ORGANIZACIÓN DEL SISTEMA DE GESTIÓN DE LA SEGURIDAD Y

The finding that HIV can directly infect resting CD4+ T cells without cellular stimulation opens new avenues for the study of HIV pathogenesis and potential applications that could be influential in the treatment of HIV infection. Despite the evidence indicating that HIV can infect resting cells directly, some differences in susceptibility between resting and stimulated cells still remain. The HIV infection kinetics in resting cells are slower than in activated cells and often, the total levels of integration in resting cells approach but do not reach the levels seen in activated cells. These differences do not appear to be fully attributable to deoxynucleoside levels; instead it appears that cellular factors, such as APOBEC, may be negatively influencing the progression of the early steps of infection in resting cells. Alternatively, it is also possible that factors that may be present in activated cells may be increasing the rate of infection. Further study on what factors, or conditions, affect the progression of HIV infection in resting cells relative to activated cells could reveal the existence of factors that could be manipulated to weaken the progression of HIV infection in CD4+ T cells. In addition, given that resting cells serve as a major reservoir for the virus in vivo, understanding the

147

requirements for establishing latency in resting cells could influence the development of future therapeutic strategies to decrease, eliminate or prevent the establishment of this reservoir.

The finding that HIV can integrate directly into resting cells also opens the doors for the development of more effective gene therapeutic strategies against HIV and other CD4+ T cell disorders. Direct gene delivery to resting cells avoids artificial manipulation of the cells, which could potentially affect the normal life span and behavior of the cells. An additional advantage of delivering genes to resting cells directly is that HIV appears to favor integration into gene dense regions less than in activated cells (38). This may further reduce the probability of the therapeutic gene interfering with normal host gene expression and normal cell behavior.

Since HIV infection of resting cells seems to differ from infection of activated cells, the biology of HIV infection in activated cells is not representative of infection of resting cells. For example, although integration takes place in both resting and activated cells, there is a significant difference in the ability of resting cells to produce viral proteins and infectious viral particles. Thus, the differences are not simple and required further study. In other words, the evidence presented here, though it indicates that resting cells are susceptible to HIV infection, also highlights that resting CD4+ T cells should be studied as separate targets of HIV independent from activated cells. Furthermore, heterogeneous HIV infection seems to occur among resting cells. We have found that a percentage of resting cells infected in vitro are capable of viral protein production without cell stimulation and without infectious particle production, while the remainder of cells do not (unpublished work). This suggests that latency may exist in different

forms. Further work on the biology of HIV infection of resting CD4+ T cells may yet reveal new aspects of HIV infection critical for understanding HIV pathogenesis in vivo.

149 Section 5.5 – References

1. Swiggard, W.J., C. Baytop, J.J. Yu, J. Dai, C. Li, R. Schretzenmair, T. Theodosopoulos, and U. O'Doherty. 2005. Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli. Journal of Virology 79:14179-14188.

2. Li, Q., L. Duan, J.D. Estes, Z.M. Ma, T. Rourke, Y. Wang, C. Reilly, J. Carlis, C.J. Miller, and A.T. Haase. 2005. Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature 434:1148-1152.

3. Blaak, H., A.B. van't Wout, M. Brouwer, B. Hooibrink, E. Hovenkamp, and H. Schuitemaker. 2000. In vivo HIV-1 infection of CD45RA+CD4+ T cells is established primarily by syncytium-inducing variants and correlates with the rate of CD4+ T cell decline. Proceedings of the National Academy of Sciences USA

97:1269-1274.

4. Kinter, A., A. Moorthy, R. Jackson, and A.S. Fauci. 2003. Productive HIV infection of resting CD4+ T cells: role of lymphoid tissue microenvironment and effect of immunomodulating agents. AIDS Research and Human Retroviruses

19:847-856.

5. Brenchley, J.M., B.J. Hill, D.R. Ambrozak, D.A. Price, F.J. Guenaga, J.P. Casazza, J. Kuruppu, J. Yazdani, S.A. Migueles, M. Connors, M. Roederer, D.C. Douek, and R.A. Koup. 2004. T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis.

Journal of Virology 78:1160-1168.

