Suppression Of Virus Infectivity By Peptides Derived From Human Immunodeficiency Virus Type 1 Vif

The global AIDS epidemic is one of the greatest challenges facing our generation. On Nov. 21, 2006, Report on Global AIDS Epidemic announced by WHO and UNAIDS (The Joint United Nations Program on HIV/AIDS) showed that the people living with HIV had reached 39.5 million, 4.3 million became newly-infected and 2.9 million died in 2006. After afferent phase and diffusion phase, the current condition of AIDS is getting much worse: the plague increases dramatically with more and more incidences. There had been 650 thousand HIV infected people in total in which incident patients occupy 75 thousands at the end of 2005 since the first AIDS case appeared in 1985 in China. The average infection rate was 0.05 percent. Till then, the amount of HIV infection had been the second place across Asia, the 14th place around the world. At present, HIV epidemic is one of the severest plague worldwidely. Anti-retroviral therapeutic of"cocktail"has effecially controlled the spread of the prevalence in developing countries. There stays stll, however, for some negative factors such as the high price, the side effect due to long-term medication, an obstacle in the way conquering this disease.HIV-1 Vif (Viral Infectivity Fetor) protein is required for establishing productive viral infection in "non-permissive" cells, but not in "permissive" cells. Vif-deficient virus particles produced by "non-permissive" cells show non-homogenous and are distinguished from wild type particles by several criteria. Vif, previously known as "sor" or "A", is one of six accessory proteins encoded by all lentivinises except equine infectious anemia virus (EIAV). Lentivirus vif coding region are very similar to one another in their genomic location and molecular weight. HIV-l Vif is a highly basic protein of 192 amino acid residues with a molecular mass of -23kD. Vif is translated from a singly spliced mRNA in a Rev-depemdent manner at the late stage of the virus replication. Computer database searches have not revealed any significant homology with any known cellular proteins. Vif is highly conserved among primary HIV-1 isolates.There is a wide consensus that the requirement of Vif for HIV-1 replication is cell-specific. Vif is indispensable for establishment productive HIV-1 infection in peripheral blood lymphocytes (PBL) and monocyles/macrophages. the major target cells for HIV-1 infection in AIDS patients. For example. Jurkat. CEM-SS, and SupTl cells do not require Vif for HIV-1 replication (permissive cells); for H9 cells, CEM cells, and primary blood-derived monocytes (PBMC), however, Vif is essential (non-permissive cells). The vif gene of primary lentivirus SIVmac can compensate for vif-negative HIV-1 phenotype .whereas vif gene from non-primate lentivinises (visna virus, feline immunodeficiency virus) can not, indicating that Vif may interact with virus-specfic gene products.Studies have shown that Vif binds to HIV-1 RNA but the mechanisms by which this might lead to the particular viral phenotype of Vif mutant viruses remained unclear. A breakthrough came in 1998, when two groups demonstrated that there seemed to be an endogenous cellular cofactor inhibitor of HIV-1. which was overcome by the viral Vif protein. This factor appeared to alter Vif mutant HIV-1 at the late stages of the viral life cycle in non-permissive producer cells. These findings were demonstrated using heterokaryon formation between permissive and non-permissive cell-types. It was demonstrated that there appeared to be a specific human gene product which inhibit HIV-1 infection but whose effects are suppressed by Vif proteins. Utilizing a novel polymerase chain reaction (PCR)-based cDNA subtraction strategy with non-permissive and permissive cell lines, which were genetically related but had different permissivity for Vif function, a cellular inhibitor representing a cofactor for Vif phenotype was demonstrated to exist. It was initially named CEM15, based on the cell line from which it was cloned. They did a relatively comprehensive job to demonstrating CEM15 presence in all non-permissive cells, but its absence in all permissive cells. Of great importance in demonstrating the specificity of this effect were the experiments which demonstrated that transfecling CEM15 in permissive cells inhibited HIV-1 vif-deleted infectivity.Studies indicate that APOBEC3G is an endogenous viral inhibitor. APOBEC3G expresses only in "non-permissive" cells and its expression in "permissive" cells will generate a "non-permissive" phenotype. Recent studes indicate that this natural antiviral factor is actually a human cytidine deaminase which exhibits broad suppressive activities against HIV-l, SIV, MLV and HBV. Specially, in the absence of HIV-1 Vif, AP0BEC3G is packaged into viral particles, mediates antiviral activity by inducing C-U mutation in newly synthesized minus-strand DNA during reverse transcnption and results in deleterious G-A hypermutation in the coding strand. HIV-1 Vif can block the AP0BEC3G virion packaging by leading its ubiquitination and degradation via the proteosome dependent pathway. Further studies show that Vif interacts with cellular proteins ElonginB, ElonginC, Cul5 and Rbxl to form an E3 ubiquitin ligase complex which is critical for the activity of Vif to target APOBEC3G and neutralize its antiviral function. CEM15 is also called APOBEC3G, belongs to a family of proteins that have cytidine deaminase activity, but its cellular function is still unknown. When expressed in E. coli, proteins