Highly active antiretroviral therapy (HAART) can effectively suppress viral replication and reduce morbidity and mortality in HIV-infected patients. However, a small population of cells carrying replication-competent latent HIV persists even after years of antiviral therapy [1–3]. Owing to its dormant state, this latent reservoir is resistant to HAART and host immune clearance. The latent reservoir has an extremely slow decay rate and may be responsible for the rapid reappearance of viremia following discontinuation of HAART . It is widely regarded as a major obstacle to the eradication of HIV. Curing HIV infection, therefore, requires a better understanding of the molecular mechanisms underlying viral latency and the development of strategies for targeting this particular population of cells [5,6]. Among the viral reservoirs proposed so far, the best established is the population of resting memory CD4+ T cells harboring replication-competent latent proviruses [7–9]. Therefore, this review article will focus on primary cell models of HIV latency in CD4+ T cells.
Latently infected cells represent only one in 1 million of resting CD4+ T cells . This rarity of latently infected cells in vivo has greatly hampered research on HIV latency. To facilitate the exploration of the molecular mechanisms governing HIV latency, a convenient and tractable in-vitro cell model system is required. Another potential application of in-vitro models of HIV latency is to serve as a platform for high-throughput screening for candidate compounds that can reverse HIV latency. As mentioned above, latent HIV is unaffected by HAART. A potential approach for eradication of HIV infection is to reactivate latent HIV, assuming that following viral reactivation, latently infected cells can be eliminated through the cytopathic effects of viral proteins or immune-mediated killing [10,11]. An ideal drug for this purpose should specifically and robustly reactivate latent HIV. The reactivation must be robust because, in principle, even a single cell with replication-competent latent HIV could become reactivated and the resultant virus would then undergo exponential expansion and cause a full-blown viral rebound after cessation of antiviral therapy.
Earlier in-vitro HIV latency models were generated in transformed cell lines and led to several important observations. However, transformed cell lines exhibit a continuously proliferating nature that is distinct from the quiescent G0 characteristics of resting CD4+ T cells . In addition, aberrant signaling pathways may exist in transformed cell lines. These fundamental differences have raised concerns about the results obtained from the transformed cell line models. Therefore, there is a pressing need for developing more appropriate primary cell models of HIV latency that can recapitulate the quiescent features of resting CD4+ T cells in vivo.
Development of a primary cell model of HIV latency is, particularly, challenging for several reasons that will be further discussed in the following section. Nevertheless, several groups have succeeded in the establishment of primary cell models of HIV latency in vitro. These models vary in their strategies of activating and maintaining CD4+ T cells in culture, the yields of latently infected cells, and degree of quiescence achieved. Thus, each individual model exhibits certain advantages and disadvantages that will be pointed out below.
HIV latency models in transformed cell lines
In late 1980s, Folks et al. first reported induction of HIV replication by certain cytokines and T-cell activating factors in the chronically infected monocytic and T-cell lines, U1 and ACH2, respectively [13,14]. These two cell lines exhibit minimal HIV replication when cultured in a cytokine-free medium, but high levels of HIV replication can be induced by the addition of certain cytokines and T-cell activation factors. Therefore, these cell lines were considered models of HIV latency. Later, it was realized that the low basal transcription of HIV in U1 and ACH2 cell lines results from the mutations in the virally encoded HIV transcription activator Tat and the Tat-associated region (TAR), respectively [15,16]. Antoni et al.  also reported that HIV latency was observed in JΔK cells that harbor an integrated HIV provirus with a deletion of nuclear factor (NF)-κB sites in the long terminal repeat (LTR). Although these mutations facilitate entry into a state of low transcription, they raise concerns about whether these cell lines can truly reflect HIV latency in vivo.
