Effect of the tri-peptide glycyl-prolyl-glycine amide on HIV-1 replication
Author: Su, Jin
Date: 2000-10-13
Location: Birkeaulan 2, F51, hus F, plan 5, Huddinge universitetssjukhus
Time: 09.00
Department: Institutionen för immunologi, mikrobiologi, patologi och infektionssjukdomar / Department of Immunology, Microbiology, Pathology and Infectious Diseases
Abstract
The human immunodeficiency virus type 1 (HIV-1) is the etiological agent responsible for AIDS worldwide. The aim of this thesis was to evaluate the anti-HIV-1 activity of short peptides and to investigate their mode of inhibition. Short peptides (2-4 amino acids) with sequence homology to the conserved RGPGR crown of the V3 loop of the viral envelope glycoprotein gpl20 were found to inhibit HIV-1 replication, among which the tri-peptide GPG-NH2 was most active. The tri-peptide showed no toxic effects in vitro, and no influence on proliferation of human eel Is. The antiviral activity was shown to be HIV specific. Replication of all the HIV-1 laboratory strains and clinical isolates tested were blocked by this peptide, including strains resistant to both RT and protease inhibitors, and strains resistant to [beta]-chemokines. The range of IC50 was 2.7-37 [my]M. The peptide GPG-NH2 with D-form praline instead of the L-form did not inhibit HIV-1 replication, and the peptide GPG-COOH, ended with carboxyl group as common peptides, showed no effects on viral replication either. GPG-NH2 added to the antiviral effect of both zidovudine (RT inhibitor) and ritonavir (protease inhibitor) as tested in vitro.
To determine whether GPG-NH2 was inhibiting the early steps of HIV-1 replication cycle, studies were performed to define the effects of GPG-NH2 on the early events including CD4 binding, coreceptor usage and penetration into cells. Whether GPG-NH2 influenced viral reverse transcription, mRNA synthesis and protein translation was also analyzed. The peptide did not affect the early events of the virus replication such as adsorption or coreceptor usage. Besides, HIV-1 having GLG instead of GPG in the V3 loop was still inhibited by the peptide. To reassure that GPG-NH2 did not affect the entry steps that mainly involved the envelope glycoproteins, we constructed the mutant HIV-1 virus deleted of GPG-motif in the V3 loop based on the infectious clone pBru-2. This mutant virus was found to be still infectious in some human cells including MT-2, C8166, C91 -PL, Molt-3, THP- 1 and PBMC derived dendritic cells, and it was still sensitive to GPG-NH2. GPG-NH2 had no discernible effect on any of the other steps in the virus replication cycle tested, including viral DNA synthesis, mRNA synthesis or splicing, viral protein translation, tat-TAR transactivation, or protease activity. However, an increased SDS-PAGE mobility of gp160/120 was observed at high concentrations of GPG-NH2. GPG-NH2 inhibited the replication of HIV-1 by an apparently new mode of action. Screening of the tri-peptide amides corresponding to the C-terminal domain of the HIV-1 capsid protein p24 for their antiviral activity revealed that more tri-peptide amides could suppress HIV-1 replication. Analyzed by affinity capillary electrophoresis, we showed that GPG-NH2, and some other amidated tri-peptides from the C-terminal domain of p24, could bind to the p24 protein therefore probably could interfere with viral assembly. In an effort to evaluate viral assembly using transmission electron microscopy (TEM), we observed that 45-52% of the viral particles produced from infected HUT78 or induced ACH-2 cells in the presence of GPG-NH2 were with various misassembled core structures as compared to 9-18% in the control untreated cultures. Another striking finding was that a unique feature of nodular structures was observed associated with cell outer membrane only in the treated cultures. In the presence of GPG-NH2, the frequency of the nodular structures was 74% in the induced ACH-2 cultures and 65% in the infected HUT78 Cultures. With immuno-EM analysis, these nodules assembled on the cell membrane bound anti-p24 antibody. Our results indicated that GPG-NH2 inhibited HIV-1 replication by interfering with the viral capsid formation and viral assembly. GPG-NH2 might prove useful by itself or as a lead compound for the treatment of drug resistant HIV-1. Highlighted by the unique mode of action, applying tri-peptide amides to target viral assembly therefore might be a new strategy for drug design and antiviral therapy.
