The Role of Macrophages in HIV-1 Persistence and Pathogenesis

1Antiretroviral therapy (ART) has dramatically improved the clinical outcome of Human Immunodeficiency Virus type 1 (HIV-1) infection. However, eradication of HIV-1 is not achieved due to persistence of a viral reservoir that harbors latent provirus that is reactivated upon discontinuation of ART. This latent viral reservoir is a major hurdle in curative treatment of HIV-1 infection and therefore new therapeutic approaches that aim to eliminate or reduce the viral reservoir are explored.

2CD4+ T cells and cells from the monocyte/macrophage lineage are considered as the most important target cells for HIV-1, and play an important role in viral persistence and the formation of the viral reservoir. While the major viral reservoir in treated HIV-1 infection is comprised of CD4+ T cells, the distribution and characteristics of the monocyte/macrophage reservoir remain largely unknown.

3Monocytes and macrophages are part of the innate immune system and respond to different signals which will direct macrophage differentiation, polarization and function. Monocytes/macrophages sense and clear pathogens, serve as antigen presenting cells priming the adaptive immune response, are involved in both pro- and anti-inflammatory responses, and tissue repair. Macrophages are found in all lymphoid as well as non-lymphoid tissues and their origin was originally described to be dependent on circulating monocytes, which would replenish the tissue-resident macrophage pool if needed. Recent studies in mice showed that the majority of tissue-resident macrophages are embryonically derived and have been implicated to be self-renewing, with the exception of the mucosal/border tissues like the intestine, the dermis and the heart, where bone marrow-derived circulating monocytes constantly replenish the aging tissue-resident macrophage pool. The proportion between the pool of tissue-resident macrophages and the pool of monocytes-derived macrophages vary during a state of homeostasis or inflammation.

4Upon differentiation of monocytes into macrophages, these cells become susceptible to HIV-1 infection and due to their ability to migrate into tissues, they contribute to the spread of viral infection to nearly every tissue of the body including gut, semen, lung, gut-associated lymphoid tissue, brain, liver, urethra, and lymph nodes. Furthermore, macrophages are considered long lived, resistant to virally induced cytopathic effects, and can reside in anatomical sanctuaries with restricted penetration of ART, which will support viral persistent even during ART.

5In this review we will discuss the role of monocytes/macrophages in HIV-1 pathogenesis, HIV-1 persistence and viral reservoir.

6Human Immunodeficiency Virus type 1 enters the body mostly through mucosal surfaces of the genital or gastrointestinal tract. The underlying lamina propria is populated with high populations of lymphocytes and macrophages, which express CD4 in combination with the coreceptor C-C chemokine receptor type 5 (CCR5) and serve as targets for HIV-1 infection. Although the CD4+ T cells in the lamina propria are considered the major target for HIV-1, infection of macrophages located in the lamina propria of the intestinal, penile urethral and vaginal mucosa has been described. This confirms a role for macrophages during the early phases of infection, however, it remains under debate whether these mucosal macrophages support HIV-1 replication and spread the infection. Indeed, HIV-1 RNA has been demonstrated to be present in macrophages located in the vagina, but not in the gut, indicating that local environmental signals required for efficient viral replication in macrophages can be lacking in local mucosal tissues under homeostatic conditions.

7Human Immunodeficiency Virus type 1 infection is characterized by high immune activation and inflammation, caused by high levels of HIV-1 replication, bacterial translocation, coinfection with other viruses (e.g., CMV, HCV, HBV) and, immune dysregulation. ART strongly reduces HIV-1 associated inflammation and immune activation, however, residual immune activation and inflammation is still observed even in effectively treated individuals. During HIV-1 infection, increased activation and differentiation of peripheral blood monocytes is also observed, which may affect their susceptibility to HIV-1 infection and their ability to migrate into the tissues, thus contributing to HIV-1 pathogenesis.

