Quiescent CD4 + T Cells Inhibit Multiple Stages of HIV ... - Springer Link

Quiescent CD4+ T Cells Inhibit Multiple Stages of HIV Infection

24

Jerome A. Zack and Dimitrios N. Vatakis

Contents

Abstract

Abstract...................................................................

253

Introduction ............................................................

253

+

CD4 T Cell Quiescence .........................................

254

Quiescent T Cells and HIV Infection ...................

255

Quiescent T Cells Exhibit Multiple Blocks of the HIV Life Cycle ............................................. Entry, Reverse Transcription and Integration........... Integration and Viral Expression..............................

256 256 257

Restriction Factors .................................................

258

Discussion................................................................

259

References ...............................................................

260

Elucidating the block of quiescent CD4+ T cells to HIV infection has been an intensely debated issue. Early studies suggested that the virus could not infect this T cell subset; latter studies demonstrated that these cells could inefficiently support HIV infection. The kinetics of infection in quiescent cells was delayed and multiple stages of the viral life cycle were marred by inefficiencies. A number of restriction factors as well as cellular protein have been implicated in the potential block. However, to this date the mechanisms of HIV infection in quiescent cells are still unclear. Further understanding will open the way for better therapeutic approaches and much improved gene therapy protocols using HIV-based vectors. Keywords

J.A. Zack Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA

CD4+ T cell quiescence • Forkhead Box class O (FOXO) factors • HIV life cycle • Lung Krupple-Like factor (LKLF) • PCR technologies • Polypyrimidine tract binding protein (PTB) • Quiescent T cells and HIV infection • Reverse transcription and integration • RNA and DNA synthesis

UCLA AIDS Institute, Los Angeles, CA, USA D.N. Vatakis (*) David Geffen School of Medicine at UCLA, Division of Hematology-Oncology, Department of Medicine, 615 Charles E. Young Drive South, BSRB 173, Mailcode: 736322, Los Angeles, CA 90095, USA e-mail: [email protected]

Introduction Some confusion still rules the literature regarding the ability of HIV to infect quiescent cells. This largely stems from major differences between the

M.A. Hayat (ed.), Tumor Dormancy, Quiescence, and Senescence, Volume 2: Aging, Cancer, and Noncancer Pathologies, Tumor Dormancy and Cellular Quiescence and Senescence, DOI 10.1007/978-94-007-7726-2_24, © Springer Science+Business Media Dordrecht 2014

253

254

monocyte/macrophage and T cell lineages in terms of their quiescent status. Studies have tried to elucidate the block of quiescent T cells to HIV infection by either examining the viral life cycle in these cells or by identifying key cellular factors that may have an impact. In this review, we will present the progress made in the field thus far and discuss yet unanswered questions.

CD4+ T Cell Quiescence Human T lymphocytes, both naive and memory, remain at a quiescent state over long periods of time. The majority of T lymphocytes present in blood, the lymph nodes and spleen are in the G0 state of the cell cycle (Tzachanis et al. 2001; Tzachanis et al. 2004). Typically following antigen stimulation a small fraction of T cells clonally expand at a high rate (Cotner et al. 1983). After stimulation, the majority of the clonally expanded lymphocytes will undergo apoptosis, leaving behind a small fraction of non-dividing memory cells that live over long periods of time and are recruited only upon antigen re-exposure. T cell quiescence was largely believed to be a default state. Recent studies, however, have demonstrated that T cell quiescence is an actively maintained state regulated by a plethora of transcription factors (Kuo et al. 1997; Buckley et al. 2001; Tzachanis et al. 2001; Haaland et al. 2005; Yusuf et al. 2008). During quiescence, T cells maintain low levels of cell metabolism, decreased RNA synthesis that is limited to basic housekeeping genes, a small cell size and very long periods of survival (Tzachanis et al. 2004). Therefore, T cell quiescence averts cellular damage from metabolism and prevents inappropriate T cell activation and expansion that could lead to lymphomas, autoimmunity, and lymphopenia due to activation induced cell death (Tzachanis et al. 2004). While the mechanisms of T cell quiescence are still under active investigation, a number of transcription factors have been shown to play a major role in this process. Lung Krupple-Like factor (LKLF) is one of the first factors identified to regulate T cell quiescence.

