The Role of HIV Integration in Viral Persistence: No More Whistling Past the Proviral Graveyard

Review Series: HIV The Journal of Clinical Investigation Series Editor: Robert F. Siliciano The role of HIV integration in viral persistence: no more whistling past the proviral graveyard

Frank Maldarelli Clinical Retrovirology Section, HIV Dynamics and Replication Program, National Cancer Institute, NIH, Frederick, Maryland, USA.

A substantial research effort has been directed to identifying strategies to eradicate or control HIV infection without a requirement for combination antiretroviral therapy (cART). A number of obstacles prevent HIV eradication, including low- level viral persistence during cART, long-term persistence of HIV-infected cells, and latent infection of resting CD4+ T cells. Mechanisms of persistence remain uncertain, but integration of the provirus into the host genome represents a central event in replication and pathogenesis of all retroviruses, including HIV. Analysis of HIV proviruses in CD4+ lymphocytes from individuals after prolonged cART revealed that a substantial proportion of the infected cells that persist have undergone clonal expansion and frequently have proviruses integrated in genes associated with regulation of cell growth. These data suggest that integration may influence persistence and clonal expansion of HIV-infected cells after cART is introduced, and these processes may represent key mechanisms for HIV persistence. Determining the diversity of host genes with integrants in HIV-infected cells that persist for prolonged periods may yield useful information regarding pathways by which infected cells persist for prolonged periods. Moreover, many integrants are defective, and new studies are required to characterize the role of clonal expansion in the persistence of replication-competent HIV.

Introduction fied clonally expanded, HIV-infected CD4+ T lymphocytes in indi- Combination antiretroviral therapy (cART) has revolutionized the viduals undergoing long-term cART (17–19), suggesting that clonal care of HIV-infected individuals (1). Although cART effectively sup- expansion may represent one mechanism of HIV persistence. The presses virus replication and prevents morbidity and mortality from replication competence of HIV in these clonally expanded popula- HIV-related complications, cART regimens do not eradicate infections has not been extensively investigated, and the precise role tion, and continuous therapy is required to prevent rebound vire- of clonal expansion in maintaining a true HIV reservoir is under mia and disease progression. Prolonged cART is associated with a investigation. Sequence analysis of HIV in peripheral blood mono- number of systemic complications (2), and regimen nonadherence cytes (PBMCs) is complex, as many infected cells harbor defective during therapy can be complicated by the development of drug or deleted HIV genomes, and the complement of HIV proviruses resistance, resulting in progressively complex regimens and poten- in PBMCs is commonly described as a “graveyard” of integration tial associated toxicity (3, 4). Consequently, a substantial research events (20–22). Nevertheless, replication-competent HIV reser- commitment has been made to eradicate HIV infection. “Cure” voirs are present among the defective clones, and new research strategies may include those therapies that completely eradicate indicates integration sites may drive clonal expansion and persis- infection as well as approaches that result in control of viremia and tence of these cells during therapy (18, 19). This review describes arrest of disease progression without ART (5). One instance of HIV HIV populations prior to and following introduction of cART, the eradication (6, 7) in the setting of bone marrow transplant spurred central role of HIV integration in long-term persistence, the evi- research into pharmacologic and immunologic approaches, but dence for clonal expansion of HIV-infected cells, and current con- gaps in understanding of HIV infection during cART prevent pre- cepts regarding the role of clonal expansion in the persistence of cisely targeted and highly effective curative therapies. replication-competent HIV. HIV persists during therapy, and populations of infected cells that give rise to infectious HIV (termed HIV reservoirs) have been HIV infection establishes genetically diverse recovered, particularly in resting CD4+ T cells (8–14); however, the populations of infected cells mechanisms of viral persistence remain incompletely understood. Rapid and error-prone HIV replication in vivo results in virus pop- Recently, analysis of HIV in plasma and in infected cells revealed ulations that are large, genetically diverse, and slow to undergo the emergence of clonal sequences after prolonged cART (15, 16). genetic change (23–30). In chronically infected individuals, genetic Advances in molecular technology and next-generation sequenc- diversity, characterized as the average pairwise nucleotide differ- ing have provided the first detailed look at proviruses and the host ences of HIV variants, is substantial, in the range of 1.5% to 2%. At genes into which they integrate. A number of reports have identi- this level of diversity, essentially no two single genomes in plasma are identical. Introduction of cART interrupts HIV replication and results in a marked decline in viral RNA levels in plasma; decreases Conflict of interest: The author has declared that no conflict of interest exists. in viremia occur with multiphase decay kinetics that correspond to Reference information: J Clin Invest. 2016;126(2):438–447. doi:10.1172/JCI80564. half-lives of virus-producing cells. Prior to cART initiation, more