8In the peripheral blood, three monocyte subpopulations can be distinguished based on the expression of the LPS receptor CD14 and the FcγIII receptor CD16. The majority of the peripheral blood monocytes (>85%) are classical monocytes that express high levels of CD14 (CD14++CD16-), whereas CD16 is co-expressed on the intermediate (CD14++CD16+) and non-classical monocytes (CD14+CD16+). Murine studies and more recently a humanized mouse study showed that monocyte precursors differentiate first into classical monocytes in bone marrow for a maturation phase of ∼ 38 h. This classical monocyte population is retained in the bone marrow and can respond to acute systemic inflammation. Most classical monocytes remain in the circulation for approximately 1–3 day before migration into the tissues. However, a small proportion matures further into intermediate monocytes within the circulation and most of these cells finally mature into non-classical monocytes before leaving the circulation. In HIV-1 infected individuals, an expansion of CD16 expressing monocytes or intermediate monocytes (CD14+CD16+) is evident during all phases of infection, especially in viremic individuals, which is normalized after initiation of ART in at least part of the patients. Moreover, monocytes express increased levels of activation markers (e.g., CD163, HLA-DR, CD69), T-cell costimulatory molecules (e.g., CD40 and CD86), adhesion molecules (e.g., CD11b, CD11c, CD91, CX3CR1), and chemokine receptors (e.g., C-C chemokine receptor type 2 (CCR2), CCR5), which is also observed during ART.

9In the peripheral blood, a small population (<0.1%) of the monocytes harbor replication competent HIV-1 provirus. While classical monocytes are relatively resistant to HIV-1 infection most likely due to low CCR5 expression, CD16+ or intermediate monocytes express higher levels of CCR5 and can be infected by HIV-1. Indeed, CD16+ or intermediate monocytes have been demonstrated to harbor proviral DNA in untreated as well as treated HIV-1 infection.

10Macrophages can be found in all lymphoid as well as non-lymphoid tissues and could therefore serve as a tissue reservoir for HIV-1 (Figure 1). In the lamina propria of mucosal tissues like the gastro intestinal tract, penile urethra and vagina, HIV-1 infection of local macrophages has been demonstrated in tissues from healthy individuals in vitro, and in vivo in HIV-1 infected untreated and ART treated individuals. Although, HIV-1 DNA has been readily detected in these mucosal tissues, viral RNA has been demonstrated only in vaginal and urethral macrophages. Moreover, replication competent HIV-1 could be isolated from urethral macrophages. In contrast, HIV-1 proviral integration could not be detected in macrophages from colon of ART treated HIV-1 infected individuals, and therefore it seems unlikely that these cells contribute to the replication competent HIV-1 reservoir.

11In lymphoid tissues like spleen and lymph node, CD4+ T cells (resting memory cells) are the predominant reservoir, however, latently infected resident macrophages have been detected at low frequencies in lymph nodes. HIV-1 residing in lymphoid tissues does not genetically differ from variants circulating in the blood, which suggest that there is no compartmentalization of HIV-1 in lymphoid tissue. However, genetic diversity of HIV-1 and independent viral evolution in monocytes and CD4+ T cells during the course of infection in treated and untreated patients suggests cellular compartmentalization.

12Tissue resident macrophages like Kupffer cells in the liver have been demonstrated to be susceptible to HIV-1 infection, and persistence of HIV-1 in Kupffer cells has been demonstrated in HIV-1 infected individuals on ART. However, virus particles produced by Kupffer cells could not be propagated in vitro using CEMx174 cells. Phylogenetic analysis of the viral quasi-species showed distinct clustering of viral variants in the liver, evident of compartmentalization of HIV-1 in the liver. Also alveolar macrophages in the lung have been identified as HIV-1 targets, and HIV-1 persistence as determined by the presence of HIV-1 DNA and HIV-1 RNA has been demonstrated in patients, even during effective ART. Analysis of the HIV quasi-species also suggested compartmentalization of macrophage tropic HIV-1 variants in the lung.

13The central nervous system (CNS) has been identified as an important viral reservoir. HIV-1 enters the CNS through trafficking of infected cells across the blood brain barrier (BBB) which occurs already shortly after primary infection. Indeed, HIV-1 associated monocyte activation increases migratory abilities of peripheral monocytes by the upregulation of adhesion molecules and chemokine receptors, in response to for instance the monocyte chemoattractant protein 1 (MCP1). In the CNS, HIV-1 persists predominantly in resident macrophages, like microglial cells and perivascular macrophages. Genetic analysis of the viral quasi-species revealed compartmentalized viral replication in the CNS.