J.A. Zack and D.N. Vatakis

The protein belongs to the Krupple-Like factors (KLFs) family of proteins. LKLF has been shown to regulate and maintain quiescence (Kuo et al. 1997; Buckley et al. 2001; Haaland et al. 2005). Studies disrupting the expression of the transcription factor LKLF resulted in increased T cell activation, proliferation and cell size (Kuo et al. 1997; Buckley et al. 2001). Furthermore, ectopic expression of the protein in cell lines such as Jurkat T cells induced quiescence (Haaland et al. 2005). Forkhead Box class O (FOXO) factors have been identified as potential players in cell quiescence and cell death, especially FOXO1, 3 and 4 (Kops et al. 2002; Ouyang et al. 2009; Barnes et al. 2010; Aksoylar et al. 2011). These factors are shown to be active in resting cells and control T cell homeostasis and tolerance, a phenotype reversed by IL-2 mediated activation (Kops et al. 2002). FOXO1 knockout mice demonstrated normal thymopoiesis, elevated numbers of activated T cells and decreased levels of naïve T cells (Ouyang et al. 2009). Furthermore, IL-7 receptor expression was completely abrogated suggesting that this receptor may be a target of FOXO1 (Ouyang et al. 2009). Recent studies have linked Gimap5 to FOXO1, 3, and 4. Gimap5 is a member of the GTPase immunity associated family of proteins (Barnes et al. 2010; Aksoylar et al. 2011). These proteins have been shown to be involved in lymphocyte survival, development, selection and homeostasis. In Gimap5 defective mouse models researchers observed severe intestinal inflammation and lack of tolerance (Barnes et al. 2010; Aksoylar et al. 2011). Gimap5 regulates FOXO 3 and 4 at the protein level by inhibiting their degradation (Aksoylar et al. 2011). Tob, another nuclear protein, has been recently shown to be involved in cellular quiescence (Tzachanis et al. 2001; Tzachanis and Boussiotis 2009). It belongs to a family of antiproliferative proteins known as APRO (Tzachanis et al. 2001). The protein is expressed in anergic and naïve T cells, is downregulated upon T cell activation and enhances TGF-β signaling pathways, thus, promoting cell quiescence (Tzachanis et al. 2001).

24 Quiescent CD4 + T Cells Inhibit Multiple Stages of HIV Infection

Knockdown of Tob results to increased activation only following only binding to TcR thereby suggesting a role in regulating thresholds of T cell activation (Tzachanis et al. 2001). This was further supported by data showing that ectopic expression of the protein resulted in inhibition of CD3-CD28 mediated proliferation and loss expression of cytokines such as IL-2, IL-4 and IFN-γ (Tzachanis et al. 2001). Recent studies have implicated Runx1 and Tsc1 as potential regulators of T cell quiescence. Runx1 has been shown to impact various stages of T cell differentiation. However, recently it was shown to be expressed at high levels in naïve T cells but it is downregulated following T cell activation (Wong et al. 2012). Knockdown of Runx1 resulted in severe immune hyperactivation, increased levels of IL-17 and IL-21 production causing fatal autoimmunity (Wong et al. 2012). While the mechanism of action is not yet clear, it is believed that the regulation of pro-inflammatory cytokine production such as IL-17 and IL-21 may be implicated (Wong et al. 2012). Similarly loss of Tsc1 expression results in increased activation, cellularity and eventual cell death. Studies have suggested that Tsc1 may be controlling T cell quiescence by regulating mTORC1, a major protein involved in cell growth, metabolism (Wu et al. 2011; Yang et al. 2011). In this chapter, we will discuss the potential HIV restriction factors present in quiescent CD4 T cells. Quiescent T cell infection by HIV has been an interesting and controversial subject that has generated a number of high profile studies in the field. While HIV infection does not require mitosis (Weinberg et al. 1991), HIV cannot efficiently infect G0 cells, as we will describe in the sections to follow. The blocks identified in quiescent cells only partly tell the story of their resistance to HIV. Interestingly, none of the aforementioned transcription factors regulating quiescence are implicated. Understanding these blocks will allow us to develop more effective ways to treat HIV and utilize lentiviral vectors in this cell type with minimal perturbation. The latter can have major implication to lentiviral vector based gene therapy protocols.

255

Quiescent T Cells and HIV Infection The infection of quiescent CD4+ T cells by the human immunodeficiency virus (HIV) has been a subject of intense debate. Unlike other retroviruses, HIV replication is not dependent on cell division and is characterized by its ability to infect non-dividing cells and establish a latent infection (Weinberg et al. 1991). Early reports suggested that HIV was able to bind to quiescent T cells but failed to infect them unless they were previously activated (Zagury et al. 1986; Gowda et al. 1989). Advances in PCR technology demonstrated that quiescent T cells are infectable by HIV (Stevenson et al. 1990; Zack et al. 1990; Spina et al. 1995). However, studies differed on the level as well as the efficiency of infection. Zack et.al demonstrated that the virus could enter quiescent T cells and initiate reverse transcription (Zack et al. 1990). However, the process was not completed resulting in the generation of labile viral cDNA intermediates that degraded over time (Zack et al. 1990). On the other hand, others demonstrated that in HIV infected quiescent T cells there was completion of reverse transcription that resulted in accumulation of viral cDNA in the cytoplasm over prolonged periods of time (Stevenson et al. 1990; Spina et al. 1995). Under this setting, infection was rescued by activation of the infected cells suggesting that there was a defect in the nuclear transport of viral cDNA in quiescent cells (Stevenson et al. 1990; Spina et al. 1995). Further studies by the Vitteta group, focused on the CD25+ and CD25- T cell populations and their ability to be infected by the virus thereby contrasting activated versus non-activated T cells (Ramilo et al. 1993; Borvak et al. 1995; Chou et al. 1997). In a series of studies, they showed that the CD25- T cells, representing nonactivated T cells, were not infectable by HIV while the CD25+ T cells were able to support infection in the absence of any stimulation. However, when total human peripheral blood mononuclear cells were infected, the nonactivated cells had copies of viral DNA. This was