6. Ostrowski, M.A., T.-W. Chun, S.J. Justement, I. Motola, M.A. Spinelli, J. Adelsberger, L.A. Ehler, S.B. Mizell, C.W. Hallahan, and A.S. Fauci. 1999. Both memory and CD45RA(+)/CD62L(+) naïve CD4(+) T cells are infected in human immunodeficiency type 1-infected individuals. Journal of Virology 73:6430-6435. 7. Plesa, G., J. Dai, C. Baytop, J.L. Riley, C.H. June, and U. O'Doherty. 2007. Addition of deoxynucleosides enhances human immunodeficiency virus type 1 integration and 2LTR formation in resting CD4+ T cells. Journal of Virology

81:13938-13942.

8. Zhang, Z.-Q., T. Schuler, M. Zupancic, S. Wietgrefe, K.A. Staskus, K.A. Reimann, T.A. Reinhart, M. Rogan, W. Cavert, C.J. Miller, R.S. Veazey, D. Notermans, S. Little, S.A. Danner, D.D. Richman, D. Havlir, J. Wong, H.L. Jordan, T.W. Schacker, P. Racz, K. Tenner-Racz, N.L. Letvin, S. Wolinsky, and A.T. Haase. 1999. Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science 286:1353-1357.

9. Manganaro, L., M. Lusic, M.I. Gutierrez, A. Cereseto, G. Del Sal, and M. Giacca. Concerted action of cellular JNK and Pin1 restricts HIV-1 genome integration to activated CD4(+) T lymphocytes. Nature Medicine 16:329-333.

10. Santoni de Sio, F.R., and D. Trono. 2009. APOBEC3G-depleted resting CD4+ T cells remain refractory to HIV1 infection. PLoS One 4:e6571.

11. Korin, Y.D., and J.A. Zack. 1999. Nonproductive human immunodeficiency virus type 1 infection in nucleoside-treated G0 lymphocytes. Journal of Virology

12. Luban, J. 2007. Cyclophilin A, TRIM5, and resistance to human immunodeficiency type 1 infection. Journal of Virology 81:1054-1061

13. Stremlau, M., C.M. Owens, M.J. Perron, M. Kiessling, P. Autissier, and J. Sodroski. 2004. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427:848-853.

14. Keckesova, Z., L.M. Ylinen, and G.J. Towers. 2004. The human and African green monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities. Proceedings of the National Academy of Sciences USA

101:10780-10785.

15. Towers, G., M. Bock, S. Martin, Y. Takeuchi, J.P. Stoye, and O. Danos. 2000. A conserved mechanism of retrovirus restriction in mammals. Proceedings of the National Academy of Sciences USA 97:12295-12299.

16. Towers, G., M. Collins, and Y. Takeuchi. 2002. Abrogation of Ref1 retrovirus restriction in human cells. Journal of Virology 76:2548-2550.

17. Besnier, C., Y. Takeuchi, and G. Towers. 2002. Restriction of lentivirus in monkeys. Proceedings of the National Academy Sciences USA 99:11920-11925. 18. Towers, G.J., T. Hatziioannou, S. Cowan, S.P. Goff, J. Luban, and P.D. Bieniasz.

2003. Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors.

Nature Medicine 9:1138-1143.

19. Best, S., P. Le Tissier, G. Towers, and J.P. Stoye. 1996. Positional cloning of the mouse retrovirus restriction gene Fv1. Nature 382:826-829.

20. Sheehy, A.M., N.C. Gaddis, J.D. Choi, and M.H. Malim. 2002. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646-650.

21. Chiu, Y.L., V.B. Soros, J.F. Kreisberg, K. Stopak, W. Yonemoto, and W.C. Greene. 2005. Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 435:108-114.

22. Chiu, Y.L., H.E. Witkowska, S.C. Hall, M. Santiago, V.B. Soros, C. Esnault, T. Heidmann, and W.C. Greene. 2006. High-molecular-mass APOBEC3G complexes restrict Alu retrotransposition. Proceedings of the National Academy of Sciences USA 103:15588-15593.