To eliminate these concerns, Jordan et al.  developed an HIV latency model in Jurkat T cells, a leukemic T-cell line, utilizing a green fluorescent protein (GFP)-encoding reporter virus with a wild-type LTR and Tat–TAR axis. Clones of infected Jurkat T cells, called J-Lat cells, which exhibited low basal expression of GFP, but were highly inducible, were selected using flow cytometry. Interestingly, these investigators found that latent HIV preferentially integrates to the regions with restrictive chromatin structure. On the contrary, by analyzing the integration sites of HIV in resting CD4+ T cells isolated from patients on HAART, Han et al.  discovered that the HIV integration favors active genes. Importantly, only a small fraction of the integrated proviruses in this population of cells are replication-competent . Lewinski et al.  then carried out a genome-wide analysis of HIV integration sites in Jurkat cells infected by a GFP-expressing HIV vector and found that latent HIV favors the regions of gene desserts, centromeric heterochromatin, and very highly expressed genes. Notably, they did not select latent clones. Instead, they compared the integration sites between populations of latently and persistently infected cells. The discrepancies among these three studies probably reflect the distinctions between transformed cell line models and primary cells, suggesting the important role of primary cell models in studies of HIV latency. Using J-Lat cells, Williams et al.  found that whereas NF-κB p50 promotes HIV latency, sustained NF-κB induction is required for reactivation of latent HIV . Lenasi et al.  also utilized the J-Lat model to demonstrate that the transcriptional interference plays a role in the establishment of HIV latency. To avoid the bias caused by clonal selection, Karn and colleagues  generated a biochemically tractable model of HIV latency using lentiviral vectors encoding a H13L mutant Tat and a destabilized GFP reporter. This system achieved a high frequency of latently infected cells. Additionally, they found that recruitment of transcription factor (TFIIH) to the HIV LTR is a rate-limiting step in the reactivation of latent HIV.
The establishment of HIV latency in primary CD4+ T cells
The mechanisms governing the establishment and maintenance of HIV latency in vivo are still not fully understood and probably involve multiple factors . It has been hypothesized that latent HIV is generated when infected CD4+ lymphoblasts survive a contraction phase following T-cell activation and revert to a resting state (for a review of HIV latency in resting CD4+ T cells, see reference ). This hypothesis is based on several observations. First, HIV preferentially infects CD4+ T lymphoblasts, and in vivo, these cells have a short life span in the productively infected state (about 1 day) . T-cell activation signals and inducible transcription factors, like NF-κB and nuclear factor of activated T cells (NFAT), are required for HIV replication [28,29]. Lack of these factors in resting T cells, thus, has been considered as one major reason of HIV latency. In addition, latent HIV genomes primarily reside in resting memory CD4+ T cells, rather than naive ones [8–10]. Therefore, it is likely that HIV latency is a consequence of a physiological process by which lymphoblasts transit to a resting memory state that is nonpermissive for viral gene expression. Given this fact, most primary cell models are generated by infecting activated T cells with wild-type HIV, mutated HIV, or lentiviral vector with tractable markers and then allowing the cells to transit to a more quiescent state (Fig. 1a).
One of the major problems with this approach is that it has not been possible until recently to generate and maintain resting memory CD4+ T cells in vitro. The generation and maintenance of memory CD4+ T cells are complicated processes in vivo and are difficult to reproduce in vitro [30,31]. Most activated T cells die quickly in culture unless simulated by trophic cytokines such as interleukin (IL)-2 or IL-7. However, many cytokines, including IL-2 and IL-7, have been shown to activate latent HIV to some degree [32–34]. Therefore, a strategy utilizing these trophic cytokines may not be appropriate for the establishment of a primary cell model of HIV latency. Ideally, the infected primary CD4+ T cells should be maintained in the presence of cytokines at a physiologic concentration to support cell viability without inducing HIV replication. However, it has been proven difficult to find this critical concentration of cytokines. Alternatively, cells should be maintained in the absence of cytokines. There is a trade-off between cell viability and HIV latency in generating a primary cell model. Notably, the generation of latently infected cells during the return to quiescence in vitro is not very efficient and may only yield a small number of latently infected cells at the end of this process. Key to these approaches is finding appropriate culture conditions or strategies to keep cells alive in a resting state, and, particularly to obtain appropriate numbers of cells for further mechanistic studies of HIV latency.