256

the first evidence of cell-to-cell mediated infection of quiescent cells. Based on these early studies, it was evident that the life cycle of HIV in quiescent CD4 T cells was quite distinct from that of activated T cells and warranted further investigation. Subsequent studies using a flow cytometry based cell cycle progression assay that assessed the levels of both cellular RNA and DNA synthesis were able to distinguish non-dividing T cells into two categories: (i) cells in the Go/G1a phase which is characterized by undetectable levels of DNA and RNA synthesis and (ii) the G1b phase which is characterized by high levels of RNA expression in the absence of DNA synthesis (Korin and Zack 1998). Cells in the G1b stage of the cell cycle, while non-dividing and thus resting, were permissive to infection. On the other hand, the Go/1a cells were deemed as truly quiescent and they were resistant to HIV infection (Korin and Zack 1998). Therefore, the above data provided a foundation for the discrepancies seen among the different groups in terms of the permissiveness of quiescent T cells to HIV infection. Furthermore, it underscored the importance of distinguishing truly quiescent cells from resting but activated T cells. However, it was not yet clear what was behind the block presented to HIV by quiescent cells and what stage of the viral life cycle was impacted.

Quiescent T Cells Exhibit Multiple Blocks of the HIV Life Cycle Entry, Reverse Transcription and Integration Studies assessing the efficiency of HIV entry in quiescent T cells have been limited, especially in comparison to activated cells. However, based on the early work on quiescent cells, it was clear that the virus could enter quiescent CD4 T cells efficiently. Our group did compare HIV entry between quiescent and activated cells and found no significant differences between the two groups (Vatakis et al. 2007). Similarly, Agosto et.al demonstrated that the use of CXCR4-expressing


envelope was more efficient than VSV-g in transducing quiescent cells (Agosto et al. 2009). As entry seemed unaffected in quiescent T cells, the next stages of the viral cycle impacted are viral uncoating and reverse transcription. The study of viral uncoating is quite challenging in primary cells such as quiescent T cells. This is further complicated by the fact that quiescent T cells are very small in size with very limited cytoplasm. The majority of studies looking at this stage of the viral life cycle have been carried out in cell lines. However, one study using lysates from activated and quiescent cells showed that in contrast to that of quiescent cells lysates from activated cells resulted in efficient uncoating of HIV virions. While the authors were able to fractionate potential factors involved in uncoating they did not further identify them (Auewarakul et al. 2005). The development of more sensitive PCR technologies as well as cell purification protocols allowed for a better characterization of HIV reverse transcription and integration in quiescent T cells. A series of elegant studies by the Siliciano group shed more light on the infection of quiescent T cells by HIV. Utilizing a linker-mediated PCR assay, they measured the rate of reverse transcription and degradation of the non-integrated linear viral DNA (Pierson et al. 2002). They showed that reverse transcription in quiescent T cells was completed in 2–3 days, whereas in activated cells it was finished within 4–6 h. The linear piece of DNA had a half-life of about 1 day. The slow rate of reverse transcription and the labile nature of the newly synthesized cDNA severely compromised the ability of the virus to establish a productive infection. These observations were supported by a follow up study showing that the linear non-integrated viral DNA was integration competent (Zhou et al. 2005). These studies pointed to a potential block at the early stages of HIV infection. Furthermore, Swiggard et.al showed that while reverse transcription was decreased in quiescent T cells, full length HIV cDNA accumulated over time, was stable for approximately 3 days, and partial viral reverse transcripts were degraded (Swiggard et al. 2004; Swiggard


et al. 2005). However, the use of an alternative method of infection, spinoculation, raised the possibility of partial cell activation, which may have improved the stability of the full-length viral cDNA. We took a more detailed look at the kinetics of HIV infection in quiescent T cells and compared this against that of stimulated cells. In our studies, not only did we see delays in reverse transcription but also significantly lower levels of reverse transcription in quiescent T cells (Vatakis et al. 2007). More specifically, initiation of reverse transcription was 30-fold lower in quiescent T cells. There was some completion of reverse transcription which was delayed by 16 h compared to that seen in activated cells. Interestingly, this inefficient infection process of quiescent T cells was not rescued with immediate stimulation (Vatakis et al. 2007). Thus, all the studies point to a strong early block to infection.

