Identification of novel host-oriented targets for Human Immunodeficiency Virus type 1 using Random Homozygous Gene Perturbation
© Mao et al; licensee BioMed Central Ltd. 2009
Received: 10 July 2009
Accepted: 29 September 2009
Published: 29 September 2009
Human Immunodeficiency Virus (HIV) is a global threat to public health. Current therapies that directly target the virus often are rendered ineffective due to the emergence of drug-resistant viral variants. An emerging concept to combat drug resistance is the idea of targeting host mechanisms that are essential for the propagation of the virus, but have a minimal cellular effect.
Herein, using Random Homozygous Gene Perturbation (RHGP), we have identified cellular targets that allow human MT4 cells to survive otherwise lethal infection by a wild type HIV-1NL4-3. These gene targets were validated by the reversibility of the RHGP technology, which confirmed that the RHGP itself was responsible for the resistance to HIV-1 infection. We further confirmed by siRNA knockdowns that the RHGP-identified cellular pathways are responsible for resistance to infection by either CXCR4 or CCR5 tropic HIV-1 variants. We also demonstrated that cell clones with these gene targets disrupted by RHGP were not permissible to the replication of a drug resistant HIV-1 mutant.
These studies demonstrate the power of RHGP to identify novel host targets that are essential for the viral life cycle but which can be safely perturbed without overt cytotoxicity. These findings suggest opportunities for the future development of host-oriented therapeutics with the broad spectrum potential for safe and effective inhibition of HIV infection.
Therapy-resistant HIV-1 strains are relentlessly emerging as a result of the error-prone HIV viral reverse transcriptase, robust viral replication and incomplete patient compliance. In some regions, viruses that are resistant to drug cocktail therapy or HAART (Highly Active Antiretroviral Therapy) were isolated from nearly 20% of AIDS patients evaluated [1, 2]. Such findings increase the urgency to identify new paradigms for the treatment of HIV/AIDS, especially mechanisms of action that are relatively insensitive to the development of resistance.
It is well established that interplay between the viruses and host cells determines the outcome of viral pathogenesis, ranging from the elimination of viruses to latent or lethal infections. HIV-1 is known to interact with host cellular proteins to aid their replication and evade immune attack. One example involves individuals who carry a defective cell surface receptor (CCR5) and have been shown to be resistant to HIV-1 infection [3, 4]. Similar interactions have been reported to encompass nearly every step of HIV-1 life cycle: from viral entry  to viral budding and release . Such findings suggest that increased understanding of the interaction of HIV-1 with host protein could improve therapeutic and prevention strategies to combat HIV/AIDS.
In light of the understood importance of host factors in HIV-1 infection, increasing investigation has begun to consider host targets for antiviral therapy. Specifically, host targets that are essential for HIV-1 replication, but not for the host cell itself, could provide a new modality of treatment. It is further postulated that certain host targets might not place direct selective pressure on the pathogen and thus minimize the acquisition of drug resistance. Host-directed therapeutics has begun to be successfully deployed against HIV/AIDS, including treatments that target the CD4 viral receptor and associated co-receptors [7, 8]. Indeed, some of the newest approved and most promising experimental therapeutic options include small molecules or biologics that target these host proteins.
Not all host molecules are suitable as therapeutic targets as many serve essential functions for the growth, function or survival of host cells. However, it is increasingly understood that viruses often circumvent the expression or function of some host proteins (in a process known as "hijacking") and this may provide an opportunity to target host molecules that are inappropriately expressed or functionally altered in HIV-infected cells. To identify such targets, our laboratory has employed a novel technology, Random Homozygous Gene Perturbation (RHGP), to select for targets that are essential for HIV infection but which are not necessary for the growth, survival or function of non-infected cells. RHGP was designed to allow the investigator to up- or down-regulate any gene in a eukaryotic cell, independent of any prior knowledge or annotation of that gene . In this manner, RHGP provides an un-biased approach to identify any target, whether up- or down-regulated, which is responsible for a desired phenotype. As one example, our laboratory has successfully used RHGP to identify and validate target genes that allow host cells to survive an otherwise lethal infection with Influenza A virus. Of 110 targets identified by this genome wide screen technology, most (106 of 110) had not been described previously or linked with influenza infection. In addition, we ascribed novel functions to previously unknown genes and orfs (open reading frames).