14Although HIV-1 infection of macrophages has been demonstrated in different tissue, their role as a replication-competent reservoir remains under debate (Figure 1). Macrophages are terminally differentiated and their contribution to the viral reservoir is mainly dependent on their lifespan, whereas latently infected resting CD4+ memory T cells which are also long lived cells, contribute to the viral reservoir through homeostatic or antigen specific proliferation. However, recent reports have clearly demonstrated that macrophages can be infected in vivo and contribute to the viral reservoir (Figure 1;;; ). In SHIV infected rhesus macaques, in vivo viral replication was sustained by tissue macrophages upon CD4 T cell depletion. Moreover, HIV-1 persistence in macrophages was confirmed in HIV-1 infected humanized myeloid only mice in which viral rebound was observed in 33% of the animals following treatment interruption.

15Monocytes and macrophages play a crucial role in several HIV-1 related comorbidities including neurological disorders, atherosclerosis and cardiovascular disease (CVD). HIV-1 associated neurocognitive disorders (HAND) describe a broad range of neurocognitive disorders from asymptomatic neurocognitive impairment, mild neurocognitive disorders (MND), and HIV-1 associated dementia (HAD). Although the use of ART has dramatically reduced development of HAD, milder forms of HAND are still highly prevalent amongst effectively treated HIV-1 infected individuals. In HIV-1 infection, activated peripheral blood monocytes have increased migratory ability and can cross the BBB, and therefore it is generally believed that HIV-1 infected peripheral monocytes are the main source of HIV-1 infection in the brain. Increased monocyte activation, expansion of the activated monocyte population as well as the level of HIV-1 DNA positive monocytes (CD14+/CD16+) have been associated with HIV-1 associated neuroinflammation and HAND. The ongoing neuroinflammation caused by monocyte activation and migration, is considered a strong mediator of neuronal damage in HAND. Moreover, persistent HIV-1 replication in resident macrophages and microglial cells of the brain, contributes to the ongoing neuroinflammation and neuronal damage. In particular the viral envelope protein gp120, the HIV-1 accessory protein Vpr and the HIV-1 transcriptional activator Tat have been identified to contribute directly or indirectly to neurotoxicity.

16HIV-1 associated inflammation and immune activation, both risk factors in the onset of atherosclerosis and CVD, likely contribute to the increased risk of CVD in the HIV-1 infected population. In early stages of atherosclerosis development, hyperlipidemia [(cholesterol, triglyceride, and low-density lipoprotein (LDL)] promotes the retention and uptake of LDL in the sub-endothelial space, which results in activation of endothelial cells and smooth muscle cells by the upregulation of adhesion molecules [intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1)] and secretion of chemokines [MCP-1 and macrophage colony-stimulating factor (M-CSF)] in the arterial wall. Cells of the immune system expressing the MCP-1 receptor CCR2, are recruited and can infiltrate the lesion. During HIV-1 infection, circulating activated monocytes express increased levels of CCR2, and have an increased ability to migrate in response to MCP1 into the arterial wall. These infiltrating monocytes further increase arterial inflammation by cytokine production [interleukin 1ß (IL-1ß) and IL-6] and uptake of (modified) lipoproteins, thereby accelerating atherosclerosis and CVD progression. Indeed, it has been shown that aortic inflammation as measured by 18fluorodeoxyglucose uptake (18F-fluorodexoyglucose Positron Emission Tomography – Computed Tomography: 18F-FDG PET-CT) was increased in effectively treated HIV-1 infected patients to a degree resembling that of uninfected individuals with known CVD. Furthermore, 18F-FDG PET-CT activity was associated with serum levels of soluble CD163 (sCD163), a marker of monocyte/macrophage activation, clearly indicating a role of these cells in accelerated onset of atherosclerosis and CVD in HIV-1 infection.

17Human Immunodeficiency Virus type 1 infection in macrophages and their role in the viral reservoir are difficult to study in vivo and therefore in vitro culture models have been developed. In these models, monocytes are isolated from peripheral blood and differentiated in an in vitro culture system toward monocyte derived macrophages. In addition, different stimuli or cytokines can be added to the cultures in order to obtain phenotypically and functionally distinct macrophages. An advantage of this in vitro culture system is that large number of cells are available for analysis and macrophage activation, differentiation, polarization and function can easily be directed. A major disadvantage is that conditions in the culture system are not representative of the local environment in the different tissues, as tissue resident macrophages continuously respond to local signals influencing their function and phenotype resulting in a heterogeneous resident macrophage population. Recently, macrophages isolated from tissues of HIV-1 infected individuals have provided a better insight in the in vivo relevance and the state of the viral reservoir (latent, activated). However, these materials are difficult to obtain and invasive for the patient.

