HIV Articles  
New HIV Targets Discovered
  Global Analysis of Host-Pathogen Interactions that Regulate Early-Stage HIV-1 Replication
Cell Sept 2008
Renate Konig,1 Yingyao Zhou,6,8 Daniel Elleder,2,8 Tracy L. Diamond,5,8 Ghislain M.C. Bonamy,6,7 Jeffrey T. Irelan,6 Chih-yuan Chiang,6 Buu P. Tu,6 Paul D. De Jesus,1 Caroline E. Lilley,3 Shannon Seidel,2 Amanda M. Opaluch,1 Jeremy S. Caldwell,6 Matthew D. Weitzman,3 Kelli L. Kuhen,6 Sourav Bandyopadhyay,4 Trey Ideker,4 Anthony P. Orth,6 Loren J. Miraglia,6 Frederic D. Bushman,5 John A. Young,2, and Sumit K. Chanda1,
1 Infectious & Inflammatory Disease Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
2 Infectious Disease Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
3 Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA 4 Department of Bioengineering, University of California, San Diego, La Jolla, CA 92037, USA
5 Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
6 The Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA
7 Hudson-Alpha Institute for Biotechnology, 127 Holmes Avenue, Huntsville, AL 35801, USA
Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.
Over the course of the last several decades, a number of host cell proteins that influence the early steps of retroviral replication have been defined (Goff, 2007). However, it is likely that many other cellular factors and processes are exploited by the virus during these stages, which involves uncoating steps that give rise to an active reverse transcription complex (RTC), movement of the viral preintegration complex (PIC) to the cell nucleus, and then integration of the linear viral DNA into a host cell chromosome to generate the provirus (Nisole et al., 2004). Recently, a genome-wide siRNA analysis revealed over 250 host cellular factors that influence HIV-1 infection (Brass et al., 2008). Notably, only a small fraction of these factors was proposed to influence the early stages of HIV-1 replication, making it likely that additional cellular factors that regulate these steps remain to be identified. Here, we present a genome-wide analysis of virus-host interactions affecting the early steps of HIV-1 infection.
Here, we describe a genome-wide assay with an arrayed siRNA library to identify genes required for early stages of HIV infection. Inhibition of the gene function of putative host factors may induce cellular responses that destabilize the viral cDNA, accelerate turnover, or otherwise indirectly interfere with viral replication. To identify those proteins that are more likely to be direct regulators of the viral life cycle, and thus be critical for HIV-1 pathogenesis, we developed further selection criteria, which took into consideration a number of additional, and statistically significant, lines of evidence, including protein interactions, mRNA expression, and gene ontology (Figure 1D; Supplemental Data). Importantly, since this approach does not only rely on identifying host factors based solely on ranking siRNA activities, we were able to more effectively mine the genetic data sets to identify those factors more likely to be immediate regulators of HIV-1 infection, and establish a network of host-pathogen interactions that coordinates HIV-1 infection. Retrospective analysis showed that evidence scores of confirmed genes are, on average, 40% higher than those that did not confirm (p = 9.7 X 10|15), indicating that our approach enriched for biologically relevant activities (Table S3; data not shown).
Further validation studies have identified over 40 host factors that regulate capsid uncoating and reverse transcription steps of early HIV-1 replication (Figure 4). Additionally, we have elucidated 15 cellular factors that facilitate nuclear entry of the HIV-1 PIC and integration of proviral DNA (Figure 5). Importantly, only genes that regulated infection with HIV-1 virus pseudotyped with both VSV-G and 10A1 envelopes were considered for further analysis, thus excluding factors that may regulate endosomal function associated with VSV-G-mediated entry (see Figure 2C legend). Taken together, these studies indicate that host cellular factors are involved in a variety of different cellular processes that influence HIV-1 reverse transcription, nuclear import, and integration.
Comparison with Reported HIV Host Factors Identified through RNAi-Based Functional Screening
A recent genome-wide RNAi analysis by Brass et al., 2008 has identified 284 genes as host cellular factors required for HIV replication. Comparison with the 295 confirmed genes presented here reveals a modest, but statistically significant, overlap of 13 genes (p = 0.00021). We speculate that this moderate concordance is largely due to differences in the analysis and experimental methodologies used. For example, the criteria Brass et al., 2008 employ to report host cellular factors are genes targeted by one or more siRNAs with activities > 2 standard deviations from the mean. Since 154 of the genes in the Brass et al. study were supported by the activity of only a single siRNA, it is likely that a fraction of these reported host factors represent false-positive readouts due to off-target RNAi activity (Echeverri et al., 2006).
If we apply the criteria used by Brass et al. to the data presented here, we can identify 60 genes that the two RNAi studies have in common (p = 0.024; Table S8). Further reinforcing the parallels, we find that, based upon protein network analysis, an additional 64 genes reported by Brass et al. directly interact with a confirmed gene in our study (p = 0.019; Table S8). In our study, we have also prioritized gene activities not only based upon siRNA activity, but also based on comparative activities in additional screens, HIV-host protein interaction data, as well as gene expression and ontology analysis. We anticipate that this approach enabled us to enrich for the most relevant host cellular factors that promote HIV infection, but this also likely contributed to the observed differences between the two host factor data sets.