Herein, we apply RHGP and identify a set of host-oriented targets that allow host cells to resist lethal HIV infection. These novel targets include both known genes and non-annotated ESTs (Expression Sequence Tags), whose functions have not been assigned. We validated these genes using unique properties of the RHGP technology as well as independent genetic targeting approaches such as siRNA. This investigation provides increased understanding of the interplay between host targets and HIV and could provide potential therapeutic targets to combat HIV/AIDS.
Cell lines and Viruses
The following cell lines, viruses and proviral molecular clones were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: MT4 cells from Dr. Douglas Richman, PM1 cells from Dr. Marvin Reitz , TZM-bl cells from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. , pNL4-3 from Dr. Malcolm Martin, HIV-1ME1 from Dr. Phalguni Gupta  and Protease-resistant HIV-1 (L10R/M46I/L63P/V82T/I84V) from Dr. Emilio Emini. MT4 and PM1 cells were grown in RPMI 1640 medium containing 10% heat-inactivated FBS (HyClone, Long, UT) supplemented with 2 mM glutamine (Invitrogen), 2-mercaptoethanol (50 μM), 100 μg/ml streptomycin (Invitrogen). TZM-bl cells were cultured in DMEM containing 10% FBS and 100 μg/ml streptomycin. HIV-1NL4-3 was made from HEK293 after transfection with the proviral DNA followed by amplification in MT4 cells.
HIV-1 Infection and Measurements of Viral Production
MT4 or PM1 cells were infected with HIV-1 at a multiplicity of infection (MOI) of 0.001 by low speed centrifugation (1,200 g) for 1 hr. The use of a relatively low MOI helped us to identify host factors whose anti-viral effects may not be robust or directly acting on virus replication and which will be more likely discovered after multiple cycles of viral replication. Supernatants collected post infection were then transferred to the TZM-bl indicator cell line for determination of infectious viral particles. Relative Luminescence Unit (RLU) was obtained on TZM-bl cells after they were treated with Bright-Glo Luciferase Assay System (Promega) 3 days post infection (dpi). Amounts of p24 in the collected supernatants were measured using HIV-1 p24 ELISA kit (Xpressbio, Thurmont, MD, USA) following the manufacturer's instructions.
Description of RHGP technology
RHGP utilizes a unique genetic element, known as a gene search vector (GSV), which is based on a retrovirus or lentivirus backbone. The GSV was designed to interrogate the entire genome and identify targets without any prior knowledge and that allow host cells to resist or survive lethal HIV-1 infection.
Construction of the MT-4 R1 Cell Lines
RheoSwitch® Mammalian Inducible Expression System was purchased from New England Biolabs (NEB). Plasmid pNEB-R1 encoding the transactivator R1 was first linearized using the restriction enzyme ScaI (NEB). MT4 cells were then transfected with the linearized pNEB-R1 by electroporation using Eppendorf Multiporator (Eppendorf, AG 22331, Hamburg, Germany) under conditions of 360 v (voltage) and 100 μs (time constant). The transfected MT4 cells were selected using G-418 (400 μg/mL) and G-418 resistant cells were cloned by serial limited dilutions. After expansion, clones were examined at least twice for luminescence (relative luminescence units (RLUs)) after transfection with an R1-responsive luciferase reporter gene (pGluc, NEB) using the Gaussia Luciferase Assay Kit (NEB). We determined the RSL1 induction folds of luminescence from these cell clones as: RLUs obtained from samples in the presence of the inducer divided by RLUs from samples without the inducer treatment. The induction fold from these clones ranged from 2-60 folds. A stable clone (#2-14) with the highest induction was chosen to create RHGP libraries.
Construction of the RHGP Gene Search Vector, pRHGP12-RSN
Production of Lentivirus Carrying GSV and Construction of RHGP Library
RHGP lentiviruses were produced using ViroPower Expression System (Invitrogen). HEK293FT cells were plated in 10 cm plates at 106 cells per plate. After 24 h incubation, the cells were transfected with 3 μg RHGP12-RSN and 9 μg ViroPower Packaging Mix using Lipofectimine 2000 (Invitrogen). The medium was changed after 5 h incubation. After 48 h, viruses in the culture medium were filtrated through a 0.45 μm filter and titrated according to the manufacturer's instruction.