Integration and Viral Expression The inefficiencies seen in reverse transcription did impact downstream events of the HIV viral life cycle. The development of a sensitive and quantitative assay allowed detection of low levels of integration in HIV infected cells, which proved very useful in the studies outlined below. Using this assay and spinoculation as the infection method, the O’Doherty group detected integrated virus in quiescent cells (Swiggard et al. 2005). Furthermore, viral expression was induced following stimulation of infected quiescent cells with IL-7 or anti-CD3/anti-CD28 co-stimulation. The results from this study demonstrated that a latent infection could be established in quiescent CD4 T cells. However, these studies did not reveal any deficiencies that may arise following reverse transcription, such as nuclear import of viral cDNA. In our studies, the delays seen in quiescent T cells impacted integration as this process was delayed by 24 h in quiescent CD4 T cells. However, the efficiency at which the viral cDNA was integrated was two to threefold less than that seen in activated cells. However, this difference

257

was within the limits of standard error and thus not significant. More interestingly, quiescent T cells expressed 48 h post-infection multiply spliced viral RNA at significantly lower levels than stimulated cells. Despite the expression of viral mRNA, there was no detectable Gag protein expression. Furthermore, the levels of integrated HIV copies remained unchanged for 5 days suggesting that these cells did not die after infection but were latently non-productively infected (Vatakis et al. 2007). Therefore, these data suggested that once reverse transcription took place, the viral cDNA was efficiently transported to the nucleus and integrated. Furthermore the provirus expressed low levels of viral mRNA, but the lack of viral protein suggested a potential posttranscriptional block. Furthermore, the above studies raised further questions regarding HIV integration site selection and viral expression. Integrated virus was found in resting cells of HIV infected individuals but this was attributed to infection of previously activated T cells that return to quiescence (Han et al. 2004). There was no indication that these cells were infected while quiescent. Furthermore, the presence of viral mRNA but the lack of detectable viral protein in quiescent T cells was quite intriguing (Vatakis et al. 2007). This raised the question of whether integration in quiescent T cells is distinct from that seen in their stimulated counterparts. As HIV preferentially integrates into actively transcribing genes and T cell quiescence is an actively maintained state, it could be inferred that a distinct distribution of integration sites could explain our observations. Others and we examined the distribution of integration sites in quiescent CD4 T cells (Brady et al. 2009; Vatakis et al. 2009). Based on our data, integration in both activated and quiescent CD4 T cells occurred mainly in transcriptionally active genes that were not affected by the T cell activation state such as housekeeping genes (Vatakis et al. 2009). However, a screening of the LTR ends in both the provirus and the 2-LTR circles revealed some interesting patterns. In quiescent CD4 T cells we observed elevated numbers of abnormal LTR-host DNA junctions. Furthermore, the levels of 2-LTR circles with

258

abnormal LTR junctions was also higher in quiescent cells; the abnormalities were primarily extensive deletions (Vatakis et al. 2009). It is suggested that the delays in reverse transcription had a detrimental effect on the ends of the viral cDNA. Similar patterns of integration were seen in studies by Brady et.al. However, in their studies, HIV integration patterns were somewhat different between stimulated and quiescent T cells (Brady et al. 2009). HIV appeared to integrate in less transcriptionally active regions in quiescent cells when compared to stimulated cells, but the observed differences were modest. To this date, there have been no studies showing viral release from HIV infected quiescent CD4 T cells in the absence of any stimulation. This can be proven quite important since in densely packed lymphoid tissues very low level viral release can be sufficient to support ongoing replication. Recent work in the SIV rhesus macaque model suggested that resting cells can spontaneously release virions (Nishimura et al. 2009). However, it is not clear if these resting cells are at the G1b stage of the cell cycle. If they are, it is expected that they will make virus as they are transcriptionally active. Additional studies have shown that the lack of the polypyrimidine tract binding protein (PTB) in resting cells results in nuclear retention of multiply spliced viral RNA limiting the production of virions (Lassen et al. 2006). However, it is unclear what the mechanism of action is and, thus, if this is an added block to infection.