Although HIV is likely to establish its latency when infected activated T cells return to a resting state, it remains possible that HIV may enter latency by directly infecting resting CD4+ T cells, particularly resting memory CD4+ T cells (Fig. 1b). Therefore, we will discuss below two strategies for the establishment of primary cell models of HIV latency.
Establishment of HIV latency following infection of activated CD4+ T cells
As discussed above, an ideal primary cell model of HIV latency should be established in CD4+ T cells that exhibit a quiescent state and memory phenotype, particularly central memory phenotype. Primary resting CD4+ T cells are characterized with certain features, including small cell size, G0 cell cycle status, low levels of activation markers, and absence of nuclear forms of certain transcription factors associated with T-cell activation, namely, NF-κB and NFAT. In addition, resting CD4+ T cells are resistant to HIV infection. An ideal model should yield enough latently infected cells for mechanistic studies and high-throughput screening. We will examine current primary cell models based on these parameters.
To overcome the obstacles mentioned above, several groups have adopted different strategies to generate primary cell models of HIV latency. Sahu et al.  developed an in-vitro primary cell model using a unique cell culture system. In their system, primary CD4+ T cells were activated with immobilized anti-CD3 antibody in the presence of IL-2 and infected by a replication-competent HIV. The HIV-infected activated primary CD4+ T cells were then co-cultured with a feeder cell line H80, a brain tumor-derived cell line. Intriguingly, the death of the activated T cells was prevented by unknown mechanisms. The activated cells gradually reverted back to a resting state, which was accompanied by the silencing of HIV replication. A fraction of infected cells became latently infected, whereas other cells still exhibited low-level replication of HIV. Although the cells were quiescent in many respects, a significant portion of cells still expressed the activation marker CD69.
Recently, Karn and colleagues have utilized this system to generate large numbers of latently infected cells using a fluorescent reporter-carrying lentiviral vector with a wild-type Tat gene or the attenuated H13L Tat mutant. Taking advantage of this system, they showed that HIV efficiently establishes a state of latency when a reporter virus encoding the H13L Tat mutant is used [36•]. They were able to obtain a population of latently infected cells with suitable yield and purity to allow the biochemical studies of HIV latency.
By mimicking the process of thymopoiesis, Zack and colleagues developed an in-vitro primary cell model by infecting the fetal CD4+CD8+ thymocytes whose differentiation was induced in vitro using IL-2/IL-4 and appropriate culture conditions . Actually, this strategy is derived from their earlier work for generation of a severe combined immunodeficiency (SCID)-hu(Thy/Liv) mouse model in which a high frequency of latently infected human CD4+ T cells can be generated during thymopoiesis in vivo . Notably, the latently infected cells in this system exhibit resting naive phenotypes, rather than memory phenotypes.
Marini et al.  utilized a more physiological way to activate primary CD4+ T cells. In this model, naive CD4+ T cells enriched from freshly isolated peripheral blood mononuclear cells (PBMCs) were activated by monocyte-derived dendritic cells loaded with the superantigen Staphylococcus enterotoxin B. The activated cells were then infected with replication-competent HIV. The resulting memory T cells were rested in culture supplemented with a low dose of IL-7, which is known to promote the transition of effector CD4+ T cells to persistent memory cells . This strategy yielded about 20% viable cells with a phenotype of central memory cells and about 2% latently infected cells at the end of the resting phase. Although a low dose of IL-7 does maintain the viability of a small number of CD4+ T cells following activation, these cells are still larger in size than the freshly isolated resting CD4+ T cells, indicating that the activation state of these cells is probably slightly different from that of the resting CD4+ T cells in vivo.