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than 99% of virus is produced by cells that have a relatively short the gut (60, 62), genital tract (63), bronchial-alveolar lavage fluid half-life (~1 day) (31, 32); this population corresponds to the num- (64), and breast milk (65). The source of these variants was uncer- ber of activated CD4+ T cells. As these cells are eliminated, vire- tain, but PPCs were detected over long periods and were present mia declines more slowly, and additional phases of decay can be in infected children (66) and in patients with low-level viremia detected. After 3 to 4 years of continuous cART, viral RNA levels (67). Comparisons with pretherapy virus populations revealed remain relatively stable, although Mellors and colleagues recently no evidence of molecular evolution in these PPC variants, even detected ongoing decay of HIV viremia over time (33). over 9 or more years of cART (16). Detection of multiple copies of A variety of cell types support HIV infection, including cells in identical sequences in individuals who initially had wide genetic lymphocyte and monocyte lineages. In tissues such as the central diversity in plasma was unexpected and suggested that the indi- nervous system, microglia and astrocytes have been reported to vidual lineages producing these viruses had undergone cell divi- be infected with HIV. Macrophages, by virtue of their longevity sion. In all of these studies, it was unclear whether the identical despite infection, may contribute to long-lived cells and persis- sequences could be the result of long-lived cells from early in the tence following introduction of cART (34–37). course of HIV infection when the viral diversity is quite restricted In contrast to the marked decline in HIV RNA, the decay of and identical sequences are common. Studies of rebound viremia HIV-infected cells is less dramatic; only 8- to 30-fold declines in after treatment interruption revealed multiple populations with HIV DNA-positive cells are detected after initiating cART, and a no evidence of molecular evolution compared with pretherapy stable level of cell-associated HIV DNA has been reported after virus (68). Many of these individual populations were present in the first 2 to 3 years of cART (38). Sensitive viral outgrowth assays multiple copies, but in these cases, it is likely that the identical (VOAs) to detect replication-competent HIV reveal very few cells sequences were the result of new and ongoing replication. In gen- with infectious HIV, and around one in a million resting cells is eral, the replication competence of PPCs or other variants present capable of producing infectious HIV after prolonged cART (9–12, in persistent viremia is unknown. 39). Many VOA studies have been performed with peripheral Further evidence supporting expansion of HIV-infected cells blood lymphocytes, but subtypes other than T cells may represent was reported in studies of cell-associated HIV DNA from indi- substantial sources of long-lived infected cells, which are not typi- viduals undergoing long-term cART. Sequence analysis of PBMC- cally prevalent in PBMCs. Primate studies have directly identified derived HIV can be complex, as many proviral sequences are these cells as critical for viral persistence (37). defective. Longitudinal studies of HIV population genetics often The origin of low-level viremia detected during prolonged avoided analysis of these graveyards, as they included cells that cART remains uncertain and is the subject of ongoing study; sup- may have been recently or remotely infected with HIV, preclud- port for a number of potential sources has been described (39–45). ing precise measurements of genetic change over time. Neverthe- Long-lived infected cells that produce HIV either on a chronic con- less, studies of HIV sequences in patients undergoing long-term stitutive basis or episodically in response to an immune activation therapy revealed examples in which multiple, identical copies of event can sustain low-level viremia. Alternatively, ongoing cycles an HIV hypermutant were repeatedly recovered over prolonged of HIV replication with infection of new host cells may occur. The periods (60, 69). As these HIV variants were clearly defective, they inability of cART to prevent such cycles of infection may be due to could not have persisted by ongoing cycles of infection and the only inadequate drug potency, insufficient drug penetration into tissues, explanation for their persistence and recovery was that cells with replication in sanctuary sites, or chronic production from long-lived these proviruses had undergone cell division. Further investigation cells. In antiretroviral intensification studies, evidence of ongoing of clonal expansion required new approaches to identifying and replication could be detected as a transient increase in 2-long termi- quantifying HIV integration events. The majority of these studies nal repeat (2-LTR) circular forms in some patient PBMCs (46, 47). have analyzed HIV in plasma and in peripheral blood-derived lym- No significant decreases in plasma HIV RNA in were detected in phocytes. Tissue sources of HIV, which harbor the majority of HIV- intensification studies (46–50), suggesting that chronically infected infected cells (70), have not been extensively investigated. cells rather than ongoing replication was the source of plasma viremia. Genetic analyses of HIV nucleic acids obtained from end-point Integration: a central event in retrovirus dilution of plasma virions or cell-associated virus have yielded sub- replication stantial data regarding the source and nature of persistence (15, 16, Like all retroviruses, HIV uses a virion-encoded reverse transcrip- 51). Despite the profound decline in viremia, HIV genetic diversity tase (RT) enzyme to synthesize a double-stranded copy of DNA persists at pretherapy levels, demonstrating that the population of (denoted cDNA) from virion RNA templates (71), and integrates HIV-producing cells remains large after cART initiation. the viral DNA into the host genome using viral integrase (IN) (72). The composition of plasma virus populations undergoes Retroviruses infect diverse vertebrate hosts and cause a wide detectable shifts over the course of therapy. Bailey and colleagues spectrum of diseases (73); in all of these conditions, viral integra- identified groups of identical sequences in these viruses, with some tion represents a critical step that is key for both virus replication of these sequences accounting for over half of all single genomes and disease pathogenesis. detected (15). Such sequences were termed predominant plasma Reverse transcription takes place early after infection and clones (PPCs) (49) and could also be detected in plasma (16, 52), generates a DNA copy of viral RNA with LTR sequences at the cerebrospinal fluid (53), PBMCs (54–57), including resting CD4+ 5′ and 3′ ends. Upon completion of viral DNA synthesis, the cells (52, 58), various CD4+ subsets (59, 60), and episomal HIV nascent viral DNA is part of a large nucleoprotein complex (61) as well as in various HIV-associated compartments, including known as the preintegration complex (PIC) (Figure 1 and refs. 72,

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Figure 1. Establishing the HIV provirus. (A) HIV infects susceptible cells, and reverse transcription takes place early after infection, following attachment and fusion. Both RNA copies are required for replication, and multiple strand transfers between the two molecules occur, resulting in frequent recombination. The terminal repeats are duplicated at each end of the integration, and the product of reverse transcription is a full-length molecule containing LTRs. As a consequence of complex interactions and strand transfers, defective copies with large internal deletions (but intact 5′ and 3′ ends) are frequent products of reverse transcription. Reverse transcripts with HIV Gag p24 and IN compose the viral PIC, which is transported to the nucleus, a process requiring specific host proteins, including nucleoporins. In the nucleus, PIC structures interact with host protein LEDGF/p75, which preferentially facilitates integration in areas of transcriptional activity. (B) IN is a dimer of dimers that creates 5-bp staggered cuts in each DNA strand of the integration site, resulting in a 5-nt duplication of the host sequence for each integrant. Integration is completed by host enzymes, which fill in the staggered cuts. Integration sites are frequently mutated or internally deleted, but have precise integration sites with duplications of the host sequence at each end of the provirus. (C) Not all of the newly synthesized viral DNA molecules are integrated into the host genome; a subset of DNA molecules form circular molecules that include one or both LTRs (1- and 2-LTR circles).

74, 75), which includes an IN molecule bound at each of the 5′ vitro and in vivo (83). The first level of preference is mediated and 3′ DNA ends. The DNA sequence specificity for IN binding by cellular cofactors binding to IN as part of the PIC (Figure 1A). to HIV DNA is conferred by nucleotides at each end, and not by In HIV, the lens epithelial-derived growth factor (LEDGF; also size or internal sequence; consequently, full-length, mutant, and known as p75) binds specifically to HIV IN, resulting in integra- defective genomes are all substrates for integration (67). HIV tion preferences for genes with transcriptional activity by teth- infects nondividing cells, and nuclear import by the PIC is spe- ering the DNA/IN/LEDGF complex (called the intasome) with- cific, requiring interactions between PIC components, includ- in areas of active transcription (84). Data from samples obtained ing the HIV capsid, and cellular proteins, such as nucleoporins from tissue culture infections as well as from infected animals (76–82). Not all of the newly synthesized viral DNA molecules do not reveal specific integration “hot spots,” but do identify are integrated into the host genome; a subset of DNA molecules general preferences for actively transcribed genes (85–88). A form circular molecules that include one or both LTRs (Figure second level of preference is entirely localized within selection 1). These LTR circles are the products of cellular ligase reactions sequences and is based on where the IN enzyme itself binds and are likely to have long half-lives in infected cells, although to cut the host sequence. In HIV, 5′-AAT, 5′-TAA, and 5′-AAA they are not sources of transcription and cannot support produc- sequences are enriched at the site of integration. This prefer- tive or ongoing replication. ence becomes part of the 5-bp repeat at each end of the integra- In general, integration site targets in the host genome are tion (89). Other retroviruses exhibit distinct preferences (90), relatively nonspecific, but the virus exhibits site preferences in suggesting such preferences are IN specific.

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Table 1. Potential sources of HIV persistence

After long-term cART Decay kinetics/half-life HIV replication competence Contribution to HIV Contribution to HIV-infected reservoirs cell population HIV-producing Activated Phase 1/~1 d + No No Activated Phase 1/~1 d - No No Intermediate Phase 2/~14 d + Unlikely Unlikely Intermediate Phase 2/~14 d – Unlikely Unlikely Long-lived Phase 3,4/>39 wk + Yes Yes Long-lived Phase 3,4/>39 wk – No Yes Quiescent Resting yr + Yes Yes Resting yr – No Yes A number of different cell types are infected with HIV, having variable half-life after infection. The majority (>99%) are short-lived, highly activated cells that do not directly represent a source of HIV persistence. Longer-lived, HIV-producing cells are eliminated slowly (intermediate) or only after prolonged periods and may contribute to long-lived reservoirs, but only if they are infected with replication-competent virus. Resting cells with infectious HIV represent a critical source of persistence. All long-lived cells with replication-competent or -incompetent virus will contribute to measures of cell- associated HIV.