To construct the RHGP library, MT4-R1 cells were transduced with RHGP viruses in the presence of polybrene (6 μg/mL) by low speed centrifugation (1,200 g) for 1 h. To minimize the potential for multiple insertions within a single cell, a low MOI (0.1) was employed during the library creation to minimize the likelihood that cells might be transduced by more than one different GSV. GSV integrated cells were selected using GBL medium (complete RPMI 1640 medium containing G418 (400 ug/ml), Blasticidin (4 ug/ml) and RSL1 ligand (0.5 uM)).
Selection of RHGP Cell Clones That Survived from HIV-1 Challenge and Confirmation via Reversibility of Viral Resistance
After challenge with HIV-1NL4-3, the MT4-R1 RHGP library was cultured in the same GBL medium described above. The individual surviving clones were established by serial limited dilutions and continuously expanded in GBL medium. Cell clones were further challenged with HIV-1NL4-3 to confirm their resistance.
To verify reversibility of RHGP induced events, the viral resistant MT4 cell clones were cultured in the GBL medium or GBL medium without RSL1 separately for at least 3 days before HIV-1 re-challenge. Viral production (infectivity and p24) in supernatants were examined as described above.
Identification of Candidate Genes from Virus Resistant Clones
The RHGP gene search vector was designed to efficiently discover target genes and determine the orientation (sense or antisense) of an integration event. The gene search vector contains an Ori-CAT reporter gene (Figure 2), which can be rescued by restriction enzyme-based genomic DNA cloning as described before. Briefly, cellular genomic DNA from each cell clone was extracted from 106 cells. Purified genomic DNA was then digested with BamHI or XbaI and self-ligated overnight using T4 ligase (Invitrogen). The ligated DNA was electroporated into DH10B ElectroMax competent cells (Invitrogen). After overnight growth, multiple colonies were isolated for plasmid DNA preparation and restriction enzyme digestion. The plasmid DNA was further used to identify the target genes by DNA sequencing and genome mapping. The resulting genomic DNA sequences flanking the RHGP vector insertion sites were subjected to genome mapping against the human genome using the UCSC Genome Browser http://www.genome.ucsc.edu/cgi-bin/hgBlat.
Validation of Identified Host Target Genes with siRNA Knockdown Assay
Human duplex siRNA (siGNOME SMARTpool) for RHGP identified genes were prepared as recommended by the manufacturer (Dharmacon). The siRNA Rab6A and HIV-1 Tat were employed as positive controls . Non-targeting siRNA (siCONTROL1) was used as a negative control. MT4 or PM1 cells were cultured in fresh complete RPMI 1640 medium overnight. The log-phase growing cells were transfected with 1.2 uM of siRNA by electroporation, according to the manufacturer's instruction (Eppendorf). The voltage and time constant for elctroporation were 360 v, 100 μs and 200 v, 200 μs for MT4 and PM1 cells, respectively. The cells were infected with HIV-1 variants 24 h post transfection. Culture media were refreshed everyday and the cell viabilities were examined daily by trypan blue dye exclusion assay. Viral production (infectivity and p24) in supernatants were examined as described above.
Western Blot Analysis
The cell pellets were washed with PBS, resuspended with lysis buffer (25 mM Tris-HCl, pH 7.6, 150 mM Nacl. 1% NP-40. 1% Sodium Deoxycholate, 0.1% SDS), and disrupted with pulse sonication. After centrifuge at ≅14000 × g for 15 minutes, the supernatant was dialyzed against PBS, and concentrated. Equal amounts of protein samples (250 μg) were loaded onto 4%-12% stacking SDS-PAGE with Dithiothreitol (DTT, 200 mM) before electrophoresis analysis. The sieved proteins were transferred on to PVDF membranes, blocked with PBS containing 5% dry non-fat milk, and blotted with 1:50 dilution of anti-Robo1 (A301-265A, Bethyl Laboratories) as primary antibody and 1: 2000 as secondary antibody HRP in PBS containing 5% dry non-fat milk, 0.1% Tween-20 and the ECL Chemiluminescence (Amersham/Pharmacia Biotech) was used to detect signals. The loading amounts controls were probed using anti-HSP (heat shock protein 90) (abcam, Cambridge, MA) and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA).