Bosque et al. developed an HIV-1 latency model by generating memory T cells in different polarizing culture conditions, including nonpolarizing conditions and T-helper cell 1 (Th1)-polarizing or Th2-polarizing conditions. Primary CD4+ T cells purified from freshly isolated PBMCs were first stimulated with beads coated with anti-CD3 and anti-CD28 antibodies. The activated cells were then maintained in culture conditions favoring memory differentiation. The resulting cells were then infected with Envelope (Env)-deficient HIV pseudotyped with HIVLAI Env. The infected cells then underwent a short 7-day resting phase in the presence of IL-2. Surprisingly, a large portion of HIV-infected cells entered a latent state of infection that could be reversed upon stimulation with certain mitogens. Unfortunately, the authors did not prove the latent virus was integrated. They further identified the signaling pathways involved in reactivation of latent HIV in their ex-vivo system. Interestingly, lymphocyte-specific protein tyrosine kinase (Lck) and NFAT, but not NF-κB, were critical for the reactivation of latent HIV in this system .
Recently, our group has also created a HIV latency model using genetically modified B cell lymphoma (Bcl)-2-transduced primary CD4+ T cells. Bcl-2 is an essential survival factor downstream of IL-7 signaling in resting CD4+ T cells in vivo, including resting naive and memory T cells [42,43]. In this system, we demonstrated that the resting Bcl-2-transduced CD4+ T cells are very similar to the freshly isolated resting CD4+ T cells in many aspects. Activation of Bcl-2-transduced cells results in the release of an array of cytokines and activation of NF-κB. While the cells are in an activated state, they can be infected with HIV vectors carrying GFP. The cells can then be cultured in the absence of activating stimuli and exogenous cytokines for weeks to months. In these cells, latency is established in a fraction of the infected cells over a course of weeks. Using this model, we carried out high-throughput screening of more than 4000 compounds and identified 5-hydroxynaphthalene-1,4-dione (5HN) as an agent that can reverse HIV latency to the same degree observed with anti-CD3 with anti-CD28 antibodies or phorbol myristate acetat (PMA) [44•]. Unfortunately, this compound is quite toxic, but the results of this small-scale screen illustrate the potential of primary cell models to identify latency-reversing agents.
Establishment of HIV-1 latency by direct infection of resting CD4+ T cells
Although HIV preferentially infects activated CD4+ T cells and the infection of resting CD4+ T cells by HIV is blocked in multiple steps [45,46], it remains possible that HIV latency can be established by direct infection of resting CD4+ T cells. By highly sensitive assays, integration of HIV-1 into host cell genomes can be detected following direct infection of resting CD4+ T cells .
O'Doherty and colleagues [47,48] first demonstrated that HIV can directly integrate into the genomes of resting CD4+ T cells, albeit inefficiently. Recently, they further showed that although direct integration of HIV into resting CD4+ T cells can occur, entry of HIV into resting naive CD4+ T cells is less efficient than entry into resting memory CD4+ T cells [49•]. These results demonstrated a strategy by which HIV latency can be established in vitro through direct infection of the resting memory CD4+ T cells with HIV. Additionally, Saleh et al.  also generated a novel model by directly infecting resting memory CD4+ T cells after exposure to the chemokines CCL19 and CCL21, indicating that the establishment of HIV latency may not necessarily require full activation through the antigen receptor. As these two chemokines can be readily detected in lymph nodes, this study also provides a plausible explanation for how HIV latency can be generated in resting memory CD4+ T cells in vivo.
In the past several years, there has been a rapid progress in the development of primary cell models of HIV latency. As discussed above, the fundamental differences between continuously proliferating cell lines and resting primary CD4+ T cells make the latter a more appropriate model of HIV latency that authentically recapitulates the quiescent features of resting CD4+ T cells in vivo. Some signaling pathways have proven to be different between cell line models and primary cell models. For example, tumor necrosis factor alpha (TNF-α) has been shown to reactivate latent HIV in cell line models, but not in primary cell models [33,36•,44•]. Previous studies also have shown that these primary cell models can be used to carry out the mechanistic studies on HIV latency. Therefore, these primary cell models provide good opportunities to verify the results obtained from the studies on HIV latency using cell line models. Having these primary cell models at hand can also greatly speed up the discovery of compounds that can reverse HIV latency for the purpose of eradication of HIV infection from patients on HAART.