23. Kamata, M., Y. Nagaoka, and I.S. Chen. 2009. Reassessing the role of APOBEC3G in human immunodeficiency virus type 1 infection of quiescent CD4+ T-cells. PLoS Pathogens 5:e1000342.

24. Berard, M., and D.F. Tough. 2002. Qualitative differences between naïve and memory T cells. Immunology 106:127-138.

25. Schlegel, R., T.S. Tralka, M.C. Willingham, and I. Pastan. 1983. Inhibition of VSV binding and infectivity by phosphatidylserine: is phosphatidylserine a VSV- binding site? Cell 32:639-646.

26. Coil, D.A., and A.D. Miller. 2004. Phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus. Journal of Virology 78:10920-10926. 27. Yu, D., W. Wang, A. Yoder, M. Spear, and Y. Wu. 2009. The HIV envelope but

not VSV glycoprotein is capable of mediating HIV latent infection of resting CD4 T cells. PLoS Pathogens 5:e1000633.

28. Camaur, D., and D. Trono. 1996. Characterization of human immunodeficiency virus type 1 Vif particle incorporation. Journal of Virology 70:6106-6111.

151

29. Liu, H., X. Wu, M. Newman, G.M. Shaw, B.H. Hahn, and J.C. Kappes. 1995. The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures. Journal of Virology 69:7630- 7638.

30. Welker, R., H. Kottler, H.R. Kalbitzer, and H.G. Krausslich. 1996. Human immunodeficiency virus type 1 Nef protein is incorporated into virus particles and specifically cleaved by the viral proteinase. Virology 219:228-236.

31. Aiken, C., and D. Trono. 1995. Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. Journal of Virology 69:5048-5056.

32. Cohen, E.A., G. Dehni, J.G. Sodroski, and W.A. Haseltine. 1990. Human immunodeficiency virus vpr product is a virion-associated regulatory protein.

Journal of Virology 64:3097-3099.

33. Dettenhofer, M., S. Cen, B.A. Carlson, L. Kleiman, and X.-F. Yu. 2000. Association of human immunodeficiency virus type 1 Vif with RNA and its role in reverse transription. Journal of Virology 74:8938-8945.

34. Warrilow, D., G. Tachedjian, and D. Harrich. 2009. Maturation of the HIV reverse transcription complex: putting the jigsaw together. Reviews in Medical Virology 19:324-337.

35. Suzuki, Y., and R. Craigie. 2007. The road to chromatin - nuclear entry of retroviruses. Nature Reviews Microbiology 5:187-196.

36. Yamashita, M., and M. Emerman. 2005. The cell cycle independence of HIV infections is not determined by known karyophilic viral elements. PLoS Pathogens 1:e18.

37. Gaddis, N.C., E. Chertova, A.M. Sheehy, L.E. Henderson, and M.H. Malim. 2003. Comprehensive investigation of the molecular defect in vif-deficient human immunodeficiency virus type 1 virions. Journal of Virology 77:5810-5820.

38. Brady, T., L.M. Agosto, N. Malani, C.C. Berry, U. O'Doherty, and F. Bushman. 2009. HIV integration site distributions in resting and activated CD4+ T cells infected in culture. AIDS 23:1461-1471.

153

A

Supplement S2.1. Generation of an integration standard (IS) cell line.

To calculate HIV integration events, we needed to compare the level of integration in our unknown samples to a standard. The standard needed to contain diverse integration sites to accurately reflect integration in vivo (26). To generate a standard with diverse integration sites, we used the strategy outlined in Supplement S2.1. pNL4- 3"env/EGFP/HygR (Supplement S2.1A) and pVSVG, an expression plasmid for vesicular stomatitis virus glycoprotein (VSV-G), were co-transfected into 293T cells to generate pseudotyped virions bearing the HIV"env genome and VSV-G glycoprotein. CEMss T cells (or 293 cells) were inoculated by spinoculation with transfection supernatant containing pseudotyped virions. The pseudotyped virions lacked HIV env, so infection was limited to a single cycle, and contained the coding sequence of EGFP and hygromycin resistance in place of the nef gene. The infected cells were grown for 28 days under hygromycin selection and sorted for EGFP expression. Soon after infection, both integrated and unintegrated DNA were detectable; however after 28 days only integrated DNA was detectable by Southern blot (not shown but previously described (130)). We then measured the level of HIV DNA/cell in this Integration Standard (IS) cell line using kinetic PCR (171) and detected ~1 copy/cell (Supplement S2.1B). Given that all of the DNA was integrated, we thus concluded that there is ~1 integration event per cell present in the IS cell line. We then used the same DNA in our two-step PCR assay to detect integration and generate a standard curve by plotting the number of proviruses vs the cycle threshold. In the first step of the PCR assay, we amplified DNA using either Alu