18In vitro, monocytes become highly susceptible to HIV-1 infection upon differentiation into macrophages. Macrophages respond to different stimuli in their environment which will shape their physiology and function. Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) polarize macrophages into an M1 or pro-inflammatory phenotype. M2 or alternatively activated macrophages are generated upon exposure to IL-4 or IL-13 (M2a), immunoglobulin G (IgG) immune complexes and toll-like receptor (TLR) agonists (M2b) or IL-10 or glucocorticoids (M2c). Polarization toward a M2a phenotype results in resistance to HIV-1 infection due to restrictions at the level of reverse transcription, whereas polarization toward M1 or M2c inhibits viral replication at a transcriptional or post-transcriptional level. This data suggests that polarizing signals can induce viral latency in macrophages at both a pre- or post-integration level (Table 1).

19Integration of HIV-1 proviral DNA into the host genome is essential for viral replication. Upon entry into the cell, HIV-1 RNA is reverse transcribed, and subsequently the pre-integration complex (PIC), consisting of viral proteins (integrase, matrix, Vpr, capsid) and double stranded DNA (dsDNA) is assembled. The PIC is transported into the nucleus and where the proviral DNA integrates into the host cell genome. Pre-integration latency results when the viral replication cycle is partially or completely blocked at steps prior to integration (Table 1). Pre-integration latency is frequently observed in CD4+ T cells but was assumed not to be clinically relevant, because in vitro studies demonstrated that the unintegrated proviral DNA only persists for 1 day in these cells. However, in patients treated with ART, including an integrase inhibitor, unintegrated HIV-1 DNA subspecies where shown to persist in CD4+ T cells and can potentially integrate when integrase inhibitor medication is discontinued. Moreover, metabolically active macrophages can contain large quantities of unintegrated viral DNA, which remains stable and biologically active in non-dividing macrophages for up to 2 months. Therefore, unintegrated HIV-1 DNA in macrophages may contribute to the viral reservoir in HIV-1 pathogenesis. Indeed, some studies have reported detection of unintegrated HIV-1 DNA in macrophages from patients, mostly in the brain. In monocytes/macrophages pre-integration latency could be a consequence of poor reverse transcription efficiency due to a reduced deoxynucleotide triphosphates (dNTP) pool or inhibition of nuclear transport of the PIC as a consequence of low adenosine triphosphate (ATP) levels. Several cellular factors have been implicated to play a role in the observed restriction in monocytes/macrophages: apolipoprotein B mRNA editing enzyme catalytic subunit 3G (APOBEC3G) and subunit 3A (APOBEC3A), a cellular deoxycytidine deaminases which induces G to A hypermutation and degradation of the HIV-1 genome; Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) which hydrolyses dNTPs into their precursors (nucleosides and triphosphates), in turn reducing the dNTPs pool and thereby limiting the reverse transcriptase activity; Myxovirus-resistance protein 2 (MX2) which inhibits HIV-1 replication post entry by hindering the nuclear accumulation and integration of proviral DNA into host chromatin; and members of the RNA polymerase II-associated factor 1 (PAF1) family, which are expressed in monocytes/macrophages and repress HIV-1 reverse transcription and proviral DNA integration.

20Post-integration latency, which is established after integration of the HIV-1 proviral DNA into the host chromatin, is in contrast to pre-integration latency very stable and only limited by the lifespan of the infected cell. Mechanisms underlying HIV-1 latency in vivo are incompletely understood, however, it is a multifactorial phenomenon. Post-integration viral latency may be maintained at a transcriptional or a post-transcriptional level inhibiting HIV-1 protein production and virus formation. Several mechanisms acting at transcriptional and post-transcriptional level inducing HIV-1 latency in CD4+ T cells are well described. However, it is unknown whether these mechanism are also effective in cells of the monocyte/macrophage lineage. Nevertheless, several mechanisms inducing HIV-1 post-integration latency have been described in monocytes/macrophages, including chromatin environment, the absence of transcriptional activation, presence of transcriptional repressors and host antiviral processes. However, not all of these mechanisms are specific for post-integration latency in monocytes/macrophages (Table 1).