The author thanks Drs. Pei-lung Chen and Yu-yi Lin for their critical comments on this manuscript. This work is supported by NTUH grant 99P02 and Liver Disease Prevention & Treatment Research Foundation.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 89).
1 Finzi D, Hermankova M, Pierson T, et al
. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 1997; 278:1295–1300.
2 Wong JK, Hezareh M, Gunthard HF, et al
. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 1997; 278:1291–1295.
3 Chun TW, Stuyver L, Mizell SB, et al
. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci U S A 1997; 94:13193–13197.
4 Siliciano JD, Kajdas J, Finzi D, et al
. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med 2003; 9:727–728.
5 Richman DD, Margolis DM, Delaney M, et al
. The challenge of finding a cure for HIV infection. Science 2009; 323:1304–1307.
6 Trono D, Van Lint C, Rouzioux C, et al
. HIV persistence and the prospect of long-term drug-free remissions for HIV-infected individuals. Science 2010; 329:174–180.
7 Chun TW, Carruth L, Finzi D, et al
. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 1997; 387:183–188.
8 Pierson T, Hoffman TL, Blankson J, et al
. Characterization of chemokine receptor utilization of viruses in the latent reservoir for human immunodeficiency virus type 1. J Virol 2000; 74:7824–7833.
9 Chomont N, El-Far M, Ancuta P, et al
. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med 2009; 15:893–900.
10 Brooks DG, Hamer DH, Arlen PA, et al
. Molecular characterization, reactivation, and depletion of latent HIV. Immunity 2003; 19:413–423.
11 Lehrman G, Hogue IB, Palmer S, et al
. Depletion of latent HIV-1 infection in vivo: a proof-of-concept study. Lancet 2005; 366:549–555.
12 Han Y, Wind-Rotolo M, Yang HC, et al
. Experimental approaches to the study of HIV-1 latency. Nat Rev Microbiol 2007; 5:95–106.
13 Folks T, Powell DM, Lightfoote MM, et al
. Induction of HTLV-III/LAV from a nonvirus-producing T-cell line: implications for latency. Science 1986; 231:600–602.
14 Folks TM, Justement J, Kinter A, et al
. Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science 1987; 238:800–802.
15 Emiliani S, Fischle W, Ott M, et al
. Mutations in the Tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line. J Virol 1998; 72:1666–1670.
16 Emiliani S, Van Lint C, Fischle W, et al
. A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency. Proc Natl Acad Sci U S A 1996; 93:6377–6381.
17 Antoni BA, Rabson AB, Kinter A, et al
. NF-kappa B-dependent and -independent pathways of HIV activation in a chronically infected T cell line. Virology 1994; 202:684–694.
18 Jordan A, Bisgrove D, Verdin E. HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 2003; 22:1868–1877.
19 Han Y, Lassen K, Monie D, et al
. Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes. J Virol 2004; 78:6122–6133.
20 Lewinski MK, Bisgrove D, Shinn P, et al
. Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription. J Virol 2005; 79:6610–6619.
21 Williams SA, Chen LF, Kwon H, et al
. NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J 2006; 25:139–149.
22 Williams SA, Kwon H, Chen LF, Greene WC. Sustained induction of NF-kappa B is required for efficient expression of latent human immunodeficiency virus type 1. J Virol 2007; 81:6043–6056.
23 Lenasi T, Contreras X, Peterlin BM. Transcriptional interference antagonizes proviral gene expression to promote HIV latency. Cell Host Microbe 2008; 4:123–133.
24 Kim YK, Bourgeois CF, Pearson R, et al
. Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency. EMBO J 2006; 25:3596–3604.