Integration site: a determining factor in viral HIV integration in persistence and clonal pathogenesis expansion of infected cells Integration site has important consequences for clonal expansion In the systems described above, where retrovirus infection does in retroviral pathogenesis. For example, human T-lymphotrophic not typically result in cell death, retroviral integration commonly virus-1 (HTLV-1) integrates into upstream regions, and several drives clonal expansion; however, it is not known whether similar thousand distinct HTLV-1–infected cells are present in infected events occur in HIV infection, where infection is frequently cyto- individuals. Many of these cells undergo clonal expansion and pathic. Many infected cells are eliminated after infection, but res- have viral integrations in genes associated with growth potential, ervoirs of infected cells persist during therapy; therefore, it is criti- but only a small subset of infected cells develop into oligoclonal cal to identify and quantify HIV integration sites, especially during T cell leukemias (91–93). In HTLV-2 infection, which does not cART, to determine whether specific sites are associated with per- cause leukemia or lymphoma, clonally expanded populations sistence or clonal expansion. Initial studies (102–104) of integra- persist for prolonged periods without the development of neo- tion sites in PBMCs from HIV-infected individuals undergoing plasia (94), indicating that clonal expansion events driven by cART were consistent with in vitro studies, and over 80% of inte- integration do not universally result in the development of neo- grants were identified in transcriptional units. Several individuals plasias. Similarly, MLV integration occurs near transcriptional studied had integrations in a single gene (BACH2), but the mech- start sites (TSS) of many genes and results in clonal expansion, anism of recurrent integration in this gene was uncertain, and it but does not invariably result in neoplasia (95), In avian retroviral was not known whether this gene represents a true hot spot for systems, viral integration is widespread, but expansions occur in integration or whether cells with BACH2 integrations have a selec- tumor and nontumor tissues (96). tive advantage favoring persistence (104). These early studies that Integration sites also affect retroviral vectors used in gene identified integrants were technically superb and accurately iden- therapy. Use of murine leukemia virus–based (MLV-based) vectors tified junction sequences, but the experimental design was unable in gene therapy for SCID resulted in integration events near TSS of to determine whether any of the integrants were present multiple human genes. Integration events occurring near enhancers resulted times and did not permit quantification of clonal expansion. Nev- in clonally expanded populations (97). Analysis of the leukemic cells ertheless, these studies confirmed in vitro studies, demonstrating revealed a selected population of genes with MLV integrations, sug- that HIV integration was widespread in transcriptional units and gesting leukemias were favored when these integrations occurred. was orientation independent. These findings reveal that clonal expansion is a characteris- Direct evidence for clonal expansion of HIV-infected cells tic of retroviral infections. As insertion sites with precise junction was obtained in several studies of individuals on long-term cART sequences accumulate, they have been catalogued for investiga- using newer techniques, including adaptations of high-through- tions of insertional mutagenesis and cancer (95, 98, 99). Such put, linker-mediated PCR assays (18, 92) and integration site loop “retroviral-tagged genes” can be used to map specific metabolic amplification (19), both of which recover substantial numbers of and processing pathways that promote growth, thereby identify- integrants. These methods, reviewed in ref. 105, identify junc- ing potential points where pathways can be interrupted to prevent tion sequences and the host gene into which HIV has integrated, or treat neoplasia (95, 98, 100, 101). determine the orientation of integration, and readily distinguish

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has also been implicated in tumorigenesis and BACH2- Table 2. Mechanisms of persistence BCL2L1 and BACH2-IGHD fusions have been reported Immune mediated Integration site mediated in lymphoma (109, 110). In mouse models of lymphoma, Homeostatic proliferation Host genes involved in cell growth regulation (orientation independent) MLV integrated in BACH2, resulting in upregulation of Antigenic stimulation Specific, orientation-dependent sites alternatively spliced variants of BACH2 in lymphomas Generalized immune activation (111). Finally, a series of observations suggest that loss of Immune mechanisms, viral integration site, or both may influence persistence and BACH2 may be linked to cellular persistence. The EBV- clonal expansion of HIV-infected cells. Homeostatic proliferation, antigen-directed transformed cell line Raji is a well-described B cell line expansion, or generalized immune activation may result in cell persistence and with substantial growth potential. DNA analyses revealed expansion. Some integrations occurring in host genes associated with cell growth will that Raji cells have an integrated copy of EBV in intron 1 result in persistence and expansion; specific integrations, e.g., BACH2 or MKL2, result in of BACH2, and transcriptome analyses detected a loss of persistence and clonal expansion that is orientation specific. BACH2 expression in this cell line (112, 113). Reintroduc- tion of BACH2 expression in these cells results in a loss of clonogenic activity (114), suggesting downregulation of BACH2 facilitated persistence, clonal expansion, or both. between true clonal expansion and artifacts arising from PCR MKL2 is also a transcription factor that is broadly expressed amplification. In these studies, repeat recovery of the identical in human tissues. Although less studied than BACH2, it has been junction sequence reflects clonal expansion, as the possibility of identified as a serum response factor (SRF) coactivator and is independent integrations into the identical nucleotide position in therefore involved in cell growth (115–117). MKL2 fusion onco- the human genome is highly statistically unfavorable. These stud- genes have also been identified, especially in chondroid lipomas, ies, which generated over 3,100 integration sites for study, again where C11orf95-MKL2 fusions are often present (118, 119). demonstrated a bias of HIV for transcriptional units (around 79% Taken together, these data suggest that the long-term fate of of all integrants in either study), but no orientation preference infected cells after cART is introduced is determined by intrinsic with respect to host transcription (18, 19). The recovery of sub- cell properties (e.g., activated vs. resting) and immune responses stantial numbers of integrants in combination with integrant copy (homeostatic proliferation, antigenic response, or nonspecific number quantification revealed a number of new findings. (a) immune activation mechanisms) as well as viral integration site After prolonged cART, approximately 40% of all integrants had (Table 1). The majority of infected cells have short half-lives undergone clonal expansion; as the sample size is relatively mod- after infection, are rapidly eliminated, and do not contribute to est compared with the infected cell population, this initial mea- long-lived HIV reservoirs. Resting cells, or those responsible for surement is likely to be an underestimate. (b) The cells with HIV low-level viremia, may persist for prolonged periods. The subset proviruses remaining after prolonged cART had integrations in infected with replication-competent virus contributes to the pop- diverse genes, but they were heavily biased toward genes involved ulation of infected cells representing the true HIV reservoir, but in cellular growth or oncogenesis. Such biases were not present in all proviruses contribute to the HIV-infected cell population. As analyses of HIV integration sites in in vitro infections, but were previously described (21, 39, 120), long-lived cells infected with detected after prolonged cART as the likely result of cells with replication-competent HIV represent a very small proportion of integrants in sites conferring a selective advantage for persistence, the total infected cell population. Mechanisms driving persistence clonal expansion, or both. (c) Cells with integrants in particular and clonal expansion include both immune functions and viral genes, notably BACH2 and MKL2, had a surprising pattern of inte- integration site (Table 2). The substantial bias that emerges over gration. After prolonged cART, the remaining integrants, some of prolonged cART for cells with integrants in genes associated with which were clonally expanded, were only detected in intron 5 of cell growth indicates that integration is an important mechanism BACH2 and intron 4 or 6 of MKL2 upstream of the translational for persistence and clonal expansion of HIV-infected cells. start site. In all cases, integrants were oriented in the same direction of transcription as the corresponding host gene. Analysis of New approaches to analyze HIV integrants prior studies of patients undergoing cART showed that integrants To date, next generation sequencing has provided only a short were similarly restricted within BACH2 and were also oriented in sequence of HIV with the junction sequence and host genome the same direction of transcription as the host gene. Very recently, sequence; matching integrants to viral variants in plasma is pos- Cohn and colleagues used a modified linker-mediated approach sible, but is not readily accomplished with high-throughput and identified BACH2 among the integration sites (17). In contrast sequencing approaches. Amplification with sequencing of longer to previous studies, their results implicated hot spots for integra- HIV sequences will aid the tracking of HIV populations expressed tion near human Alu sequences, with less bias for integration in during HIV infection. transcriptionally active genes. With the advent of new methods to quantify the diversity of BACH2 is a basic region, leucine zipper family transcription fac- HIV-infected cells, additional statistical approaches will be need- tor that regulates transcription of both B and T lymphocytes through ed to analyze alterations (or stability) in these populations over enhancer binding sequences. In T cells, BACH2 contributes to T cell time, particularly with regard to integrant diversity and clonality homeostasis (106, 107). In transgenic mouse models, loss of BACH2 during therapy. Bangham and colleagues developed an oligoclo- was associated with increases in effector T cell (Teff) function and nality index (92, 94) to quantify clonal expansion of HTLV-1/2– loss of regulatory T lymphocyte (Treg) function (108). BACH2 infected populations, which will also likely be useful in analyzing