Restriction Factors As stated earlier, quiescent cells are characterized by low metabolic rates and low levels of cellular RNA transcription. HIV, like any other virus, relies on cellular resources to replicate efficiently. Thus, it is very reasonable to infer that the lack of cellular substrates or raw materials can have a negative impact on viral replication. One such limited resource is the total nucleotide pool as it is important for viral reverse transcription. Pretreatment of quiescent T cells with exogenous nucleosides improved reverse transcription, increased the levels of integrated provirus and


2-LTR circles but still failed to rescue infection (Korin and Zack 1999; Plesa et al. 2007). Furthermore, the low metabolic rates of quiescent T cells would result in decreased amounts of available ATP. As the processes of reverse transcription and integration are energy dependent, it is expected that this could limit infection. However, there have not been studies looking into the impact of ATP levels on the efficiency of HIV infection. This suggested that the presence of inhibitory factors or the absence of supportive processes were responsible for this phenotype. siRNA technology has enabled researchers to identify the role of a number of genes using siRNA-mediated knockdown. This has also been used to study the effect of cellular factors on HIV replication. Ganesh et.al used this technology to identify a potential HIV restriction factor in quiescent T cells (Ganesh et al. 2003). Murr1 is a protein involved in copper regulation and inhibition of NFκB activity through inhibition of IκB degradation by the proteasome (Ganesh et al. 2003). When knocked down in cell lines, it decreased levels of IκB-α and enhanced NFκB activity. Furthermore, Murr1 is highly expressed in T cells. When the authors knocked down Murr1 using siRNA, they observed enhanced infection of quiescent T cells demonstrated by increased Gag expression. This suggested Murr1 may regulate HIV infection in quiescent CD4 T cells (Ganesh et al. 2003). The authors used nucleofection as means to introduce the siRNA. While the expression of CD25, CD69 and HLA-DR T cell activation markers was not observed, the process of nucleofection may have had an impact on the infection. However, no additional studies were performed to further elucidate the role of this protein. Apolipoprotein B mRNA editing catalytic peptide like 3G (APOBEC3G) has been shown to have strong anti-viral activity and is classified as an innate antiviral factor. APOBEC3G (A3G) is a cellular cytidine deaminase expressed in T cells and was initially found to be a potent antiviral factor against vif deficient HIV-1 (Sheehy et al. 2002; Mangeat et al. 2003; Zhang et al. 2003). Studies showed that in the absence of APOBEC3G causes severe hypermutation of the viral genome resulting resulting in non-productive infection.


Vif sequesters APOBEC3G mediating its degradation by the proteasome (Sheehy et al. 2002). Additional studies showed that A3G is found in a catalytically active low molecular mass form in quiescent T cells and in an inactive high molecular mass form in activated T cells, thus impacting permissiveness to infection between cellular activation states (Chiu et al. 2005). A3G knockdown in quiescent T cells initially indicated that the restriction to infection in quiescent T cells is abrogated in the absence of A3G. By nucleofecting an siRNA against A3G into quiescent T-cells, Chiu et al. demonstrated that these cells could be infected once A3G is eliminated even though there was no apparent indication of cellular activation (Chiu et al. 2005). However, two groups have independently published results that would indicate otherwise. Using identical techniques with the same, and two additional, siRNAs against A3G, Kamata et al. could not reproduce the results of the initial experiments (Kamata et al. 2009). Further, Santoni et al. knocked down A3G using both stable shRNA and ectopic vif expression in activated T-cells then allowed them to return to a resting state before infecting them with HIV-1. They found no difference in infection between cells with, and those without, A3G, nor did they find a correlation between viral restriction and high vs. low molecular mass A3G complexes (Santoni de Sio and Trono 2009). Thus, while A3G remains a major antiviral factor, its impact on quiescent T cell resistance to HIV does not seem to be major. Manganaro and colleagues shifted the focus on the lack of a cellular protein as a factor for the block to productive infection in quiescent T cells. In recent studies they demonstrated that c-Jun N-terminal kinase (JNK) phosphorylates viral integrase, which in turn interacts with the peptidylprolyl-isomerase enzyme Pin1 causing a conformational change in integrase (Manganaro et al. 2010). This results in increased integrase stability and completion of viral integration. Since quiescent T cells do not express JNK, they argued that the process is not efficient in quiescent cells (Manganaro et al. 2010). These findings do provide support for the earlier studies that suggested the presence of a preintegrated viral DNA in resting cells that acts as an inducible