21The host chromatin is organized into heterochromatin which is densely packed and transcriptionally silent and euchromatin which is loosely packed and transcriptionally active. After nuclear import a transcriptional co-activator, lens epithelium-derived growth factor (LEDGF)/p75, which interacts with HIV-1 integrase, targets the PIC mainly to intronic regions of actively transcribed genes. The majority of silent HIV-1 provirus is integrated into the euchromatin, which seems paradoxical considering the fact that euchromatin is transcriptionally active. Different mechanisms of transcriptional interference (e.g., steric hindrance, promotor occlusion and enhancer trapping) have been suggested as possible explanation for integrated proviral DNA expression suppression. Most of the data on HIV-1 integration and latency has been derived from CD4+ T cells. However, the integration of HIV-1 proviruses has also been studied in primary macrophages. Barr et al., for instance, studied HIV-1 DNA integration sites in primary macrophages by sequencing 754 unique integration sites and found that, similar to CD4+ T cells, HIV-1 also integrates preferentially into the transcriptionally active regions in macrophages. In CD4+ T cells transcription gene silencing is the most favored mechanism for the establishment and maintenance of HIV-1 latency, whether this is also the case in monocytes/macrophages still has to be determined. However, it is well established that chromatin organization and reorganization influences the gene expression and that HIV-1 proviral DNA follows the same rules that apply to host genes. The role of histone H3 lysine 9 trimethylation (H3K9me3) in heterochromatin formation and the transcriptional silencing of integrated HIV-1 has been described. In addition, there are two nucleosomes, nuc-0 and nuc-1, that interact with the HIV-1 promoter irrespective of the HIV-1 integration site in the host genome. HIV-1 gene transcription from proviral DNA is only possible with the displacement of nuc-1. These studies were performed in the monocytic U1 cell line and collectively suggest that chromatin remodeling is an essential mechanism of HIV-1 latency establishment and regulation in monocytes/macrophages, however, these observations have not yet been confirmed in primary monocytes/macrophages.

22The integrated HIV-1 proviral DNA is flanked by the long terminal repeats (LTR). The 5′LTR has binding sites for several transcription factors including specificity protein 1 (Sp1), activator protein 1 (AP1), nuclear factor of activated T-cells (NFAT), nuclear factor-κB (NF-κB), c-myc, chicken ovalbumin upstream promoter (COUP), upstream stimulatory factor (USF), CCAAT box transcription factor/nuclear factor 1 (CTF/NF1), T cell factor 1α (TCF-1α) and the glucocorticoid receptor. These transcription factors act together to regulate HIV-1 proviral DNA expression (Table 1). Sp1, for instance, recruits c-myc to the 5′LTR of proviral DNA, which in turn recruits histone deacetylase 1 (HDAC1). HDAC1 then induces chromatin remodeling, which results in the suppression of HIV-1 gene expression. In addition, HDACs were also shown to be recruited to the proviral promoter by COUP transcription factor interacting protein 2 (CTIP2), Ying Yang 1 (YY1), C-promoter binding factor-1 (CBF-1) and Late SV40 Factor (LSF), thereby promoting the establishment of latency. Similar results were observed in microglial cells, where CTIP2 has been shown to recruit both HDAC1 and HDAC2 to the 5′LTR of HIV-1 proviral DNA. Moreover, CTIP2 also interacts with suppressor of variegation 3–9 homolog 1 (SUV39H1), a methyl transferase for H3K9me3 which subsequently promotes the recruitment of heterochromatin protein 1 (HP1) leading to local heterochromatization and induction of viral latency. The role of CTIP2 was further confirmed by who analyzed the expression of CTIP2 in postmortem brain tissue of HIV-1 infected patients with HIV-1 encephalitis (HIV-1 DNA and RNA positive) and HIV-1 positive patients with latent HIV-1 (HIV-1 DNA positive and HIV-1 RNA negative) and no detectable HIV-1 (HIV-1 DNA negative) in the CNS. Higher amounts of CTIP2 were observed in the HIV-1 patients with latent HIV-1 in the CNS as compared to the other groups. Notably, CTIP2 was detected in microglial cells of patients with latent HIV-1, indicating that CTIP2 may play an important role in the regulation of viral latency in microglial cells. Furthermore, lysine-specific demethylase 1 (LSD1) was found to regulate HIV-1 gene expression in a synergistic way with CTIP2 in microglial cells. LSD1 also assists in the recruitment of CTIP2 and human SET1/WD40-repeat protein 5 (hSET1/WDR5) at the Sp-1 binding sites of the HIV-1 proximal promoter, resulting in increased H3K4 trimethylation (H3K4me3), which in turn represses viral gene expression. This property of LSD1 seems to be highly specific for cells of the monocyte/macrophage lineage.