Construction of the RHGP library of CD4+ T cell line MT4
To identify novel targets that render T cells resistant to HIV infection, we utilized the human MT4 cell model, which provided an HIV-1 permissive, CD4 positive T lymphocyte cell line. The use of a natural target line for a wild-type strain of HIV-1 provided a model to identify targets that are physiologically relevant to the HIV life cycle. In addition, MT4 cells were selected for these studies, in part, after confirming that this model was highly sensitive to HIV-1 infection. Specifically, challenge of MT4 cells with HIVNL4-3, at a relatively low initial MOI (0.001), was sufficient to eliminate MT4 cells in the absence of RHGP-mediated gene perturbation.
We then utilized RHGP to interrogate the genome of human T lymphocytes to identify targets that allow these cells to survive an otherwise lethal infection with HIV-1. To accomplish this, cultures of MT4-R1 cells were transduced with the GSV (pRHGP12; Figure 2), which contains an expression cassette consisting of a constitutive promoter driving a Blasticidin resistance gene. Blasticidin selection allowed us to establish an "RHGP library" of MT4-R1 cells with different genetic perturbations rendered by random GSV integrations (Figure 3, step 2). To maintain stable R1 expression and GSV integration, the MT4-R1 RHGP library was continuously incubated with G418 and Blasticidin. RSL1 was also included in the culture medium to ensure that the activated GSV promoter was able to generate anticipated RHGP effects by producing transcripts. To control for the quality of the library, we confirmed that downstream gene expression from the GSV was induced only upon incubation with RSL1 but not when RSL1 was absent (data not shown). Statistical analyses of gene expression and genome size were implemented to ensure that a sufficient number of GSV integration events would be analyzed to thoroughly evaluate the human genome, both for gain or loss of target expression. Specifically, we calculated that a library of MT4-R1 cells with 105 GSV integration events would ensure coverage of the human genome.
Isolation of cell clones resistant to HIV-1 infection
The cell library containing the different RHGP perturbation MT4 cells was then challenged with HIVNL4-3, infected at an initial MOI of 0.001 (Figure 3, step 3). Analysis of Trypan blue exclusion examination indicated that non-transduced MT4-R1 cells were greater than 99% depleted following HIV-1NL4-3 challenge. As indicated above, we also confirmed that the inclusion of RSL1 in non-transduced cells did not alter cell sensitivity to HIV-1 infection. As an additional control, parallel cultures of mock-transduced cells were treated identically and no survivors were observed after 5 days. These controls confirmed that surviving cells arose as a result of the RHGP perturbation and not as an artifact of spontaneous resistance to HIV-1. The small number of surviving cells was cloned and expanded. The resulting clones were then subjected to multiple rounds of challenge to eliminate any susceptible cells. Ultimately, we obtained 25 different cell clones that were insensitive to the lethal HIV-1 challenge.
To preclude that the act of the GSV integration into the MT4 genome might itself impart a nonspecific impact on HIV-1 replication, we tested naïve MT4 RHGP clones that had never previously been challenged with HIV-1. As a representative example, Clone H6 (in which the GSV integrates into the human SC6A3 gene) demonstrated no resistance to HIV-1, producing levels of HIV-1 production comparable to parental MT4 cells (Figure 6B). Likewise, HIV-1 infected H6 cells were completely depleted after infection (data not shown), thus confirming the specificity of the HIV-resistance demonstrated by the RHGP strategy.