25 Lassen K, Han Y, Zhou Y, et al
. The multifactorial nature of HIV-1 latency. Trends Mol Med 2004; 10:525–531.
26 Pierson T, McArthur J, Siliciano RF. Reservoirs for HIV-1: mechanisms for viral persistence in the presence of antiviral immune responses and antiretroviral therapy. Annu Rev Immunol 2000; 18:665–708.
27 Perelson AS, Essunger P, Cao Y, et al
. Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 1997; 387:188–191.
28 Kinoshita S, Su L, Amano M, et al
. The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells. Immunity 1997; 6:235–244.
29 Nabel G, Baltimore D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 1987; 326:711–713.
30 MacLeod MK, Kappler JW, Marrack P. Memory CD4 T cells: generation, reactivation and re-assignment. Immunology 2010; 130:10–15.
31 Marrack P, Kappler J. Control of T cell viability. Annu Rev Immunol 2004; 22:765–787.
32 Chun TW, Engel D, Mizell SB, et al
. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J Exp Med 1998; 188:83–91.
33 Brooks DG, Arlen PA, Gao L, et al
. Identification of T cell-signaling pathways that stimulate latent HIV in primary cells. Proc Natl Acad Sci U S A 2003; 100:12955–12960.
34 Wang FX, Xu Y, Sullivan J, et al
. IL-7 is a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART. J Clin Invest 2005; 115:128–137.
35 Sahu GK, Lee K, Ji J, et al
. A novel in vitro system to generate and study latently HIV-infected long-lived normal CD4+ T-lymphocytes. Virology 2006; 355:127–137.
36• Tyagi M, Pearson RJ, Karn J. Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction. J Virol 2010; 84:6425–6437.
37 Burke B, Brown HJ, Marsden MD, et al
. Primary cell model for activation-inducible human immunodeficiency virus. J Virol 2007; 81:7424–7434.
38 Brooks DG, Kitchen SG, Kitchen CM, et al
. Generation of HIV latency during thymopoiesis. Nat Med 2001; 7:459–464.
39 Marini A, Harper JM, Romerio F. An in vitro system to model the establishment and reactivation of HIV-1 latency. J Immunol 2008; 181:7713–7720.
40 Li J, Huston G, Swain SL. IL-7 promotes the transition of CD4 effectors to persistent memory cells. J Exp Med 2003; 198:1807–1815.
41 Bosque A, Planelles V. Induction of HIV-1 latency and reactivation in primary memory CD4+ T cells. Blood 2009; 113:58–65.
42 Akashi K, Kondo M, von Freeden-Jeffry U, et al
. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell 1997; 89:1033–1041.
43 Maraskovsky E, O'Reilly LA, Teepe M, et al
. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice. Cell 1997; 89:1011–1019.
44• Yang HC, Xing S, Shan L, et al
. Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation. J Clin Invest 2009; 119:3473–3486.
45 Pierson TC, Zhou Y, Kieffer TL, et al
. Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection. J Virol 2002; 76:8518–8531.
46 Zack JA, Arrigo SJ, Weitsman SR, et al
. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 1990; 61:213–222.
47 Agosto LM, Yu JJ, Dai J, et al
. HIV-1 integrates into resting CD4+ T cells even at low inoculums as demonstrated with an improved assay for HIV-1 integration. Virology 2007; 368:60–72.
48 Swiggard WJ, Baytop C, Yu JJ, et al
. Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli. J Virol 2005; 79:14179–14188.
49• Dai J, Agosto LM, Baytop C, et al
. Human immunodeficiency virus integrates directly into naive resting CD4+ T cells but enters naive cells less efficiently than memory cells. J Virol 2009; 83:4528–4537.
50 Saleh S, Solomon A, Wightman F, et al
. CCR7 ligands CCL19 and CCL21 increase permissiveness of resting memory CD4+ T cells to HIV-1 infection: a novel model of HIV-1 latency. Blood 2007; 110:4161–4164.