23Recently, the host factor dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) was shown to control HIV-1 replication by regulating HIV-1 provirus transcription in macrophages and CD4+ T cells. DYRK1A was shown to regulate HIV-1 transcription via phosphorylation of the NFAT, thereby promoting NFAT translocation from the nucleus to the cytoplasm resulting in decreased viral transcription and latency. Chemical inhibition of DYRK1A resulted in an increased NFAT binding to the viral LTR and reactivating latent provirus.

24Furthermore, it has been reported the 5′LTR of the HIV-1 provirus contains multiple T cell factor 4 (TCF-4) binding sites. TCF-4 interacts with β-catenin and the scaffold matrix attachment region-binding protein 1 (SMAR1) at the 5′LTR and this complex has been shown to repress HIV-1 proviral gene expression in different cell types including lymphocytes and astrocytes. β-catenin/TCF4 signaling has also been demonstrated to regulate HIV-1 replication in cells of the monocyte/macrophage lineage, which makes it plausible that these proteins also play a role in viral latency in these cells.

25Apart from cellular factors, HIV-1 latency is also influenced by viral factors. HIV-1 replication requires the viral trans-activator protein Tat for efficient transcription and virus production. In the absence of Tat, only low level of HIV-1 transcription of mostly short abortive transcripts is observed. These short RNA transcripts contain a trans-activation response (TAR) element which is the binding site for the Tat protein. The positive transcription elongation factor (P-TEFb) is a critical cellular cofactor of Tat, that favors the generation of complete transcripts from HIV-1 proviral DNA. P-TEFb is composed of a catalytic subunit, cyclin-dependent kinase 9 (CDK9), and a regulatory subunit, cyclin T1 (CycT1). Tat binds to the TAR region of the RNA transcript and directs P-TEFb to the RNA polymerase II resulting in transcription of full-length HIV-1 RNA. The CDK2 can phosphorylate Ser90 on CDK9, thereby assisting in HIV-1 transcription. Monocytes express very low levels of CycT1, which transiently increases during differentiation into macrophages. In contrast CDK9 expression levels remain constant in monocytes and macrophages. The low levels of CycT1 in monocytes result in low functional levels of P-TEFb, resulting in low HIV-1 transcription and thus viral latency in monocytes.

26Human Immunodeficiency Virus type 1 latency can also be regulated at a post-transcriptional level (Table 1). miRNAs are small single stranded non-coding RNAs that can regulate host gene expression at a post-transcriptional level. miRNAs have also been reported to influence HIV-1 gene expression. For instance, the miRNA cluster miR-17/92 is actively inhibited by HIV-1 to support efficient replication in different cell types including lymphocytes, monocytes and macrophages. Furthermore, miR-28; miR-150; miR-223; and miR-382 have been demonstrated to target HIV-1 and play a role in HIV-1 susceptibility of monocytes and macrophages. Inhibition of these miRNAs in monocytes results in increased HIV-1 replication, while increased expression of these miRNAs in macrophages decrease of HIV-1 replication. These data indicate that miRNAs can play an important role in viral latency in macrophages.