442 jci.org Volume 126 Number 2 February 2016 The Journal of Clinical Investigation Review Series: HIV clonality of HIV-infected cells. With evidence of an increase in genes, facilitating persistence and expansion without produc- the number of integrants in host genes involved in cell growth and tion of virus. The roles of HIV genes other than IN remain uncer- persistence, bioinformatic approaches that classify host cell gene tain, and additional studies will determine contributions of HIV function with integrants will be quite useful. genes to persistence or expansion. Expression of HIV regulatory Further characterization of specific integrants, such as those genes such as Tat and Rev does not require intact genomes and integrated into BACH2 and MKL2, is essential. Expanded popula- may affect expression of HIV and chimeric host:HIV transcripts. tions with proviruses in BACH2 or MKL2 are striking, but they rep- The HIV Vpr gene has been implicated in cell cycle arrest. While resent a relatively small proportion of all the proviruses in infected it may seem paradoxical that proviruses with Vpr may undergo individuals and will likely be difficult to identify and study directly. clonal expansion, cell activation can occur without HIV transcrip- Site-specific recombination approaches to introduce individual pro- tion, and clonal expansion may occur in the absence of HIV tran- viruses within genes of interest will provide tractable in vitro sys- scription. Clonal expansion itself can affect proviral structure. tems to study cellular functions. As integration sites are identified, During DNA replication, LTR sequences at each end of a provirus it will be necessary to assemble these data into common, publicly can undergo homologous recombination, resulting in deletion of available databases. Much groundwork for this process has already internal sequences that yield highly deleted proviruses, including been provided for other retroviruses (121, 122), and Bushman, Shao, those consisting of a single LTR sequence. Analogous to numerous and coworkers have established interfaces (123, 124) that will allow animal systems and endogenous retroviruses, where such “solo investigators to take advantage of the increasing number of integra- LTRs” have become common over millions of years of evolution tion sites for in silico analyses with statistical power. (126, 127), all of these deleted proviral structures may influence persistence and expansion. Characterizing the role of clonal expansion in The studies reported thus far represent very early steps in HIV infection the investigation of a role for clonal expansion in HIV persis- Data accumulated to date indicate that HIV-infected cells persist tence. Recent studies have identified integrants in a cross sec- for prolonged periods and undergo clonal expansion; it remains tion of patients with HIV infection of variable duration. As HIV uncertain to what degree clonally expanded populations contrib- integrates broadly in transcribed genes, many more junction ute to the population of replication-competent cells, which repre- sequences will be necessary for comprehensive analysis of inte- sent the relevant reservoir that requires eradication or long-term gration sites during prolonged therapy. The number of integrants control. In an exhaustive analysis of 213 uninduced proviruses, recovered from patients (in the few thousands) is low relative to Ho and colleagues found that only 11.3% were full length (21). the number of integrants obtained from model systems or in vitro It is not known whether any of these proviruses were members HIV infection, in which many thousands of junction sequences of a clonally expanded population, but the data indicate that few can be unequivocally identified. A larger compendium of inte- integrants, expanded or not, are replication competent. Cohn and gration sets will be essential to identifying whether the persist- colleagues (17) analyzed 75 proviruses from expanded popula- ing populations have integrants in specific growth or apoptotic tions and did not identify any full-length HIV proviruses, suggest- pathways after prolonged cART. ing the frequency of clonally expanded populations with replica- Further longitudinal data will be essential in understanding tion-competent virus is low and may not contribute significantly the contribution of clonally expanded populations to the total HIV to the HIV reservoir. integrant population. Interestingly, a number of studies have indi- In contrast to the findings described above, direct demon- cated gradual shifts in the genetic composition of HIV populations stration of replication-competent HIV from a clonally expanded during prolonged periods on cART. Moreover, precise quantita- population has been reported (125). Persistent low-level PPC tive analysis by Besson (38) revealed that the relative levels of HIV was identified, sequenced, and matched to an identical full- proviruses in PBMCs (quantified using an internal IN sequence) length provirus with all HIV reading frames intact in PBMCs; an were stable over time. These data suggest that the HIV population unequivocal chromosomal assignment was not possible because is dynamic, undergoing population shifts and clonal expansion, the provirus was in an ambiguous region of the genome. The pro- while the total amount of PBMC-associated HIV DNA remains virus was amplified from patient-derived PBMC DNA and found relatively constant. Longitudinal studies of integrant distribution to be infectious in in vitro infection experiments. Moreover, combined with quantification of relative levels of HIV integrants infectious virus could be recovered from patient-derived PBMCs will help to determine whether maintaining a stable population of stimulated in vitro. These results indicate that clonally expanded proviral DNA represents a steady state of expansion and elimina- HIV-infected cells can produce infectious HIV. The frequency tion of variants. Investigations into the roles of specific immune of such infectious, clonally expanded cells is unknown, and the responses and homeostatic proliferation will help elucidate the relative contribution of clonally expanded populations to the HIV mechanisms of persistence of infected cells. reservoir remains to be determined. Analyses of a number of natural history cohorts will provide Further analysis of proviral structures in highly expanded pop- useful insights into the emergence of clonally expanded popula- ulations will be necessary to characterize the HIV reservoir. Ini- tions. For example, investigation of pre- and on-therapy PBMCs tial studies detected complex deletions in expanded populations will help determine the degree of clonal expansion prior to intro- and many defective proviruses with internal defects (17, 21, 120). duction of antiretrovirals. Similarly, longitudinal analysis of acute Integrants with intact promoter or other regulatory sequences infection, early infection, and perinatally acquired HIV-infected (e.g., splice donor sequences) can have profound effects on host patients undergoing cART will yield critical information regard-