Identification of the host gene by genomic DNA cloning
Summary of properties of identified HIV-1 resistant cellular genes
Main known function
CASD1 CAS1 domain-containing protein 1 precursor
Multi-pass membrane protein
Antisense intron 4 Down-regulation
HECW2 HECT, C2 and WW domain-containing protein 2
NEDD4-like E3 ubiquitin-protein ligase 2
Sense intron15 ATG(sc) exon 7 Down-regulation (DN)
ROBO1 roundabout protein homolog 1
axon guidance receptor and cell adhesion receptor
Type I membrane protein
Sense intron 3 ATG(sc) exon 3 Down-regulation (DN)
NLGN1 member of the neuroligin family
splice site-specific ligands for beta-neurexins and may be involved in the formation and remodeling of central nervous system synapses
Neuronal cell surface
Antisense intron 3 Down-regulation
DZIP3 (DAZ-interacting protein 3)
E3 Ubiquitin ligase proteins. Specifically bind RNA. Also called huRUL138 (RNA-binding ubiquitin ligase of 138 kDa)
Antisense intron 1 Down-regulation
CAMSAP1L1 calmodulin regulated spectrin-associated protein
may be involved in spectrin's function as a cytoskeletal protein providing a scaffolding and maintenance of plasma membrane
Antisense intron 2 Down-regulation
GSTCD glutathione S-transferase (GST), C-terminal domain
structural domain of GST, which conjugates reduced glutathione to a variety of targets to facilitate detoxification of the targets
Antisense intron 5 Down-regulation
CPSF1 Cleavage and polyadenylation specificity factor
It recognizes the AAUAAA signal in the pre-mRNA and facilitates both RNA cleavage and polyA synthesis
Antisense intron 2 Down-regulation
GDAP2 ganglioside induced differentiation associated protein 2
a signal transduction pathway during neuronal development
Sense last exon Down-regulation (DN)
TNRC6A trinucleotide repeat containing 6 protein
post-transcriptional gene silencing through the RNAi and microRNA pathways.
Sense intron 4 ATG(sc) exon 1 Down-regulation (DN)
TTC21B tetratricopeptide repeat domain 21B
may negatively modulate SHH (Sonic Hedgehog) signal transduction and may play a role in retrograde intraflagellar transport in cilia
Antisense intron 4 Down-regulation
ATP8A1 aminophospholipid transporter (APLT), Class I, type 8A, member 1
transport of aminophospholipids from the outer to the inner leaflet of various membranes and the maintenance of asymmetric distribution of phospholipids, mainly in secretory vesicles
Sense intron 4 ATG(sc) exon 1 Down-regulation (DN)
BE066906, AW817767, EB388641
Validation of target genes using naïve cells
The studies above demonstrated that RHGP could identify novel host targets that conferred resistance to HIV-1 infection. We then sought to verify these candidates using an independent experimental system to exclude outcomes that might arise as spontaneous mutation or unanticipated artifacts of the RHGP technology. Thus, duplex siRNAs targeting these candidates were obtained. Each siRNA preparation contained a pool of 4 individual siRNAs, all of which selectively target the gene of interest. Non-targeting siRNAs provided a matched control for the transfection and a reference standard. siRNA constructs specific for viral Tat and a cellular target, Rab6A, provided positive controls based on recent reports that these siRNA were able to efficiently inhibit HIV-1 infection .
An important goal of our present studies was to identify targets that are broadly applicable to HIV-1 infection. We also sought to confirm that targets identified using RHGP would not be unique to any particular cell system. To address both issues, we asked if the host gene candidates that rendered MT4 cells insensitive to challenge by HIV-1NL4-3 (a CXCR4-tropic virus) would similarly allow a different cell system to become insensitive to challenge by a CCR5 tropic HIV-1 virus. For this, the same siRNA approach as used with MT4 cells was used to target relative molecules in PM1 T cells. PM1 was selected since it expresses both CXCR4 and CCR5 co-receptors and thus can provide a model for both R5 and X4-tropic viruses . Similar to our findings with CXCR4-tropic viruses, targeting in PM1 cells demonstrated that this same set of 12 siRNAs was able to inhibit viral replication of the R5-tropic HIV-1ME1. Viral production of HIV-1ME1 strain was significantly inhibited in the cells treated with specific siRNA targeting each of these 12 gene targets (Figure 8B). These results confirmed our findings that the targets identified using RHGP are important for the replication of both X4 and R5 tropic HIV-1 viruses.
To validate the targets identified using RHGP, we sought to reproduce the perturbation in a "naïve" cell that has not been modified by the GSV. To verify that the siRNA targeting Robo1 in naïve T cells significantly reduced viral production during HIV-1 infection (Figure 8), we next examined whether Robo1 expression was successfully knocked down upon siRNA treatment using western blot. Indeed, reduced amounts of Robo1 were found in the siRNA treated cells (Figure 9B).