from this family have been shown to induce DNA mutations and may show a preference for single-stranded DNA. Vif-mutant, but not wild-type, HIV-1 viruses produced in the presence of APOBEC3G undergo hypermutations in newly synthesized viral DNA, presumably due to C-to-U modification during minus-strand viral DNA synthesis. Although it was shown initially that APOBEC3G was incorporated efficiently into wild-type and Vif-mutant virions, recent studies have shown that HIV-1 Vif could efficiently prevent virion incorporation of APOBEC3GThese findings make Vif-E3-ligase be an ideal target for the development of anti-HIV therapeutics. However, the molecular mechanism of Vif in viral life cycle remains to be further determined and structure of Vif-E3-complex is still unknown. So. it is quite difficult to generate a small molecule inhibitor to block Vif function at the present time. It has been observed that HIV-1 Vif binds with ElonginC through a unique SOCS-box including the SLQ motif which is also required for the function of E3 ligase. So an interesting question is raised whether a barrier against interaction between these proteins can act as antiviral inhibitors in cells.In this report, we reveal that peptides derived from the binding regions of Vif and ElonginC are able to suppress HIV-1 infectivity in "non-permissive" cells but not in "permissive cells". Moreover, a Vif-derived peptide, peptide 10. containing the SOCS-box motif is found to be the most potent inhibitor and further research for inhibition mechanism is also shown in the work. Thus, we suggest that Vif-ElonginC interaction could be a promising target for anti-HIV agent development and these peptides may be used as lead compounds to design novel HIV-1 infectivity inhibitors. The SOCS box domain of HIV-1 Vif is critical for its binding to ElonginC and recruitment of a Cul5 E3 ligase, which is required to neutralize APOBEC3G through proteasome mediated degradation and support the production of infectious particles.In this study we identified the peptide 10, corresponding to Vif144-155, can halt the degradation of APOBEC3G induced by Vif and result in loss of virus infectivity produced only in non-permissive cells. We also demonstrated peptide 10 can bind spectificialy with ElonginC and block the Vif-ElonginC interaction. Furthermore, peptide 10 mimiced the structure of Vif SOCS box domain in a simulation system with a high binding affinity to the hydrophobic interface of ElonginC. With the change of several key sites, the peptide 10 showed weak interaction to ElonginC. This model was consistent with the finding that the site-mutant peptides had a less ability to act as an HIV-I inhibitor. In line with our results, it was reasonable to assume that viral infectivity inhibition induced by peptidel 0 occured, at least in part, through competing with Vif on the ElonginC binding sites. Moreover, it abolished Vif function as a APOBEC3G neutralizer.Based on the recent study, we also synthesized the peptides (3mer to 15 mer) derived from C-terminal of ElonginC (95-109) and analysed the ability of these peptides to serve as an inhibitor. We note that these peptides have less ability of suppressing virus infectivity than peptidelO. The difference may be induced for this domain was not the main binding region of ElonginC to Vif. This hypothesis was proved with our three-dimensional simulation result of Vif-ElonginC-peptidelO complex structure, showing that peptidelO formed potent interactions with 79Tyr and 93Phe of ElonginC. Also we found there was a large negative electricity potential region containing three Glu and one Asp in which the peptide 10 just inserted. It was conceivable that although all these ElonginC binding sites were sequentially remote, they were spatially close and could therefore form an active site which played a pivotal role in regulating the proteins interaction. It was also unknown for us whether the binding of peptide to ElonginC induced structural modification. Answers to these questions were dependent on resolving thoroughly the three-dimensional structure of the Vif protein and E3 complex.Interestingly, the increased stability of HIV-1 Vif in the presence of peptidel0 suggested that the same Cul5 E3 ligase could serve a dual purpose of regulating both Vif and APOBEC3G This result was also supported by the experiment of cells, with treatment of peptide 10, transferred with vectors expressing both APOBEC3G and Vif. Our data were consistent with a recent study showing that polyubiquhination of HIV-1 Vif, reduced in the presence of Cul5 mutants, was reduced in a BC-box mutant of Vif that does not associate with E3 ligase. In accordance with our findings, it can be expected that peptidelO treatment would reduce the package of APOBEC3G into HIV-1 virus from the transfected cells. However, we failed to demonstrate any effect on that. We suggested therefore that elimination of APOBEC3G by proteasome degradation may not be the sole prerequisite for production of infectious virus.Because of the essential role of Vif in HIV-1 replication, we believe that it is a particularly appealing therapeutic target for anti-virus agent development. Indeed, preliminary reports have indicated that peptides from several Vif motifs, including the PPLP motif nearby SOCS box, are effective inhibitor through different ways. This report has provided a novel strategy for development of HIV-1 inhibitor by abolishing Vif-ElonginC interaction. It is also interesting to further investigate the structural mechanism of peptidelO as inhibitor and develop more potent nonpeptide inhibitors.