jci.org Volume 126 Number 2 February 2016 443 Review Series: HIV The Journal of Clinical Investigation ing establishment of HIV-infected cells early in infection and T cell repertoire will provide useful comparative analyses. It is not their persistence during therapy. HIV-infected individuals with known whether extensive clonal expansions occur in the setting of low CD4 cell numbers who initiate cART may experience immune a diverse or restricted repertoire; thus, analysis of integration sites reconstitution syndrome. Analysis of these individuals may help may represent a tool for understanding the immunologic response determine whether the exuberant immune response includes as much as for characterizing the virology of the infection itself. marked clonal expansion of HIV-infected cells. Individuals undergoing cART, but with incomplete reconstitution, represent Summary an important subset of treated patients; mechanisms preventing Recent studies of HIV integration have been made possible by robust CD4 reconstitution in these immune nonresponders is advances in the identification of HIV-host junction sequences and unknown, but early studies of a few individuals (18) indicate that determination of the relative extent of clonal expansion of cells con- immune nonresponders have potential for extensive clonal expan- taining HIV proviruses. Initial analyses of clonally expanded popula- sion. Comprehensive analyses of immune responders and nonre- tions demonstrate a substantial role for the integration site in shap- sponders may yield useful insights into the mechanism of incom- ing the population of cells persisting after prolonged cART. Detailed plete reconstitution. analysis of HIV in clonally expanded populations has revealed com- In addition to natural history studies, specific interventional plex proviral structures with many deletions and insertions, but also studies will yield critical and strategically useful information replication-competent virus. Thus, the population of cells with inte- regarding clonal expansion during suppressive cART. A number grated HIV is no longer just a “graveyard” of proviruses, analysis of of ongoing studies investigating the effects of latency reversing which can be naively or conveniently avoided. Rather than whistling agents (LRAs) and other modalities to eliminate HIV reservoirs past this graveyard, analysis of proviruses, their integration sites, and (128–133) may also benefit from analysis of clonal populations. In their clonal expansion represents a new approach to characterizing shock and kill strategies, LRAs induce HIV by activating infected, HIV infection during cART, which will be useful in evaluating the quiescent T cells. These cells presumably undergo cell death, but effects of strategies to eradicate the infection in humans. may undergo expansion as well, preventing eradication. Specific integration site data will be useful in identifying integrants that Acknowledgments are activated, eliminated, or expanded during latency reversal. I am grateful to the study participants who contributed to research Sensitive integration assays are likely to represent useful adjunct described and to F.R. Simonetti, S.H. Hughes, J. Coffin, J. Mellors, analyses to characterize proviral populations in eradication strate- M.F. Kearney, W. Shao, X. Wu, T. Uldrick, R. Yarchoan, C. Lane, gies, especially as these approaches begin to incorporate multiple M. Polis, and J. Kovacs for insightful discussions. This work was agents to activate and eliminate infected cells. The effects of pre- supported in part by the NIH Bench-to-Bedside Program. ventative or therapeutic vaccination and other biologically active agents on clonal expansion will also be of interest in understand- Address correspondence to: Frank Maldarelli, Clinical Retrovirol- ing immune function during cART. ogy Section, HIV Dynamics and Replication Program, National Ultimately, data describing the persistence and expansion of Cancer Institute, NIH, Building 535, Room 108E, 1050 Boyles cells containing HIV proviruses should be analyzed in the context St., Frederick National Cancer Laboratory, Frederick, Maryland of overall CD4 cell persistence and expansion. Studies analyzing 21702, USA. Phone: 301.846.5611; E-mail: [email protected].

1. Piot P, Legido-Quigley H. Global perspectives on CCR5 Δ32/Δ32 stem-cell transplantation. N Engl 14. Siliciano JD, Siliciano RF. A long-term latent res- Human Immunodeficiency Virus infection and J Med. 2009;360(7):692–698. ervoir for HIV-1: discovery and clinical implica- Acquired Immunodeficiency Syndrome. In: Ben- 8. Chun TW. Tracking replication-competent HIV tions. J Antimicrob Chemother. 2004;54(1):6–9. nett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, reservoirs in infected individuals. Curr Opin HIV 15. Bailey JR, et al. Residual human immunode- and Bennett’s Principles and Practice of Infectious AIDS. 2013;8(2):111–116. ficiency virus type 1 viremia in some patients Diseases. 8th ed. Philadelphia, Pennsylvania, 9. Chun TW, et al. Quantification of latent tissue on antiretroviral therapy is dominated by USA: Elsevier; 2015:1469–1482. reservoirs and total body viral load in HIV-1 a small number of invariant clones rarely 2. Morse CG, Kovacs JA. Metabolic and skeletal infection. Nature. 1997;387(6629):183–188. found in circulating CD4+ T cells. J Virol. complications of HIV infection: the price of suc- 10. Chun TW, Finzi D, Margolick J, Chadwick 2006;80(13):6441–6457. cess. JAMA. 2006;296(7):844–854. K, Schwartz D, Siliciano RF. In vivo fate of 16. Kearney MF, et al. Lack of detectable 3. Calvo KR, Daar ES. Antiretroviral therapy: treat- HIV-1-infected T cells: quantitative analysis HIV-1 molecular evolution during suppres- ment-experienced individuals. Infect Dis Clin of the transition to stable latency. Nat Med. sive antiretroviral therapy. PLoS Pathog. North Am. 2014;28(3):439–456. 1995;1(12):1284–1290. 2014;10(3):e1004010. 4. Solomon DA, Sax PE. Current state and limita- 11. Finzi D, et al. Latent infection of CD4+ T cells 17. Cohn LB, et al. HIV-1 integration landscape tions of daily oral therapy for treatment. Curr provides a mechanism for lifelong persistence of during latent and active infection. Cell. Opin HIV AIDS. 2015;10(4):219–225. HIV-1, even in patients on effective combination 2015;160(3):420–432. 5. Blankson JN, Siliciano JD, Siliciano RF. Finding a therapy. Nat Med. 1999;5(5):512–517. 18. Maldarelli F, et al. HIV latency. Specific HIV cure for human immunodeficiency virus-1 infec- 12. Finzi D, et al. Identification of a reservoir for integration sites are linked to clonal expan- tion. Infect Dis Clin North Am. 2014;28(4):633–650. HIV-1 in patients on highly active antiretroviral sion and persistence of infected cells. Science. 6. Yukl SA, et al. Challenges in detecting HIV therapy. Science. 1997;278(5341):1295–1300. 2014;345(6193):179–183. persistence during potentially curative interven- 13. Siliciano JD, et al. Long-term follow-up stud- 19. Wagner TA, et al. HIV latency. Proliferation tions: a study of the Berlin patient. PLoS Pathog. ies confirm the stability of the latent reservoir of cells with HIV integrated into cancer genes 2013;9(5):e1003347. for HIV-1 in resting CD4+ T cells. Nat Med. contributes to persistent infection. Science. 7. Hutter G, et al. Long-term control of HIV by 2003;9(6):727–728. 2014;345(6196):570–573.