and gag primers or only the gag primer (see the PCR conditions section of Materials and methods for explanation). In the second step of the PCR assay, we used HIV-1 specific primers, which detect Alu-gag and gag-only amplicons, but do not detect Alu-Alu

155

Supplement S2.2. Integration of HIV-1 in CD4+ _{T cells after spinoculation also}

exhibits a dose dependent decrease in integration with each dilution.

ppCD4+ T cells were inoculated with three-fold dilutions of supernatant containing VSV- G pseudotyped pNL4-3. Cells were inoculated by spinoculation for 2hr at 25°C, then washed and the level of cell associated virus (viral binding) was determined at the most concentrated and most dilute doses by p24 ELISA. The cells were cultured for 3 days, DNA was prepared, and reverse transcription and integration were measured. Similar to Figure 2.4, a dose-dependent decrease in integration and RU5 reverse transcripts was observed with serial dilution of the viral inoculum. The results are presented in provirus per cell (A), log (3) of provirus per cell (B), RU5 per cell (C) and log(3) RU5 per cell (D). AZT inhibited reverse transcription (78%) and integration (99%) (added at the top dose). Error bars represent the standard deviation of 16 measurements of integration and 2-4 measurements of reverse transcripts, for each virus dilution depending on sample availability. The number of proviruses/cell and RU5/cell are written above each point (B, D).

Supplement S3.1. Viral fusion to resting CD4+ T cells in the presence of the fusion inhibitor enfurvitide cannot be detected by the BlaM-Vpr assay.

Resting CD4+ T cells were spinoculated with X4-HIV (pNL4-3) particles containing BlaM-Vpr in the presence or absence of 10ug/ml of the fusion inhibitor enfurvitide (T- 20). The gate that measures the level of fusion was set based on a sample infected with X4-HIV that did not contain BlaM-Vpr and a background signal of 0.5%.

157

Supplement S4.1. Unstimulated CD4+ T cells are viable and a minimal number of viable cells are lost after spinoculation with HIVvector(VSV-G).

Viability and numbers of unstimulated CD4+ T cells were assessed at 0, 24, and 48hr post-inoculation with the trypan blue cell viability dye. Percent cell recovery was calculated as the percent of viable cells recovered from the cell input at time 0hr. Cell samples were diluted 1:10 in trypan blue and cells were counted using a hemacytometer. Percent cell recovery is representative of the average of 2 cell counts.

Supplement S4.2. Plasmid DNA is responsible in large part for the reverse transcription signal obtained at 0hr post-inoculation.

Unstimulated CD4+ T cells were spinoculated with pNL4-3 transfection supernatant in the presence or absence of the reverse transcription inhibitor efavirenz (NNRTI). Late reverse transcription was measured at the indicated time points by quantitative PCR using primers that detect the second strand transfer step of reverse transcription (SST). Error bars represent the standard deviation of 3 measurements of reverse transcription.

159

Supplement S4.3. Early reverse transcription, like late reverse transcription, is not detected in unstimulated CD4+ T cells inoculated with HIVvector(VSV-G).

(A) Unstimulated CD4+ T cells (uCD4T) or (B) an activated T cell line, CEMss, were spinoculated with pNL4-3 (wtHIV), VRX494 (HIVvector(VSV-G)), or pNL4-3 in the presence of the reverse transcriptase inhibitor efavirenz (+NNRTI). Early reverse transcription was measured by quantitative PCR using primers that recognize the R-U5 region of the HIV-LTR. Error bars represent the standard deviation of 3 measurements of reverse transcription. The figure is representative of 3 experiments.