Protein kinase C-delta regulates HIV-1 replication at an early post-entry step in macrophages.

BACKGROUND: Macrophages, which are CD4 and CCR5 positive, can sustain HIV-1 replication for long periods of time. Thus, these cells play critical roles in the transmission, dissemination and persistence of viral infection. Of note, current antiviral therapies do not target macrophages efficiently. Previously, it was demonstrated that interactions between CCR5 and gp120 stimulate PKC. However, the PKC isozymes involved were not identified. RESULTS: In this study, we identified PKC-delta as a major cellular cofactor for HIV-1 replication in macrophages. Indeed, PKC-delta was stimulated following the interaction between the virus and its target cell. Moreover, inhibition of PKC-delta blocked the replication of R5-tropic viruses in primary human macrophages. However, this inhibition did not have significant effects on receptor and co-receptor expression or fusion. Additionally, it did not affect the formation of the early reverse transcription product containing R/U5 sequences, but did inhibit the synthesis of subsequent cDNAs. Importantly, the inhibition of PKC-delta altered the redistribution of actin, a cellular cofactor whose requirement for the completion of reverse transcription was previously established. It also prevented the association of the reverse transcription complex with the cytoskeleton. CONCLUSION: This work highlights the importance of PKC-delta during early steps of the replicative cycle of HIV-1 in human macrophages.

Fusion intermediates of HIV-1 gp41 as targets for antibody production: design, synthesis, and HR1-HR2 complex purification and characterization of generated antibodies.

The objective of this project was to study the interaction between HR1 and HR2, the stability of the complex formed, and to characterize the antibodies produced against monomeric HR1 and HR2 peptides as well as the HR1-HR2 complex. In this work, HR1 was mimicked by peptide N36, and HR2 was mimicked by peptide C34L and its analogues C34M2, C34M3, and C34D. Whereas C34M2 and C34M3 are partially composed of D-amino acids, C34D has same sequence as C34L, but is assembled entirely of D-amino acids. Using CD analysis, SPR assays, and gel filtration chromatography, we demonstrate the physical interaction between N36 and C34L and its analogues C34M2 and C34M3, but not C34D. We show that the HR1-HR2 complex is formed rapidly (<1 min) and remains stable, as demonstrated by its inability, in contrast to each free peptide, to inhibit the formation of syncytia. To generate antibodies with predetermined specificity against the transiently exposed intermediate that corresponds to the six-helix bundle structure, purified preformed HR1-HR2 complex was used, in parallel with monomeric HR1 and HR2 peptides, as immunogens in mice. Although the produced antibodies recognize total HIV-1 envelope glycoproteins in ELISA, they are unable to neutralize HIV-1-mediated fusion at 37 °C. However, if the incubation with these antibodies is carried out at 27 °C, a temperature that allows stabilization of the transient intermediate complex, anti-peptide antibodies are able to bind their corresponding domains in HeLa cells expressing HIV-1 gp41 in co-culture with HeLa CD4-CCR5/CXCR4 during the dynamic mechanism of membrane fusion. In agreement with the latter results, these antibodies, if previously incubated for 2 h at 27 °C, are able to strongly neutralize HIV-1 entry by membrane fusion, as shown by their ability to block the formation of syncytia.

Structure-antigenicity of the V3 region of SIVmac envelope glycoprotein.

The objective of this study was to analyze the immunogenicity and antigenicity of the V3 domain (Cys313-Cys346) of the external envelope glycoprotein gp125 of SIVmac251. The corresponding peptide was synthesized and characterized as linear and cyclic peptides. Our results showed that this region, as for HIV-1, contained an immunodominant epitope. The antigenicity was similar for the linear and cyclic peptides when tested against a panel of 15 sera from SIV infected macaques. Similarly, both peptide structures presented similar immunogenicity as shown by the characterization of the anti-peptide antibodies produced in rabbits against the cyclic and linear forms. But, unexpectedly, the antibodies produced against linear peptides recognized with a relatively higher intensity the native envelope gp140 than those produced against the cyclic structure. Furthermore, we showed that these antibodies recognized better the deglycosylated form of the glycoprotein. But, in contrast to the neutralizing activity obtained with anti-V3 peptides from HIV-1, no antiviral activity was obtained with antibodies generated against linear or cyclic SIVmac V3 peptides.

Development and characterization of peptidic fusion inhibitors derived from HIV-1 gp41 with partial D-amino acid substitutions.

The aim of this study was to design synthetic peptides with D-amino acid substitutions that mimic the human immunodeficiency virus (HIV) gp41 HR2 region. The objective was to develop new and active C34 analogue peptides by introducing D-amino acid point substitutions at nonessential sites for HR1-HR2 interaction without disrupting the structure of the peptide. Herein we report a study with C34L peptide analogues, including the enantiomer peptide C34D, the retro-inverso analogue (RI), and two peptides with D-amino acid point substitutions (C34M2 and C34M3). Our results show that, with the exception of RI, these peptides adopt an alpha-helical structure and are, like C34L, able to interact with HR1, mimicked by the N36 peptide. Furthermore, we show that modifications introduced in C34M2, but not in C34M3, enhance its resistance to trypsin-mediated hydrolysis and increase the stability of C34M2 in physiological medium. Interestingly, our results show that C34 peptide analogues C34M2 and C34M3, but not C34D and its RI analogue, retain their ability to inhibit HIV-1 replication with an efficiency similar to that of the C34L peptide. These data underscore the interest in using D-amino acids at specific sites in the C34 peptide sequence and may lead to a new strategy for the development of more stable and active anti-HIV-1 peptidic drugs.