259

reservoir (Stevenson et al. 1990; Spina et al. 1995). However, they do not explain the recent findings by our group as well as others that show integration occurring in quiescent T cells and that the defects seen in these cells are prior to that event in the viral life cycle (Swiggard et al. 2004; Vatakis et al. 2007; Brady et al. 2009; Vatakis et al. 2009). In addition, recent work by the Chow group (Briones et al. 2010), has implicated integrase HIV viral core stability. It is possible that JNK and Pin1 may be improving core stability, thus positively impacting reverse transcription. If this is the case, then JNK and Pin1 will have an impact on the early rather than later events of HIV life cycle. Finally, Glut1 has recently been added to the list of potential cellular factors that may facilitate HIV infection. Like JNK and Pin1, the absence of this cellular factor seems to impact HIV infection (Loisel-Meyer et al. 2012). More specifically, Glut1 is a major glucose transporter in T cells both mature T cells and thymocytes (LoiselMeyer et al. 2012). The expression of the protein is upregulated following exposure to IL-7 and conventional T cell activation. When knocked out, HIV infection was abrogated in activated T cells (Loisel-Meyer et al. 2012). Furthermore, its expression was linked to increased permissiveness to HIV, since double positive thymocytes expressing high levels of Glut1 were more likely to be infected by HIV than their low Glut1 expressing counterparts. This is the first study linking T cell metabolism to productive HIV infection. In summary, while it is accepted that there is a plethora of cellular factors that can enhance or restrict HIV replication in quiescent CD4 T cells, it is becoming more evident that the concerted action of multiple events is responsible for the lack of productive infection seen in quiescent CD4 T cells.

Discussion The permissiveness of quiescent CD4 T cells to HIV infection has been quite controversial. However, the development of more quantitative and sensitive techniques has provided us with some concrete answers as to the nature of HIV infection in this cell type. It is now clear that


260

quiescent T cells can become infected very inefficiently by HIV. It seems that the blocks present are focused on the early stages of infection. However, blocks in post-transcriptional events may also be implicated. While there has been major progress, it is still unclear how quiescent T cells block infection. A number of restriction factors have been presented as a possible explanation but their role is at best incremental. Similarly, the lack of key cellular factors and/or raw materials while important does not seem to tell the whole story. The major hurdle for the virus seems to be localized at the early stages of the life cycle, uncoating and reverse transcription. Poor uncoating in quiescent cells can prove detrimental to infection but studies thus far are very limited due to the inherent difficulties studying the viral capsid (Auewarakul et al. 2005). Moreover, quiescent T cell physiology is quite intriguing. They are characterized by very small size, a small cytoplasm and a very large nucleus. Thus, a more careful examination of quiescent cell physiology may yield important answers. At the post-integration level, quiescent cells still pose a very important enigma. Are they capable of making virus or is there another block? The defect we saw in LTR ends of infected quiescent cells were mainly the result of inefficiencies in reverse transcription. Epigenetic studies on these cells are imperative as they can shed some light on the state of the provirus. In addition, since we have detected viral RNA, the potential of posttranscriptional events such as the lack of PTB may pose a late stage block to infection. In summary, identifying the blocks in HIV infection of quiescent T cells remains quite important as increased knowledge can have major benefits in the area of study; one is the development of improved means to block infection especially at the early stages of the viral life cycle. Second, as HIV-based vectors are becoming more prevalent in gene therapy protocols, understanding fully the infection processes will allow for the development of more effective gene therapy vectors. Finally, HIV infected quiescent T cells can shed some more light as to the potential of our immune system to detect and eliminate non-activated cells as targets. Are dormant cells

whether viral infected or cancerous effective at evading our immune system? Acknowledgements This work was supported by in part by NIH/NIAID AI 070010-06A1, NIH Martin Delaney Collaboratory(to J.A.Z.), UCLA Center for AIDS Research NIH/National Institute of Allergy and Infectious Diseases Grant AI028697, NIH/NIDA R21 DA031036-01A1 (D.N.V.).

References Agosto LM, Yu JJ, Liszewski MK, Baytop C, Korokhov N, Humeau LM, O’Doherty U (2009) The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope. J Virol 83(16):8153–8162 Aksoylar HI, Lampe K, Barnes MJ, Plas DR, Hoebe K (2011) Loss of immunological tolerance in Gimap5deficient mice is associated with loss of foxo in CD4+ T cells. J Immunol 188(1):146–154 Auewarakul P, Wacharapornin P, Srichatrapimuk S, Chutipongtanate S, Puthavathana P (2005) Uncoating of HIV-1 requires cellular activation. Virology 337(1): 93–101 Barnes MJ, Aksoylar H, Krebs P, Bourdeau T, Arnold CN, Xia Y, Khovananth K, Engel I, Sovath S, Lampe K, Laws E, Saunders A, Butcher GW, Kronenberg M, Steinbrecher K, Hildeman D, Grimes HL, Beutler B, Hoebe K (2010) Loss of T cell and B cell quiescence precedes the onset of microbial flora-dependent wasting disease and intestinal inflammation in Gimap5deficient mice. J Immunol 184(7):3743–3754 Borvak J, Chou CS, Bell K, Van Dyke G, Zola H, Ramilo O, Vitetta ES (1995) Expression of CD25 defines peripheral blood mononuclear cells with productive versus latent HIV infection. J Immunol 155(6):3196–3204 Brady T, Agosto LM, Malani N, Berry CC, O’Doherty U, Bushman F (2009) HIV integration site distributions in resting and activated CD4+ T cells infected in culture. AIDS 23(12):1461–1471 Briones MS, Dobard CW, Chow SA (2010) Role of human immunodeficiency virus type 1 integrase in uncoating of the viral core. J Virol 84(10):5181–5190 Buckley AF, Kuo CT, Leiden JM (2001) Transcription factor LKLF is sufficient to program T cell quiescence via a c-Myc–dependent pathway. Nat Immunol 2(8): 698–704 Chiu YL, Soros VB, Kreisberg JF, Stopak K, Yonemoto W, Greene WC (2005) Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 435(7038):108–114 Chou CS, Ramilo O, Vitetta ES (1997) Highly purified CD25- resting T cells cannot be infected de novo with HIV-1. Proc Natl Acad Sci U S A 94(4):1361–1365