27RNA binding host proteins like GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) and the matrix-associated RNA binding protein Matrin 3 (MATR3), have been shown to play a role in the post-transcriptional regulation of HIV-1 latency. G3BP1 is a single-strand-specific endonuclease that binds mRNA transcripts at the 3′UTR inside stress granules to control mRNA translation during cellular stress. G3BP1 was shown to interact with HIV-1 mRNA transcripts thus preventing viral protein translation and restricting HIV-1 replication in T cells and macrophages. Resting T cells and IFNγ and TNFα polarized macrophages, express high levels of G3BP1 indicating a role of this host factor in HIV-1 latency. MATR3 is a nuclear matrix protein that has the ability to bind DNA and RNA, and is involved in the regulation of gene expression and controls mRNA export from the nucleus. MATR3 also plays an essential role in Rev-mediated export of HIV-1 mRNA transcript from the nucleus, through the interaction with the polypyrimidine tract binding protein and associated splicing factor (PSF) and the viral protein Rev. Depletion of MATR3 was demonstrated to inhibit nuclear export of Rev responsive element (RRE)-containing viral RNAs resulting in decreased HIV-1 replication. Resting T cells only express low levels of MATR3 which increases upon activation, indicating a role of this protein in the establishment of latency in resting cells. Since MATR3 is also expressed in monocytes, it can be expected that it is also involved in the posttranscriptional regulation of HIV-1 in these cells.

28Even though ART has significantly increased life expectancy of HIV-1 infected individuals, complete eradication of the virus is not achievable without targeting the HIV-1 latent reservoirs. Currently, several therapeutic strategies are explored and early findings suggest that different strategies may be required to eliminate the different cellular viral reservoirs: memory CD4+ T cells which importantly maintain the viral reservoir through homeostatic- or antigen specific proliferation, and long lived cells like macrophages residing in the tissue.

29The latent viral reservoir is not recognized by the immune system and therefore these cells are not eliminated. The “shock and kill” strategy aims to induce viral protein production by reactivation of the latent HIV-1 provirus to prompt elimination of the infected cells through recognition by the immune system or cell death as a result of viral replication. The “shock and kill” strategy has been investigated primarily using CD4+ T cells and T cell lines. Histone deacetylases (HDACs), for instance, are important for suppression of HIV-1 proviral DNA expression, and several HDAC inhibitors including SAHA, vorinostat, oxamflatin, metacept-1, and metacept-3 have been studied for their ability to reactivate and eliminate latently infected cells. Although HDAC inhibitors have shown activity to reactivate HIV-1 from latent reservoirs in patients on ART, no decline in HIV-1 proviral DNA in CD4+ T cells was observed. In contrast, in vitro studies in both the monocytic U1 cell line and monocyte-derived macrophages show that HDAC inhibitors not only reactivate and decrease HIV-1 release, but also degrades the viral particles through the canonical autophagy pathway. Moreover, several other compounds targeting different pathways have also been shown to effectively reactivate latent HIV-1, these include protein kinase C agonists (in vitro in both monocytic and T cell lines) (e.g., prostratin and ingenols), histone methyltransferases inhibitors (in vitro in T cell lines and primary monocytes/macrophages and CD4+ T cells from HIV-1 infected ART treated patients), anti-miRNA inhibitors, NF-κB activators, and cytokine therapy in vitro and in vivo in CD4+ T cells as well as in cells of the monocyte/macrophage lineage. Although the efficacy of the majority of these latency reversing agents (LRAs) has been demonstrated, a major hurdle in this treatment approach is that HIV-1 reactivation only occurs in a small subset of the latently infected cells, which might be overcome at least in part by the use of combinations of these LRAs. Furthermore, targeting of cellular factors and pathways identified to be involved in the regulation of HIV-1 latency at a transcription or post-transcription level, such as DYRK1A, β-catenin/TCF4 and Tat/pTEFb (;;;;; Table 1) may improve the efficacy of latency reversal. Moreover, LRAs alone are mostly not sufficient to induce cell death and therefore an effective immune response is essential for the elimination of the reactivated cells and this is lacking in the majority of HIV-1 infected patients. Furthermore, HIV-1 infected macrophages can reside in immune privileged compartments like the CNS and can therefore not be reached by cytotoxic T cells.

