3-Hydroxyphthalic Anhydride-Modified Bovine Beta-Lactoglobulin to Block Human Papillomavirus Entry Into the Host Cell.

Hua Chen C Zhu Yun Y Wu Congquan C Si Lulu L Wang Qian Q Sui Long L Jiang Shibo S

Frontiers in microbiology 20190926

We have previously demonstrated that 3-hydroxyphthalic anhydride (3HP)-modified bovine beta-lactoglobulin (3HP-β-LG) is highly effective in inhibiting entry of pseudovirus (PsV) of high- and low-risk human papillomavirus (HPV) into the target cell. Intravaginally applied 3HP-β-LG-containing vaginal gel could significantly inhibit HPV infection and reduce viral load in the cervical region. However, we still do not understand the underlying molecular mechanism by which 3HP-β-LG is able to inhibit ...[more]

PMID: 31611852

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Project description:Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. Beta-lactoglobulin A has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCI at pH 7.5, producing a modified beta-lactoglobulin (TNB-bIg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-bIg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-bIg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the alpha-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-bIg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-bIg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-bIg were monomeric. The unfolding transition of TNB-bIg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-bIg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-bIg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-bIg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.

Project description:Like other human-pathogenic arenaviruses, Lujo virus (LUJV) is a causative agent of viral hemorrhagic fever in humans. LUJV infects humans with high mortality rates, but the susceptibilities of other animal species and the molecular determinants of its host specificity remain unknown. We found that mouse- and hamster-derived cell lines (NIH 3T3 and BHK, respectively) were less susceptible to a replication-incompetent recombinant vesicular stomatitis virus (Indiana) pseudotyped with the LUJV glycoprotein (GP) (VSVΔG*-LUJV/GP) than were human-derived cell lines (HEK293T and Huh7). To determine the cellular factors involved in the differential susceptibilities between the human and mouse cell lines, we focused on the CD63 molecule, which is required for pH-activated GP-mediated membrane fusion during LUJV entry into host cells. The exogenous introduction of human CD63, but not mouse or hamster CD63, into BHK cells significantly increased susceptibility to VSVΔG*-LUJV/GP. Using chimeric human-mouse CD63 proteins, we found that the amino acid residues at positions 141 to 150 in the large extracellular loop (LEL) region of CD63 were important for the cellular entry of VSVΔG*-LUJV/GP. By site-directed mutagenesis, we further determined that a phenylalanine at position 143 in human CD63 was the key residue for efficient membrane fusion and VSVΔG*-LUJV/GP infection. Our data suggest that the interaction of LUJV GP with the LEL region of CD63 is essential for cell susceptibility to LUJV, thus providing new insights into the molecular mechanisms underlying the cellular entry of LUJV and the host range restriction of this virus. IMPORTANCE Lujo virus (LUJV) infects humans with high mortality rates, but the host range of LUJV remains unknown. We found that rodent-derived cell lines were less susceptible to LUJV infection than were human-derived cell lines, and the differential susceptibilities were determined by the difference of CD63, the intercellular receptor of LUJV. We further identified an amino acid residue on human CD63 important for efficient LUJV infection. These results suggest that the interaction between LUJV glycoprotein and CD63 is one of the important factors determining the host range of LUJV. Our findings on the CD63-regulated susceptibilities of the cell lines to LUJV infection provide important information for the development of anti-LUJV drugs as well as the identification of natural hosts of LUJV. Importantly, our data support a concept explaining the molecular mechanism underlying viral tropisms controlled by endosomal receptors.

Dataset Information

The Underlying Mechanism of 3-Hydroxyphthalic Anhydride-Modified Bovine Beta-Lactoglobulin to Block Human Papillomavirus Entry Into the Host Cell.

Publications

The Underlying Mechanism of 3-Hydroxyphthalic Anhydride-Modified Bovine Beta-Lactoglobulin to Block Human Papillomavirus Entry Into the Host Cell.

Similar Datasets

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