24 Quiescent CD4 + T Cells Inhibit Multiple Stages of HIV Infection Cotner T, Williams JM, Christenson L, Shapiro HM, Strom TB, Strominger J (1983) Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content. J Exp Med 157(2): 461–472 Ganesh L, Burstein E, Guha-Niyogi A, Louder MK, Mascola JR, Klomp LW, Wijmenga C, Duckett CS, Nabel GJ (2003) The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes. Nature 426(6968):853–857 Gowda SD, Stein BS, Mohagheghpour N, Benike CJ, Engleman EG (1989) Evidence that T cell activation is required for HIV-1 entry in CD4+ lymphocytes. J Immunol 142(3):773–780 Haaland RE, Yu W, Rice AP (2005) Identification of LKLF-regulated genes in quiescent CD4+ T lymphocytes. Mol Immunol 42(5):627–641 Han Y, Lassen K, Monie D, Sedaghat AR, Shimoji S, Liu X, Pierson TC, Margolick JB, Siliciano RF, Siliciano JD (2004) 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 78(12): 6122–6133 Kamata M, Nagaoka Y, Chen IS (2009) Reassessing the role of APOBEC3G in human immunodeficiency virus type 1 infection of quiescent CD4+ T-cells. PLoS Pathog 5(3):e1000342 Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang TT, Bos JL, Medema RH, Burgering BM (2002) Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419(6904):316–321 Korin YD, Zack JA (1998) Progression to the G1b phase of the cell cycle is required for completion of human immunodeficiency virus type 1 reverse transcription in T cells. J Virol 72(4):3161–3168 Korin YD, Zack JA (1999) Nonproductive human immunodeficiency virus type 1 infection in nucleosidetreated G0 lymphocytes. J Virol 73(8):6526–6532 Kuo CT, Veselits ML, Leiden JM (1997) LKLF: a transcriptional regulator of single-positive T cell quiescence and survival. Science 277(5334):1986–1990 Lassen KG, Ramyar KX, Bailey JR, Zhou Y, Siliciano RF (2006) Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells. PLoS Pathog 2(7):e68 Loisel-Meyer S, Swainson L, Craveiro M, Oburoglu L, Mongellaz C, Costa C, Martinez M, Cosset FL, Battini JL, Herzenberg LA, Atkuri KR, Sitbon M, Kinet S, Verhoeyen E, Taylor N (2012) Glut1-mediated glucose transport regulates HIV infection. Proc Natl Acad Sci U S A 109(7):2549–2554 Manganaro L, Lusic M, Gutierrez MI, Cereseto A, Del Sal G, Giacca M (2010) Concerted action of cellular JNK and Pin1 restricts HIV-1 genome integration to activated CD4+ T lymphocytes. Nat Med 16(3): 329–333 Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, Trono D (2003) Broad antiretroviral defence by