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20. Grant RM, Abrams DI. Not all is dead in HIV-1 Sci U S A. 2001;98(2):658–663. forms in the plasma of patients undergoing suc- graveyard. Lancet. 1998;351(9099):308–309. 38. Besson GJ, et al. HIV-1 DNA decay dynamics cessful HAART. Arch Virol. 2010;155(6):895–903. 21. Ho YC, et al. Replication-competent noninduced in blood during more than a decade of sup- 56. Wagner TA, McKernan JL, Tobin NH, Tapia KA, proviruses in the latent reservoir increase barrier pressive antiretroviral therapy. Clin Infect Dis. Mullins JI, Frenkel LM. An increasing propor- to HIV-1 cure. Cell. 2013;155(3):540–551. 2014;59(9):1312–1321. tion of monotypic HIV-1 DNA sequences during 22. Kesic M, Green PL. Retroviruses and insights into 39. Siliciano JM, Siliciano RF. The remarkable antiretroviral treatment suggests proliferation of cancer: retroviral regulatory/accessory genes and stability of the latent reservoir for HIV-1 in HIV-infected cells. J Virol. 2013;87(3):1770–1778. cancer. In: Dudley J, ed. Retroviruses And Insights resting memory CD4+ T cells. J Infect Dis. 57. Buzon MJ, et al. Deep molecular characterization Into Cancer. New York, New York, USA: Springer; 2015;212(9):1345–1347. of HIV-1 dynamics under suppressive HAART. 2011:163–190. 40. Maldarelli F. Targeting viral reservoirs: ability of PLoS Pathog. 2011;7(10):e1002314. 23. Domingo E, Sheldon J, Perales C. Viral qua- antiretroviral therapy to stop viral replication. 58. Brennan TP, Woods JO, Sedaghat AR, Siliciano sispecies evolution. Microbiol Mol Biol Rev. Curr Opin HIV AIDS. 2011;6(1):49–56. JD, Siliciano RF, Wilke CO. Analysis of human 2012;76(2):159–216. 41. Palmer S, Josefsson L, Coffin JM. HIV reservoirs immunodeficiency virus type 1 viremia and 24. Maldarelli F, et al. HIV populations are large and and the possibility of a cure for HIV infection. provirus in resting CD4+ T cells reveals a novel accumulate high genetic diversity in a nonlinear J Intern Med. 2011;270(6):550–560. source of residual viremia in patients on antiret- fashion. J Virol. 2013;87(18):10313–10323. 42. Siliciano JD, Siliciano RF. Recent developments roviral therapy. J Virol. 2009;83(17):8470–8481. 25. Rambaut A, Posada D, Crandall KA, Holmes EC. in the search for a cure for HIV-1 infection: tar- 59. Chomont N, et al. HIV reservoir size and persis- The causes and consequences of HIV evolution. geting the latent reservoir for HIV-1. J Allergy Clin tence are driven by T cell survival and homeostat- Nat Rev Genet. 2004;5(1):52–61. Immunol. 2014;134(1):12–19. ic proliferation. Nat Med. 2009;15(8):893–900. 26. Rouzine IM, Coffin JM. Linkage disequilibrium 43. Siliciano RF. What do we need to do to cure HIV 60. Josefsson L, et al. The HIV-1 reservoir in test implies a large effective population num- infection. Top HIV Med. 2010;18(3):104–108. eight patients on long-term suppressive anti- ber for HIV in vivo. Proc Natl Acad Sci U S A. 44. Siliciano RF, Greene WC. HIV latency. Cold retroviral therapy is stable with few genetic 1999;96(19):10758–10763. Spring Harb Perspect Med. 2011;1(1):a007096. changes over time. Proc Natl Acad Sci U S A. 27. Rouzine IM, Coffin JM, Weinberger LS. Fifteen 45. Archin NM, Sung JM, Garrido C, Soriano-Sarabia 2013;110(51):E4987–E4996. years later: Hard and soft selection sweeps con- N, Margolis DM. Eradicating HIV-1 infection: 61. Sharkey M, Babic DZ, Greenough T, Gulick R, firm a large population number for HIV in vivo. seeking to clear a persistent pathogen. Nat Rev Kuritzkes DR, Stevenson M. Episomal viral cDNAs PLoS Genet. 2014;10(2):e1004179. Microbiol. 2014;12(11):750–764. identify a reservoir that fuels viral rebound after 28. Rouzine IM, Rodrigo A, Coffin JM. Transition 46. Buzon MJ, et al. HIV-1 replication and immune treatment interruption and that contributes to treat- between stochastic evolution and deterministic dynamics are affected by raltegravir intensifica- ment failure. PLoS Pathog. 2011;7(2):e1001303. evolution in the presence of selection: general tion of HAART-suppressed subjects. Nat Med. 62. Imamichi H, et al. Lack of compartmentaliza- theory and application to virology. Microbiol Mol 2010;16(4):460–465. tion of HIV-1 quasispecies between the gut and Biol Rev. 2001;65(1):151–185. 47. Hatano H, et al. A randomized controlled trial peripheral blood compartments. J Infect Dis. 29. Coffin JM. Genetic diversity and evolution assessing the effects of raltegravir intensification 2011;204(2):309–314. of retroviruses. Curr Top Microbiol Immunol. on endothelial function in treated HIV infection. 63. Bull M, et al. Compartmentalization of HIV-1 1992;176:143–164. J Acquir Immune Defic Syndr. 2012;61(3):317–325. within the female genital tract is due to mono- 30. Coffin JM. HIV viral dynamics. AIDS. 48. Dinoso JB, et al. Treatment intensification does typic and low-diversity variants not distinct viral 1996;10(suppl 3):S75–S84. not reduce residual HIV-1 viremia in patients populations. PLoS One. 2009;4(9):e7122. 31. Ho DD, Neumann AU, Perelson AS, Chen W, on highly active antiretroviral therapy. Proc Natl 64. Heath L, et al. Evidence for limited genetic com- Leonard JM, Markowitz M. Rapid turnover of Acad Sci U S A. 2009;106(23):9403–9408. partmentalization of HIV-1 between lung and plasma virions and CD4 lymphocytes in HIV-1 49. Gandhi RT, et al. No effect of raltegravir inten- blood. PLoS One. 2009;4(9):e6949. infection. Nature. 1995;373(6510):123–126. sification on viral replication markers in the 65. Gantt S, et al. Genetic analyses of HIV-1 env 32. Perelson AS, Neumann AU, Markowitz M, Leonard blood of HIV-1-infected patients receiving anti- sequences demonstrate limited compartmental- JM, Ho DD. HIV-1 dynamics in vivo: virion clear- retroviral therapy. J Acquir Immune Defic Syndr. ization in breast milk and suggest viral replica- ance rate, infected cell life-span, and viral genera- 2012;59(3):229–235. tion within the breast that increases with masti- tion time. Science. 1996;271(5255):1582–1586. 50. McMahon D, et al. Short-course raltegravir inten- tis. J Virol. 2010;84(20):10812–10819. 33. Riddler SA, et al. Continued slow decay of the sification does not reduce persistent low-level 66. Shiu C, et al. Identification of ongoing human residual plasma viremia level in HIV-1-infected viremia in patients with HIV-1 suppression dur- immunodeficiency virus type 1 (HIV-1) replica- adults receiving long-term antiretroviral therapy ing receipt of combination antiretroviral therapy. tion in residual viremia during recombinant [published online ahead of print September 2, Clin Infect Dis. 2010;50(6):912–919. HIV-1 poxvirus immunizations in patients with 2015]. J Infect Dis. doi:jiv433. 51. Simonetti FR, Kearney MF. Review: Influence clinically undetectable viral loads on durable sup- 34. Verollet C, Le Cabec V, Maridonneau-Parini I. of ART on HIV genetics. Curr Opin HIV AIDS. pressive highly active antiretroviral therapy. HIV-1 infection of T lymphocytes and macro- 2015;10(1):49–54. J Virol. 2009;83(19):9731–9742. phages affects their migration via Nef. Front 52. Anderson JA, et al. Clonal sequences recovered 67. Tobin NH, et al. Evidence that low-level viremias Immunol. 2015;6:514. from plasma from patients with residual HIV-1 during effective highly active antiretroviral 35. Abbas W, Tariq M, Iqbal M, Kumar A, Herbein viremia and on intensified antiretroviral therapy therapy result from two processes: expression G. Eradication of HIV-1 from the macro- are identical to replicating viral RNAs recovered of archival virus and replication of virus. J Virol. phage reservoir: an uncertain goal? Viruses. from circulating resting CD4+ T cells. J Virol. 2005;79(15):9625–9634. 2015;7(4):1578–1598. 2011;85(10):5220–5223. 68. Joos B, et al. HIV rebounds from latently 36. Crowe S, Zhu T, Muller WA. The contribution of 53. Evering TH, Kamau E, St Bernard L, Farmer CB, infected cells, rather than from continuing monocyte infection and trafficking to viral persis- Kong XP, Markowitz M. Single genome analysis low-level replication. Proc Natl Acad Sci U S A. tence, and maintenance of the viral reservoir in reveals genetic characteristics of Neuroadap- 2008;105(43):16725–16730. HIV infection. J Leuk Biol. 2003;74(5):635–641. tation across HIV-1 envelope. Retrovirology. 69. Imamichi H, et al. Lifespan of effector mem- 37. Igarashi T, et al. Macrophage are the principal 2014;11:65. ory CD4+ T cells determined by replication- reservoir and sustain high virus loads in rhesus 54. Chun TW, et al. HIV-infected individuals receiv- incompetent integrated HIV-1 provirus. AIDS. macaques after the depletion of CD4+ T cells by ing effective antiviral therapy for extended 2014;28(8):1091–1099. a highly pathogenic simian immunodeficiency periods of time continually replenish their viral 70. Svicher V, Ceccherini-Silberstein F, Antinori A, virus/HIV type 1 chimera (SHIV): Implications reservoir. J Clin Invest. 2005;115(11):3250–3255. Aquaro S, Perno CF. Understanding HIV com- for HIV-1 infections of humans. Proc Natl Acad 55. Lopez CA, et al. Characterization of HIV-1 RNA partments and reservoirs. Curr HIV/AIDS Rep.