To determine whether GPG-NH2 was inhibiting the early steps of HIV-1 replication cycle, studies were performed to define the effects of GPG-NH2 on the early events including CD4 binding, coreceptor usage and penetration into cells. Whether GPG-NH2 influenced viral reverse transcription, mRNA synthesis and protein translation was also analyzed. The peptide did not affect the early events of the virus replication such as adsorption or coreceptor usage. Besides, HIV-1 having GLG instead of GPG in the V3 loop was still inhibited by the peptide. To reassure that GPG-NH2 did not affect the entry steps that mainly involved the envelope glycoproteins, we constructed the mutant HIV-1 virus deleted of GPG-motif in the V3 loop based on the infectious clone pBru-2. This mutant virus was found to be still infectious in some human cells including MT-2, C8166, C91 -PL, Molt-3, THP- 1 and PBMC derived dendritic cells, and it was still sensitive to GPG-NH2. GPG-NH2 had no discernible effect on any of the other steps in the virus replication cycle tested, including viral DNA synthesis, mRNA synthesis or splicing, viral protein translation, tat-TAR transactivation, or protease activity. However, an increased SDS-PAGE mobility of gp160/120 was observed at high concentrations of GPG-NH2. GPG-NH2 inhibited the replication of HIV-1 by an apparently new mode of action. Screening of the tri-peptide amides corresponding to the C-terminal domain of the HIV-1 capsid protein p24 for their antiviral activity revealed that more tri-peptide amides could suppress HIV-1 replication. Analyzed by affinity capillary electrophoresis, we showed that GPG-NH2, and some other amidated tri-peptides from the C-terminal domain of p24, could bind to the p24 protein therefore probably could interfere with viral assembly. In an effort to evaluate viral assembly using transmission electron microscopy (TEM), we observed that 45-52% of the viral particles produced from infected HUT78 or induced ACH-2 cells in the presence of GPG-NH2 were with various misassembled core structures as compared to 9-18% in the control untreated cultures. Another striking finding was that a unique feature of nodular structures was observed associated with cell outer membrane only in the treated cultures. In the presence of GPG-NH2, the frequency of the nodular structures was 74% in the induced ACH-2 cultures and 65% in the infected HUT78 Cultures. With immuno-EM analysis, these nodules assembled on the cell membrane bound anti-p24 antibody. Our results indicated that GPG-NH2 inhibited HIV-1 replication by interfering with the viral capsid formation and viral assembly. GPG-NH2 might prove useful by itself or as a lead compound for the treatment of drug resistant HIV-1. Highlighted by the unique mode of action, applying tri-peptide amides to target viral assembly therefore might be a new strategy for drug design and antiviral therapy.
List of papers:
I. Su J, Andersson E, Horal P, Naghavi MH, Palm A, Wu Y-P, Eriksson K, Jansson M, Wigzell H, Svennerholm B, Valne A (2000). The non-toxic tripeptide glycyl-prolyl-glycine amide inhibits the replication of HIV-1. J Hum Virol. 4(1):1-7.
Pubmed
II. Su J, Palm A, Wu Y, Sandin S, Hoglund S, Vahlne A (2000). Deletion of the GPG motif in the HIV type 1 V3 loop does not abrogate infection in all cells. AIDS Res Hum Retroviruses. 16(1):37-48.
Pubmed
III. Su J, Vahlne A (2000). Coreceptor usage of HIV-1 after deletion of Gly317-Pro318-Gly319 motif in the gp120 V3 loop. AIDS. 14(1):91-92.
Pubmed
IV. Su J, Naghavi MH, Jejcic A, Horal P, Furuta Y, Wu Y-P, Li S-L, Hall WH, Goobar-Larsson L, Svennerholm B, Vahlne A (2000). The tri-peptide GPG-NH2 does not affect the early steps of the HIV-1 replication. J Hum Virol. 4(1):8-15.
Pubmed
V. Höglund S, Su J, Sandin S, Vegvari A, Hjertén S, Sintorn I-M, Foster H, Wu Y, Goobar-Larsson L, Nyström I, Vahlne A (2000). Tri-peptide interference with viral capsid assembly; a new strategy for antiviral therapy. [Submitted]
I. Su J, Andersson E, Horal P, Naghavi MH, Palm A, Wu Y-P, Eriksson K, Jansson M, Wigzell H, Svennerholm B, Valne A (2000). The non-toxic tripeptide glycyl-prolyl-glycine amide inhibits the replication of HIV-1. J Hum Virol. 4(1):1-7.
Pubmed
II. Su J, Palm A, Wu Y, Sandin S, Hoglund S, Vahlne A (2000). Deletion of the GPG motif in the HIV type 1 V3 loop does not abrogate infection in all cells. AIDS Res Hum Retroviruses. 16(1):37-48.
Pubmed
III. Su J, Vahlne A (2000). Coreceptor usage of HIV-1 after deletion of Gly317-Pro318-Gly319 motif in the gp120 V3 loop. AIDS. 14(1):91-92.
Pubmed
IV. Su J, Naghavi MH, Jejcic A, Horal P, Furuta Y, Wu Y-P, Li S-L, Hall WH, Goobar-Larsson L, Svennerholm B, Vahlne A (2000). The tri-peptide GPG-NH2 does not affect the early steps of the HIV-1 replication. J Hum Virol. 4(1):8-15.
Pubmed
V. Höglund S, Su J, Sandin S, Vegvari A, Hjertén S, Sintorn I-M, Foster H, Wu Y, Goobar-Larsson L, Nyström I, Vahlne A (2000). Tri-peptide interference with viral capsid assembly; a new strategy for antiviral therapy. [Submitted]
Issue date: 2000-09-22
Publication year: 2000
ISBN: 91-628-4326-5
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