Abstract
HIV-1 progeny are released from infected cells as immature particles that are unable to infect new cells. Gag-Pol polyprotein dimerization via the reverse transcriptase connection domain (RTCDs) is pivotal for proper activation of the virus protease (PR protein) in an early event of the progeny virus maturation process. Thus, the RTCD is a potential therapeutic target for a broadly effective anti-HIV agent through impediment of virus maturation. In this study, human single-chain antibodies (HuscFvs) that bound to HIV-1 RTCD were generated using phage display technology. Computerized simulation guided the selection of the transformed Escherichia coli-derived HuscFvs that bound to the RTCD dimer interface. The selected HuscFvs were linked molecularly to human-derived-cell-penetrating peptide (CPP) to make them cell-penetrable (i.e., become transbodies). The CPP-HuscFvs/transbodies produced by a selected transformed E. coli clone were tested for anti-HIV-1 activity. CPP-HuscFvs of transformed E. coli clone 11 (CPP-HuscFv11) that presumptively bound at the RTCD dimer interface effectively reduced reverse transcriptase activity in the newly released virus progeny. Infectiousness of the progeny viruses obtained from CPP-HuscFv11-treated cells were reduced by a similar magnitude to those obtained from protease/reverse transcriptase inhibitor-treated cells, indicating anti-HIV-1 activity of the transbodies. The CPP-HuscFv11/transbodies to HIV-1 RTCD could be an alternative, anti-retroviral agent for long-term HIV-1 treatment.
Highlights
Laboratory-adapted HIV-1DA5 (CXCR4 tropic CRF01_AE strain), originally isolated from HIV-infected Thai subjects [19], and H9 cell line were obtained from the Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
The plasmid pRARE2 was from E. coli Rosetta 2 (DE3), while the plasmid pYidC was constructed by cloning yidC into a pACYC vector backbone (Figure S1)
The reverse transcriptase (RT) activity of the cell-penetrating peptide (CPP)-HuscFv11-exposed virus particles, derived from the treatThe RT activity of the CPP-HuscFv11-exposed virus particles, derived from the treatment assay, was reduced to 48.4% compared to the viruses in medium alone (Medium)
Summary
HIV is a retrovirus that belongs to the genus Lentivirus within the family Retroviridae. HIV infects vital cells of the human immune system, monocytes/macrophages and CD4+ T cells, by using cell surface CD4 molecules as a primary receptor along with the human chemokine receptors, CCR5 and CXCR4, as co-receptors [1,2,3]. The infection subverts dendritic cell functions to enhance the virus entry to key target cells and evade immune mechanisms for virus perpetuation and transmission [4]. Long-term HIV infection without treatment leads to progressive immune deficiency and fatal opportunistic infections and/or cancer, called acquired immunodeficiency syndrome (AIDS). The great majority of infection globally is caused by HIV-1 [5]. HIV-1 was found to be more virulent and more infectious than
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