jci.org Volume 126 Number 2 February 2016 445 Review Series: HIV The Journal of Clinical Investigation

2014;11(2):186–194. 90. Demeulemeester J, De Rijck J, Gijsbers R, Deby- critical for regulating CD4 T-cell senescence 71. Hu WS, Hughes SH. HIV-1 reverse tran- ser Z. Retroviral integration: site matters: mecha- and cytokine homeostasis. Nat Commun. scription. Cold Spring Harb Perspect Med. nisms and consequences of retroviral integration 2014;5:3555. 2012;2(10):a006882. site selection. Bioessays. 2015;37(11):1202–1214. 108. Roychoudhuri R, et al. BACH2 represses effector 72. Craigie R, Bushman FD. HIV DNA inte- 91. Cook LB, Rowan AG, Melamed A, Taylor GP, programs to stabilize T(reg)-mediated immune gration. Cold Spring Harb Perspect Med. Bangham CR. HTLV-1-infected T cells contain homeostasis. Nature. 2013;498(7455):506–510. 2012;2(7):a006890. a single integrated provirus in natural infection. 109. Turkmen S, Riehn M, Klopocki E, Molkentin M, 73. Goff SP. Retroviridae. In: Knipe DM, Howley P, Blood. 2012;120(17):3488–3490. Reinhardt R, Burmeister T. A BACH2-BCL2L1 eds. Fields Virology. 6th ed. Philadelphia, Penn- 92. Gillet NA, et al. The host genomic environ- fusion gene resulting from a t(6;20)(q15;q11.2) sylvania, USA: Lippincott Wiliams and Wilkins; ment of the provirus determines the abun- chromosomal translocation in the lymphoma 2013:1424–1473. dance of HTLV-1-infected T-cell clones. Blood. cell line BLUE-1. Genes Chromosomes Cancer. 74. Engelman A. Isolation and analysis of HIV-1 2011;117(11):3113–3122. 2011;50(6):389–396. preintegration complexes. Methods Mol Biol. 93. Niederer HA, Bangham CR. Integration site 110. Kobayashi S, et al. Identification of IGHCdelta- 2009;485:135–149. and clonal expansion in human chronic ret- BACH2 fusion transcripts resulting from cryptic 75. Li M, Craigie R. Nucleoprotein complex roviral infection and gene therapy. Viruses. chromosomal rearrangements of 14q32 with intermediates in HIV-1 integration. Methods. 2014;6(11):4140–4164. 6q15 in aggressive B-cell lymphoma/leukemia. 2009;47(4):237–242. 94. Melamed A, et al. Clonality of HTLV-2 in natural Genes Chromosomes Cancer. 2011;50(4):207–216. 76. Krishnan L, et al. The requirement for cellular infection. PLoS Pathog. 2014;10(3):e1004006. 111. Liu J, Sorensen AB, Wang B, Wabl M, Nielsen transportin 3 (TNPO3 or TRN-SR2) during 95. Du Y, Jenkins NA, Copeland NG. Insertional AL, Pedersen FS. Identification of novel Bach2 infection maps to human immunodeficiency mutagenesis identifies genes that promote the transcripts and protein isoforms through tagging virus type 1 capsid and not integrase. J Virol. immortalization of primary bone marrow pro- analysis of retroviral integrations in B-cell lym- 2010;84(1):397–406. genitor cells. Blood. 2005;106(12):3932–3939. phomas. BMC Mol Biol. 2009;10:2. 77. Lee K, et al. Flexible use of nuclear import pathways 96. Justice Jt, et al. The MET gene is a common 112. Cao S, et al. High-throughput RNA sequencing- by HIV-1. Cell Host Microbe. 2010;7(3):221–233. integration target in avian leukosis virus sub- based virome analysis of 50 lymphoma cell lines 78. Matreyek KA, Engelman A. The requirement for group J-induced chicken hemangiomas. J Virol. from the Cancer Cell Line Encyclopedia project. nucleoporin NUP153 during human immunode- 2015;89(9):4712–4719. J Virol. 2015;89(1):713–729. ficiency virus type 1 infection is determined by 97. Dave UP, et al. Murine leukemias with retro- 113. Wang X, et al. Integration of Epstein-Barr virus the viral capsid. J Virol. 2011;85(15):7818–7827. viral insertions at Lmo2 are predictive of the into chromosome 6q15 of Burkitt lymphoma cell 79. De Iaco A, Luban J. Inhibition of HIV-1 infection leukemias induced in SCID-X1 patients fol- line (Raji) induces loss of BACH2 expression. by TNPO3 depletion is determined by capsid and lowing retroviral gene therapy. PLoS Genet. J Virol. 2004;164(3):967–974. detectable after viral cDNA enters the nucleus. 2009;5(5):e1000491. 114. Sasaki S, et al. Cloning and expression of human Retrovirology. 2011;8:98. 98. Akagi K, Suzuki T, Stephens RM, Jenkins NA, B cell-specific transcription factor BACH2 80. Di Nunzio F, et al. Nup153 and Nup98 bind the Copeland NG. RTCGD: retroviral tagged cancer mapped to chromosome 6q15. Oncogene. HIV-1 core and contribute to the early steps of gene database. Nucleic Acids Res. 2004;32(Data- 2000;19(33):3739–3749. HIV-1 replication. Virology. 2013;440(1):8–18. base issue):D523–D527. 115. Lee SM, Vasishtha M, Prywes R. Activation and 81. Hulme AE, Kelley Z, Foley D, Hope TJ. Comple- 99. Kustikova OS, et al. Retroviral vector insertion repression of cellular immediate early genes by mentary assays reveal a low level of CA associ- sites associated with dominant hematopoi- serum response factor cofactors. J Biol Chem. ated with viral complexes in the nuclei of HIV-1- etic clones mark “stemness” pathways. Blood. 2010;285(29):22036–22049. infected cells. J Virol. 2015;89(10):5350–5361. 2007;109(5):1897–1907. 116. Cen B, Selvaraj A, Prywes R. Myocardin/MKL 82. Wong RW, Mamede JI, Hope TJ. Impact of 100. Dave UP, Jenkins NA, Copeland NG. Gene family of SRF coactivators: key regulators of nucleoporin-mediated chromatin localization therapy insertional mutagenesis insights. Science. immediate early and muscle specific gene and nuclear architecture on HIV integration site 2004;303(5656):333. expression. J Cell Biochem. 