Resistance of RHGP cell clones to drug-resistant HIV-1
In our present study, we applied RHGP technology to conduct a genome-wide screen for host factors required for HIV-1 virus infection and identified novel host-based targets that render cells resistant to an otherwise lethal challenge with HIV-1 virus. In addition, we ascribed novel anti-HIV-1 functions to previously-known genes as well as non-annotated ESTs. These targets were validated first using an inducible promoter incorporated within the RHGP vector to reverse the phenotype and then in naïve cells using the conventional siRNA approach. We further found that the resultant targets were broadly applicable to different HIV variants, including CCR5 and CXCR4-tropic viruses. We further showed that cell clones with the gene targets disrupted by RHGP were resistant to viral challenge by a drug resistant HIV-1 mutant.
An independent study from our group recently identified host targets that allow host cells to survive in the face of an otherwise lethal infection with influenza virus. That study, as well as the work herein, employed a lentiviral system to overcome the prior limitation of low GSV production, which had been a problem associated with Moloney murine leukemia virus (MMLV)-based strategies . These improvements allowed us to sample the entire host genome and identify 12 gene targets from the 10^5 integration events invoked by the improved GSV. Consistent with prediction that a lentiviral vector favors single site insertion into sites of active gene transcription , all integration occurred in regions with active gene expression.
Based on MMLV-derived vectors which randomly integrate into the host chromosome, insertional mutagenesis was described as a high-throughput forward genetics approach to inactivate and thus discover cellular genes. Using these vectors cellular genes were identified that are required for replication of HIV-1 and other viruses, but not for host cell survival [22, 23]. With a built-in inducible promoter in GSV to drive transcript production from a host gene, our RHGP can also generate activation, often over-expression, of genes in mammalian cells, depending on the location and direction of GSV insertion relative to the candidate gene. Although not observed in our current study, over-expression of a subset of targets including the B-cell CLL/lymphoma 2 (BCL2) allowed MDCK cells to survive influenza infection during discovery of host genes against influenza virus infection. Since target expression is under control of the inducible promoter, the causal relationship between the phenotype (resistance to HIV-1 infection) and the perturbed gene can be confirmed by withdrawal of the inducer. By validating these targets within the same experiment, this feature markedly increases the efficiency of discovery of therapeutic candidates. Indeed, all the targets from resistant clones were successfully confirmed in the subsequent siRNA studies with naïve cells. Instead of transient knock down effects generated by conventional approaches (e.g., siRNA), the RHGP phenotype is highly stable, which can allow mechanistic studies to continuously characterize the roles of these perturbed genes in HIV-1 replication.
Increasing evidence suggests that the concept of host-oriented therapeutics may be particularly useful for identifying improved opportunities to combat HIV/AIDS. To identify relevant host targets, recent siRNA or shRNA based genome-wide studies have successfully identified host targets associated with HIV-1 infection [17, 24–26]. Unfortunately, siRNA is intrinsically limited by the need for strong and stable over-expression of the siRNA. Moreover, the outcomes of some siRNA findings have been clouded by recent questions of whether the siRNAs might non-specifically alter host defense mechanisms , which could be particularly problematic for applications of siRNA technology to therapies against viral diseases. Any siRNA screen is likely to generate false positive and false negative data due to the potential "off-target effects" along with variability both in siRNA efficacy and protein half-lives. This variability will ultimately lead to different levels of protein knockdown. We believe RHGP could provide an alternative since it is not limited by these same constraints.
Notably, the genes indentified herein are not included in the list discovered by the three recent siRNA-based HIV-1 studies [17, 24, 25]. We postulate this discrepancy may reflect that the different cell systems, viruses and approaches were used during screening coupled with contrasting experimental designs. Consistent with this postulation, there was very limited overlap between the HIV dependency factors identified in these investigations. These earlier studies did not utilize CD4+ T lymphocytes, a natural cell target for HIV infection. To achieve efficient siRNA delivery and investigate viral replication in those studies, adherent HeLa -derived cells were reconstructed to express CD4 and CXCR4 (such as TZM-bl cells used in the Brass et al screen  or P4/R5 cells in the Zhou et al study ). In a third investigation, a mutant HIV-1 virus pseudotyped with VSV-G was used to allow virus entry . This raises a question as to whether such differences might alter the mechanism of viral replication. Consistent with this, defective viral budding was observed in TZM-bl cells (our unpublished data). Using CD4+ T lymphocytes and wild type HIV-1 virus, we were able to interrogate very natural interactions between the entire repertoire of host factors and viral proteins during complete cycles of viral replication. We also noted that while we sampled the whole genome, a relatively low number of host genes (<20) were identified in our study. This subset of candidates might have resulted from the fact that our strategy of cloning surviving cells might have precluded targets that impact cell growth rates or viability. Likewise, the use of cell models and laboratory adapted viruses might have limited or biased the repertoire of host targets identified using RHGP. Thus, future studies should seek to adapt this technology using virus isolates obtained from primary cells.