30Recently more research is done into the “Prime, Shock, and Kill” method, where latent reservoirs are made sensitive to apoptosis (Prime), followed by reactivation of HIV-1 (Shock) and subsequently leading to cell death of the infected cell (Kill). HIV-1 infection of CD4+ T cells and macrophages have been shown to induce an anti-apoptotic gene profile and resistance to apoptosis. Several viral proteins have been associated with the resistance to apoptosis: Vpr upregulates expression of anti-apoptotic proteins like B-cell lymphoma 2 (Bcl-2) in T cells only, thereby promoting cell survival. In monocytes/macrophages vpr facilitates HIV-1 replication. Tat, however, does upregulate Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) in both monocytes/macrophages and CD4+ T cells; Nef upregulates the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-Xl) in macrophages, while nef inactivates the Bcl-2-associated death promoter protein (BAD), a pro-apoptotic Bcl-2 family member, in a phosphoinositide 3-kinase (PI3K)– and p21-activated kinase (PAK)-dependent manner in T cells; Furthermore, gp120 has been demonstrated to increase in expression of M-CSF in HIV-1 infected macrophages resulting in the upregulation of anti-apoptotic proteins [e.g., induced myeloid leukemia cell differentiation protein 1 (Mcl-1) and Bcl-2-related protein A1 (Bfl-1)]. Therefore, targeting of the HIV-1 induced anti-apoptotic pathways during proviral reactivation strategies may promote cell death of the latently infected cells. Multiple agents targeting the anti-apoptotic pathways including PI3K/Protein kinase B (AKT) inhibitors, Bcl2 inhibitors and second mitochondria-derived activator of caspase (SMAC) mimetics/XIAP inhibitors, are explored for their ability to clear latently infected cells. SMAC mimetics/XIAP inhibitors, for instance, function by sensitizing macrophages to Vpr-induced cell death. In addition, SMAC mimetics/XIAP inhibitors can trigger transcription of latent proviruses via the non-canonical NF-κB pathway. Furthermore, in vitro studies showed that combinations of HDAC inhibitors and SMAC/XIAP mimetics have synergistic activities in T cells. However, all these compounds do not specifically target the viral reservoir, and targeting of non-HIV-1 infected bystander cells will likely occur especially in ART-treated individuals with increased levels of immune activation, inflammation, and high prevalence of coinfections.

31Indeed, an apoptosis resistant gene expression signature was observed in monocytes from HIV-1 infected individuals. The circulating monocyte population is largely uninfected, which indicates that the anti-apoptotic expression profile may not be due to direct effects of viral infection. Moreover, in vitro infection of monocytes/macrophages demonstrated that HIV-1 infection, as well as exposure to HIV-1 or immune modulators increased apoptosis resistance.

32Despite effective treatment, HIV-1 infected patients are not cured because the virus is not eradicated and latently infected cells persist. The latent viral reservoir mainly resides in resting CD4+ T cells, however, latent HIV-1 proviruses have also been detected in cells of the monocyte/macrophage lineage. HIV-1 infected macrophages can be found in virtually every tissue including the CNS. Viral latency in cells of the monocyte/macrophage lineage can be regulated by diverse molecular mechanisms either at a transcriptional or post-transcriptional level and this is most likely dependent on the activation and differentiation state of these cells. Current eradication strategies rely on reactivation of the latent virus and subsequent kill of the infected cell. The majority of LRAs under study, have been shown to reactivate only small proportions of the latent HIV-1 reservoir, and therefore there is an urgent need for the development of new compounds that target different mechanisms of viral latency both in CD4+ T cells and monocytes/macrophages. Another hurdle is the lack of kill of latently infected cells upon reactivation, which is mainly due to an inefficient antiviral immune response and HIV-1 induced resistance to apoptosis. Apoptosis inducing compounds have been developed for cancer therapy, and their efficacy to specifically induce apoptosis in HIV-1 infected cells is currently under study.

33Cure research has mainly focused on CD4+ T cells, because these cells contribute to maintenance pf the viral reservoir through their long lifespan as well as their ability to proliferate under homeostatic conditions or in response to antigens. In contrast, macrophages are terminally differentiated and their contribution to the viral reservoir is mainly dependent on their lifespan and distribution to immune privileged tissues like the CNS. Although, these terminally differentiated latently infected macrophages cannot proliferate, they could be able to spread HIV-1 infection to CD4+ T cells upon reactivation of the virus by LRAs. Therefore, development of eradication strategies combining both novel LRAs and apoptosis inducing agents, targeting either specific cell types or ideally target both latently infected CD4+ T cells and monocytes/macrophages are needed.

34ZK and NK wrote and edited the review.

35The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.