2004;93(1):74–82. selection. J Virol. 2015;89(19):9702–9705. 101. Du Y, Spence SE, Jenkins NA, Copeland NG. 117. Cen B, et al. Megakaryoblastic leukemia 1, a 83. Mitchell RS, et al. Retroviral DNA integration: Cooperating cancer-gene identification through potent transcriptional coactivator for serum ASLV, HIV, and MLV show distinct target site oncogenic-retrovirus-induced insertional muta- response factor (SRF), is required for serum preferences. PLoS Biol. 2004;2(8):E234. genesis. Blood. 2005;106(7):2498–2505. induction of SRF target genes. Mol Cell Biol. 84. Kvaratskhelia M, Sharma A, Larue RC, Serrao E, 102. Mack KD, et al. HIV insertions within and proximal 2003;23(18):6597–6608. Engelman A. Molecular mechanisms of retrovi- to host cell genes are a common finding in tissues 118. Huang D, et al. C11orf95-MKL2 is the result- ral integration site selection. Nucleic Acids Res. containing high levels of HIV DNA and macro- ing fusion oncogene of t(11;16)(q13;p13) in 2014;42(16):10209–10225. phage-associated p24 antigen expression. J Acquir chondroid lipoma. Genes Chromosomes Cancer. 85. Craigie R, Bushman FD. Host factors in Immune Defic Syndr. 2003;33(3):308–320. 2010;49(9):810–818. retroviral integration and the selection of 103. Han Y, et al. Resting CD4+ T cells from human 119. Flucke U, et al. Presence of C11orf95-MKL2 integration target sites. Microbiol Spectr. immunodeficiency virus type 1 (HIV-1)-infected fusion is a consistent finding in chondroid 2014;2(6):MDNA3-0026-2014. individuals carry integrated HIV-1 genomes lipomas: a study of eight cases. Histopathology. 86. Engelman A, Cherepanov P. The lentiviral inte- within actively transcribed host genes. J Virol. 2013;62(6):925–930. grase binding protein LEDGF/p75 and HIV-1 2004;78(12):6122–6133. 120. Ho Y-C. Replication-competent non-induced replication. PLoS Pathog. 2008;4(3):e1000046. 104. Ikeda T, Shibata J, Yoshimura K, Koito A, Mat- proviruses in the latent reservoir increase barrier 87. Ferris AL, et al. Lens epithelium-derived sushita S. Recurrent HIV-1 integration at the to HIV-1 cure [dissertation]. Baltimore MD: Johns growth factor fusion proteins redirect HIV-1 BACH2 locus in resting CD4+ T cell populations Hopkins University. 2013. https:Jscholarship. DNA integration. Proc Natl Acad Sci U S A. during effective highly active antiretroviral library.jhu.edu/handle/1774.2/37077. 2010;107(7):3135–3140. therapy. J Infect Dis. 2007;195(5):716–725. 121. Brady T, Agosto LM, Malani N, Berry CC, 88. Marshall HM, et al. Role of PSIP1/LEDGF/p75 105. Maldarelli F. HIV-infected cells are frequently O’Doherty U, Bushman F. HIV integration site in lentiviral infectivity and integration targeting. clonally expanded after prolonged antiretroviral distributions in resting and activated CD4+ T cells PLoS One. 2007;2(12):e1340. therapy. J Virus Erad. 2015;1:237–244. infected in culture. AIDS. 2009;23(12):1461–1471. 89. Serrao E, Ballandras-Colas A, Cherepanov P, 106. Uittenboogaard LM, et al. BACH2: a marker of 122. Brady T, et al. Quantitation of HIV DNA inte- Maertens GN, Engelman AN. Key determinants DNA damage and ageing. DNA Repair (Amst). gration: effects of differential integration site of target DNA recognition by retroviral inta- 2013;12(11):982–992. distributions on Alu-PCR assays. J Virol Methods. somes. Retrovirology. 2015;12:39. 107. Kuwahara M, et al. The Menin-Bach2 axis is 2013;189(1):53–57.

446 jci.org Volume 126 Number 2 February 2016 The Journal of Clinical Investigation Review Series: HIV

123. Bushman FD. Integration SIte Pipeline and Data- 2004;101(6):1668–1672. 130. Kent SJ, et al. The search for an HIV cure: base. UPenn Web site. http://www.bushmanlab. 127. Subramanian RP, Wildschutte JH, Russo C, tackling latent infection. Lancet Infect Dis. org. Accessed December 14, 2015. Coffin JM. Identification, characterization, and 2013;13(7):614–621. 124. Shao W, et al. NCI Retrovirus Integration Data- comparative genomic distribution of the HERV-K 131. Rajasuriar R, Wright E, Lewin SR. Impact of base (RID). DRP-RID Web site. https://fr-s-bsg- (HML-2) group of human endogenous retrovi- antiretroviral therapy (ART) timing on chronic hgi-0.ncifcrf.gov. Accessed December 14, 2015. ruses. Retrovirology. 2011;8:90. immune activation/inflammation and end-organ 125. Simonetti FR, et al. Clonally expanded CD4+ 128. Badley AD, Sainski A, Wightman F, Lewin SR. damage. Curr Opin HIV AIDS. 2015;10(1):35–42. T-cells can produce infectious HIV-1 in vivo. Proc Altering cell death pathways as an approach to 132. Archin NM, Margolis DM. Emerging strategies to Natl Acad Sci U S A. In press. cure HIV infection. Cell Death Dis. 2013;4:e718. deplete the HIV reservoir. Curr Opin Infect Dis. 126. Hughes JF, Coffin JM. Human endogenous 129. Evans VA, Khoury G, Saleh S, Cameron PU, 2014;27(1):29–35. retrovirus K solo-LTR formation and inser- Lewin SR. HIV persistence: chemokines and 133. Margolis DM, Hazuda DJ. Combined tional polymorphisms: implications for human their signalling pathways. Cytokine Growth Factor approaches for HIV cure. Curr Opin HIV AIDS. and viral evolution. Proc Natl Acad Sci U S A. Rev. 2012;23(4–5):151–157. 2013;8(3):230–235.

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