A central tenet of the RHGP technology is that it is not biased by prior knowledge of the target. Consistent with this, two (of 14) that render host cells resistant to HIV infection were ESTs that had not yet been annotated (Table 1). Analysis of target function via the PANTHER Classification System , indicated little (CASD1, GDAP2, TNRC6A, and TTC21B,) or no (CAMSAP1L1 and GSTCD) knowledge of target function. The function for four targets have been ascribed (ROBO1, NLGN1, CPSF1, ATP8A1), but none of them have been linked with viral infection. The biological processes that these targets are involved in diversely include mRNA polyadenylation (CPSF1), cation transport (ATP8A1) and cell adhesion mediated signaling (ROBO1 and NLGN1).
HECW2 and DZIP3 are members of the E3 ubiquitin ligase family. This finding is intriguing since the ubiquitin ligase pathway has been shown to be necessary for the budding and release of HIV-1 and other viruses. For example, the TSG101 host protein is a ubiquitin-like ligase that interacts with HIV-1-encoded p6 Gag and is "hijacked" to facilitate viral egress from the cell surface . Likewise, Nedd4 is another ubiquitin ligase that regulates viral budding and release including HIV-1 [30, 31]. It is notable that HECW2 shares considerable homology with Nedd4 . It is thus tempting to postulate that HECW2 and DZIP3 may be also important for HIV-1 maturation and egress.
Robo1 is also essential for HIV replication. Viral production was markedly inhibited in both RHGP promoter "on" cells and siRNA treated naïve cells where knockdown of Robo1 expression were observed (Figure 8 and 9). This consistency was demonstrated again in RHGP cells in the subsequent reversibility assay when inducible promoter turned off and where levels of viral replication increased with the reemergence of Robo1 expression (Figure 9A). Robo1 is a type I transmembrane protein with an extracellular N-terminus comprising of 5 immunoglobulin and 3 fibronectin domains . It was originally identified as axon guidance receptor during neuronal development and was recently shown to regulate T cell chemotaxis [33, 34].
Our work thereby provides potential insights into new opportunities for host-directed therapeutics. Specifically, novel technologies like RHGP provide an opportunity to identify and prioritize host molecules that might provide safe and effective targets for drug intervention. In light of the increasing evidence that different virus types can share essential host pathways during their replication cycles, the therapeutics developed from genes identified in this study might also have broad application to other viruses as well.
Using RHGP, we have successfully identified novel host cell targets that are essential for HIV-1 replication but which can be safely targeted to preclude damage to normal cells. These targets emphasize safety while effectively blocking viral propagation. Most current HIV drugs target the HIV virus and therefore are vulnerable to the development of drug resistance through viral mutation. In contrast, therapeutics based on these newly identified human host targets will prevent HIV virus from using the host's cellular mechanism for its life cycle and are insensitive to drug resistance. Moreover, by targeting cellular pathways shared by HIV variants and even viruses other than HIV, these therapies have potentially broad spectrum anti-viral activities.
This work in this manuscript was supported by an SBIR grant from the National Institute of Allergy and Infectious Diseases (1R43 AI074484-01). The authors thank Dr. Stanley Cohen from Stanford University for providing plasmid reagents and expert advice for RHKO technologies and Dr. Bruce Torbett from the Scripps Research Institute for suggestions on research and critical review of the manuscript. The authors also thank Drs. Y. Abdul and L. Diaz for helpful comments and Ms. Ke Weng (Functional Genetics, Inc.) for guidance on cell cloning and siRNA transfection.
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