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1. Title In Here: Title In Here: Title In Here: Title in Here: Title In Here – put your title in here Author Number one*, Author Number Two and Author Number Three Department of Mayo Foundation, Mayo Clinic, Rochester MN 55905 USA Subspeciality Area or other afflilation if necessary Results INTRODUCTION After 7 months in culture (with 21 cell-free infection steps), we obtained viruses which replicated more efficiently than parental NL4-3 in MT4-TRIM5arh clones. PCR of the MA-CA region of the virus revealed common mutations (H87Q and V86A) in the cyclophilin-binding loop of HIV-1 Gag. We and others have shown that TRIM5αrh-expressing lymphocytes are strongly resistant to HIV-1 replication, thus making TRIM5αrh a promising therapeutic gene for AIDS gene therapy. Several groups have shown that the replacement of HIV-1 capsid sequence with the corresponding SIVmac sequence allows the virus to become resistant to TRIM5arh-mediated post-entry restriction. However, it remains elusive whether HIV-1 can naturally acquire TRIM5arh-resistant mutations. In this study, we determined whether or not HIV-1 could achieve escape mutations through long-term cultivation in a restrictive cellular environment. Serial cell-free infections of NL4-3 in TRIM5arh-expressing MT4 cells Mutations in the Gag -- H87Q Capsid mutation (3/8) -- V86A/H87Q Capsid mutation (5/8) Mutations in the Vif -- No mutation in the vif gene (N=3) In order to accelerate the evolution of HIV-1 to escape the TRIM5a-mediated antiviral activities, we serially passaged HIV-1 (NL4-3) in a TRIM5arh-expressing MT4 cell clone, which was 20-fold more resistant to a HIV-1 vector than in parental MT4 cells. Based on these results, we created an HIV-1 NL4-3 mutant with V86A and H87Q substitutions (NLesc). Procedure Replication Infection Control and TRIM5arh expressing MT4 clones were infected with NL4-3, NLesc or NLSCyp equivalent to 0.4 ng of RT at day 0. At various time points, supernatants were collected and subjected to RT-ELISA. NLSCyp was used as an HIV-1 control, which has the CA sequence of SIV. NL4-3 and NLesc viral stocks were made from 293T by proviral transfection, then viral titers in the supernatant were determined as RT copy#/mg of genome. Control and TRIM5arh expressing MT4 clones were infected with various RT amounts of NL4-3 or NLesc. Eight hours post-infection, cellular DNA was subjected to quantative PCR to determine the number integrated proviral copies. NLesc replicated more efficiently than NL4-3 in TRIM5arh expressing MT4 cells. NLesc was more resistant to the TRIM5arh-mediated early-restriction. Since the infectivity of NLesc was similar to that of NL4-3 in the absence of TRIM5arh (in MT-G12 cells), the resistance is likely due to the escape from TRIM5arh recognition, rather than increased general infectivity of the mutant. Production Transient expression of TRIM5arh in 293T cells Stable expression of TRIM5a in MT4 cells Statistics 293T cells were transfected with pNL4-3 or pNLesc proviral plasmid. Forty-eight hours post-transfection, virions were collected by centrifugation through 20% sucrose layer. Then, cell and virion lysates were subjected to immunoblot analysis. 293T cells transfected with pNL4-3, pNLesc or pNLSCyp together with pRhT5a (Rh), pHuT5a (Hu) or pcDNA3.1 (Ct). Forty-eight hours post-transfection, viral titers in the supernatant were assayed in GHOST cells. SCyp is an NL4-3 mutant made by replacing the CA region with the corresponding region of SIVmac. TRIM5arh stably expressing MT4 clones were established by MLV-vector transduction followed by G418 selection. Expression levels of TRIM5arh was verified by western blotting (left panels). Left panels have the data of additional control (GFP) and TRIM5arh expression clones, G-X and T-X respectively. Then clones were transfected with a proviral plasmid by Lipofectamine LTX. Forty-eight hours post-transfection, viral titer in supernatants were determined as RT activity (right graph). In the absence of TRIM5arh, NLesc released higher levels of HIV-1 Gag in the supernatants, suggesting that the resistance to the late restriction is due to increased viral fitness with V86A/H87Q mutations. NLesc was more resistant to the TRIM5arh-mediated late-restriction. Conclusion Acknowledgements Serial cell-free passages of NL4-3 resulted in HIV-1 mutants which replicated more efficiently in TRIM5arh-expressing MT4 cells. Common H87Q or V86A/H87Q CA substitutions were found in the mutants. NLesc was more resistant to TRIM5arh-mediated early-restriction than parental NL4-3. NLesc showed more Gag production than wild-type NL4-3. We thank Dr. J. Sodroski and Dr. M. Stremlau for TRIM5arh, expression plasmid, Dr. V.N. KewalRamani and Dr. D.R. Littman for GHOST (3) R3/X4/R5 cells, Dr. J. Allan for AG3.0, and Dr. B. Chesebro and Dr. K. Wehrly for 183-H12-5C. This work was supported by NIH 1R56AI074363-01A1 (to Y.I.), the Mayo Foundation (to Y. I.) and Siebens Ph.D. Training Fellowship (to S.O.). Our results suggest that NL4-3 became more resistant to TRIM5arh-mediated antiviral activities through the acquisition of H87Q or V86A/H87Q CA substitutions. The V86A/H87Q substitutions appear to make the HIV-1 CA less visible to TRIM5arh during viral infection, while they increased general viral fitness during viral production.