| Indentification of potential target for antiviral drugs and development ofeffective HIV/AIDS vaccine are main goals worldwide in the pursuit of theprevention and eradication of HIV-1infection as well as blocking of its transmission.HIV-1viral infectivity factor (Vif) protein is essential for viral replication. Vifrecruits cellular ElonginB/C-Cullin5E3ubiquitin ligase to target the host antiviralprotein APOBEC3G (A3G) for proteasomal degradation. In the absence of Vif, A3Gis packaged into budding HIV-1virions and introduces multiple mutations in thenewly synthesized minus-strand viral cDNA to restrict virus replication. Thus, theA3G-Vif-E3complex represents an attractive target for development of novelanti-HIV drugs.In the first part of this study, we identified a potent small molecularcompound (VEC-5) by virtual screening and validated its anti-Vif activity throughbiochemical analysis. We showed that VEC-5inhibited viral replication only inA3G-positive cells. Treatment with VEC-5increased cellular A3G levels when Vifwas coexpressed and enhanced A3G incorporation into HIV-1virions to reduce viralinfectivity. Coimmunoprecipitation and computational analysis further attributed theanti-Vif activity of VEC-5to the inhibition of Vif from direct binding to theElonginC protein. Thus, VEC-5was the first HIV-1Vif inhibitor that targets theVif-ElonginC interaction. Structural modification of VEC-5was conductedsystematically.207novel compounds were synthesized and evaluated by antiviralactivity. The structure-activity relationship revealed that the hydrophobic groups onthe both sides of VEC-5played key roles in its antiviral activity. The π-π interactionbetween the naphthyl and the Tyr76was necessary for preservation of activity.Theactivity decreased when the left phenyl ring was replaced by unaryl rings or thecarbonyl group was transformed to other groups. The activity was also reduced when the naphthyl was replaced by substituted phenyl rings. However, the activity wasrecovered when a methanamine group was introduced into the structure, in which thearyl ring stretched into the hydrophobic pocket. The activity could also be preservedby substitution on both sides. The indolizine core structure of VEC-5served as alinker to support the structure of the compound. These findings support the notionthat suppressing Vif function can liberate A3G to carry out its antiviral activity anddemonstrate that regulation of the Vif-ElonginC interaction is a novel target for smallmolecule inhibitors of HIV-1.Following peak viremia, HIV-1viral loads decrease to the initial set point6months after infection and remain at this level for a long time during the course ofchronic infection. Viral fitness has been considered as an important factor for thepathogenesis and transmission of HIV-1.Reduced viral fitness may associate withlower viral load and slow disease progression. However, dynamic change of viralfitness over the course of disease has not been precisely elucidated. In the secondpart of this study, we examined the fitness difference between multiple pairs of6-month virus (6mo),2-year virus (2yr) and their parental transmitted/founder (T/F)virus to determine the correlation between viral fitness and viral load.From14infected individual,12pairs of T/F and6mo viruses as well as2pairs of T/F and2yrviruses were generated. A direct comparison between the T/F viruses and6mo or2yrviruses from the same subjects was then performed to measure the relative fitnessdifferences between the acute and later viruses. In9of12pairs,6mo viruses wereoutcompeted by their cognate T/F within a single passage. In2pairs (CH42andCH77), T/F virus did not show a competitive advantage over6mo virus in singlepassage. However, when the competition was performed in multiple passages, thesetwo T/F viruses evidently outcompeted6-month viruses. In4pairs of viruses (CH40,CH58, CH77and CH470), this outgrowth of T/F over6-month viruses was observedeven with10-fold excess of the6-month viruses in the initial inoculum, indicatingsignificant fitness lost in6-month virus. These resultes suggest that the low viralfitness of6mo viruses play an important role in maintenance of low set point viral loads in HIV-1infection. Surprisingly, the2yr viruses were more fit than its cognateT/F. The2yr viruses almost completely dominated over the T/F. The data showedthat some viruses could gain fitness during chronic infection and became even morefit than their cognate T/F viruses. Such increased fitness might play a critical role inmaintenance of relative high viral loads by fighting against immune selectionpressure and final progress to AIDS. Based on these findings, a U shape curve wasproposed to elucidate the dynamic change of viral fitness over the course of disease.Viral fitness declines within6months of infection and rebound to a higher level inchronic infection. These data show that HIV-1fitness constantly changing during thecourse of infection, which may be a determining factor in disease progression. |
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