261

human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424(6944):99–103 Nishimura Y, Sadjadpour R, Mattapallil JJ, Igarashi T, Lee W, Buckler-White A, Roederer M, Chun TW, Martin MA (2009) High frequencies of resting CD4+ T cells containing integrated viral DNA are found in rhesus macaques during acute lentivirus infections. Proc Natl Acad Sci U S A 106(19):8015–8020 Ouyang W, Beckett O, Flavell RA, Li MO (2009) An essential role of the Forkhead-box transcription factor Foxo1 in control of T cell homeostasis and tolerance. Immunity 30(3):358–371 Pierson TC, Zhou Y, Kieffer TL, Ruff CT, Buck C, Siliciano RF (2002) Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection. J Virol 76(17):8518–8531 Plesa G, Dai J, Baytop C, Riley JL, June CH, O’Doherty U (2007) Addition of deoxynucleosides enhances human immunodeficiency virus type 1 integration and 2LTR formation in resting CD4+ T cells. J Virol 81(24):13938–13942 Ramilo O, Bell KD, Uhr JW, Vitetta ES (1993) Role of CD25+ and CD25-T cells in acute HIV infection in vitro. J Immunol 150(11):5202–5208 Santoni de Sio FR, Trono D (2009) APOBEC3G-depleted resting CD4+ T cells remain refractory to HIV1 infection. PLoS ONE 4(8):e6571 Sheehy AM, Gaddis NC, Choi JD, Malim MH (2002) Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418(6898):646–650 Spina CA, Guatelli JC, Richman DD (1995) Establishment of a stable, inducible form of human immunodeficiency virus type 1 DNA in quiescent CD4 lymphocytes in vitro. J Virol 69(5):2977–2988 Stevenson M, Stanwick TL, Dempsey MP, Lamonica CA (1990) HIV-1 replication is controlled at the level of T cell activation and proviral integration. Embo J 9(5): 1551–1560 Swiggard WJ, O’Doherty U, McGain D, Jeyakumar D, Malim MH (2004) Long HIV type 1 reverse transcripts can accumulate stably within resting CD4+ T cells while short ones are degraded. AIDS Res Hum Retroviruses 20(3):285–295 Swiggard WJ, Baytop C, Yu JJ, Dai J, Li C, Schretzenmair R, Theodosopoulos T, O’Doherty U (2005) Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli. J Virol 79(22): 14179–14188 Tzachanis D, Boussiotis VA (2009) Tob, a member of the APRO family, regulates immunological quiescence and tumor suppression. Cell Cycle 8(7): 1019–1025 Tzachanis D, Freeman GJ, Hirano N, van Puijenbroek AA, Delfs MW, Berezovskaya A, Nadler LM, Boussiotis VA (2001) Tob is a negative regulator of activation that is expressed in anergic and quiescent T cells. Nat Immunol 2(12):1174–1182

262 Tzachanis D, Lafuente EM, Li L, Boussiotis VA (2004) Intrinsic and extrinsic regulation of T lymphocyte quiescence. Leuk Lymphoma 45(10):1959–1967 Vatakis DN, Bristol G, Wilkinson TA, Chow SA, Zack JA (2007) Immediate activation fails to rescue efficient human immunodeficiency virus replication in quiescent CD4+ T cells. J Virol 81(7):3574–3582 Vatakis DN, Kim S, Kim N, Chow SA, Zack JA (2009) HIV integration efficiency and site selection in quiescent CD4+ T cells. J Virol. doi:10.1128/ JVI.00356–09 Weinberg JB, Matthews TJ, Cullen BR, Malim MH (1991) Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes. J Exp Med 174(6):1477–1482 Wong WF, Kohu K, Nakamura A, Ebina M, Kikuchi T, Tazawa R, Tanaka K, Kon S, Funaki T, SugaharaTobinai A, Looi CY, Endo S, Funayama R, Kurokawa M, Habu S, Ishii N, Fukumoto M, Nakata K, Takai T, Satake M (2012) Runx1 deficiency in CD4+ T cells causes fatal autoimmune inflammatory lung disease due to spontaneous hyperactivation of cells. J Immunol 188(11):5408–5420 Wu Q, Liu Y, Chen C, Ikenoue T, Qiao Y, Li CS, Li W, Guan KL, Zheng P (2011) The tuberous sclerosis complex-mammalian target of rapamycin pathway

J.A. Zack and D.N. Vatakis maintains the quiescence and survival of naive T cells. J Immunol 187(3):1106–1112 Yang K, Neale G, Green DR, He W, Chi H (2011) The tumor suppressor Tsc1 enforces quiescence of naive T cells to promote immune homeostasis and function. Nat Immunol 12(9):888–897 Yusuf I, Kharas MG, Chen J, Peralta RQ, Maruniak A, Sareen P, Yang VW, Kaestner KH, Fruman DA (2008) KLF4 is a FOXO target gene that suppresses B cell proliferation. Int Immunol 20(5):671–681 Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS (1990) HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61(2):213–222 Zagury D, Bernard J, Leonard R, Cheynier R, Feldman M, Sarin PS, Gallo RC (1986) Long-term cultures of HTLV-III–infected T cells: a model of cytopathology of T-cell depletion in AIDS. Science 231(4740):850–853 Zhang H, Yang B, Pomerantz RJ, Zhang C, Arunachalam SC, Gao L (2003) The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424(6944):94–98 Zhou Y, Zhang H, Siliciano JD, Siliciano RF (2005) Kinetics of human immunodeficiency virus type 1 decay following entry into resting CD4+ T cells. J Virol 79(4):2199–2210