Several experimental differences must also be considered when comparing these studies (also see Table S8). Two independent RNAi libraries, constructed by using separate design criteria and arrayed in different formats, were used to conduct these screens. Also, we have employed a single-cycle, replication-defective HIV vector pseudotyped with a VSV-G envelope in HEK293 cells and measured viral infection at a 24 hr time point. Brass et al., 2008 monitored the replication of a wild-type X4 strain of HIV in CD4/CXCR4-expressing HeLa cells over the course of 48 hr. Thus, we would not be able to identify factors involved in CD4-mediated viral entry, as well as host molecules that regulate late-stage HIV replication. In contrast, however, we monitored single-cycle virus infectivity at an early (24 hr) time point, which likely enabled us to elucidate a more comprehensive set of host factors specifically involved in the early stages of replication, including uncoating, reverse transcription, and integration. These also encompassed proteins that regulate the kinetics of these processes (Figure 4A). Thus, whereas false-positive activities are an inherent part of large-scale analyses, it is likely that variations in both experimental and data analysis techniques can largely account for the differences between our results and those reported by Brass et al., 2008.
The Role of Cytoskeletal Proteins in Early HIV Replication
The actin cytoskeleton was previously implicated in regulating the initiation of HIV-1 reverse transcription (Bukrinskaya et al., 1998) as well as in the movement of intracellular viral nucleoprotein complexes (NPCs) (Arhel et al., 2006). Consistently, the present study has revealed important roles in the earliest steps of HIV-1 infection for AKAP13, a RhoA-specific guanine nucleotide exchange factor (GEF) that regulates actin stress fiber formation; for NCKAP1, which associates with WAVE proteins that regulate actin nucleation/organization; and for TAGLN-2, a putative actin crosslinking/gelling protein (Table 1). Microtubules, previously shown to be involved in the intracellular movement of HIV-1 RTCs (Arhel et al., 2006, Naghavi et al., 2007), may also play a regulatory role in reverse transcription, since viral DNA levels were influenced by RP3-355C18.2, a predicted tubulin tyrosine-ligase, and MID1IP1, involved in bundling and stabilizing microtubules (Table 1). MID1IP1 was also identified recently by Elledge and colleagues (Brass et al., 2008), but its role in HIV-1 replication was not defined.
Involvement of the DNA-Damage Repair Pathway in HIV Uncoating/Reverse Transcription
Cellular DNA repair machinery has been implicated in playing roles in viral DNA integration and in the completion of viral DNA synthesis after integration (Goff, 2007). An unanticipated finding here was that proteins involved in DNA-damage response and repair also influence the initiation of reverse transcription and the accumulation of HIV-1 DNA products prior to integration (Table 1). Moreover, we have found two locally dense networks of proteins containing host factors that participate in DNA repair (Figure 3C, DNA transcription/repair; Figure 3F, DNA damage/replication). Both clusters contain multiple confirmed factors, including POLR2A, XAB2, and ERCC5, which have been mapped to early steps in the viral life cycle (Table 1). The viral interface for this host-pathogen interaction is mediated by Vpr, a component of the RTC/PIC that was recently being linked to the DNA-damage response pathway (Schrofelbauer et al., 2007). Several other DNA repair proteins, including MUS81, ERCC1, and MRE11, involved in nucleotide excision repair were also implicated in the early events of HIV-1 infection (Figure 2C).
Nucleic Acid-Binding Proteins Participate in the Early Stages of HIV Replication
Retroviral reverse transcription presumably involves the unwinding of RNA-RNA, RNA-DNA, and DNA-DNA strands, suggesting, that one or more cellular helicase may participate in viral DNA synthesis. Our results indicate that DHX15, a putative ATP-dependent RNA helicase that plays a role in pre-mRNA splicing, is important for early viral DNA synthesis in target cells (Table 1). Other nucleic acid-binding proteins, including RBM17, were also implicated in regulating HIV-1 DNA synthesis (Table 1). DHX15 and RBM17 are contained in a densely connected network (Figure 3H), supporting their status as proximal regulators of HIV-1 replication. In addition, several factors involved in transcription or mRNA splicing that regulate viral uncoating or reverse transcription were also identified (Table 1). Correspondingly, two MCODE protein complexes, which regulate transcription, splicing, and nucleic acid binding, were identified through our network analysis (Figure 3D and 3E). The latter cluster consists of 11 confirmed factors, of which 2 were required for reverse transcription, suggesting that these proteins collectively coordinate either the uncoating or DNA synthesis steps of HIV replication. It remains to be determined whether these host proteins act by interacting directly with the viral nucleic acids, or whether they regulate other host factors required for HIV infection.
The Ubiquitin-Proteasome Pathway Is Required for Capsid Uncoating and Viral DNA Synthesis
The ubiquitin-proteasome pathway has previously been associated with the early steps of HIV replication, where it acts negatively to destroy incoming viral replication complexes (Butler et al., 2002, Schwartz et al., 1998). Our studies have also revealed a positive role for this pathway. The ubiquitin ligases UBE2B (RAD6) and TRIAD3, as well as the proteasome component PSMB2, were each important for HIV-1 reverse transcription (Table 1). Network analysis also revealed that the viral Integrase and Vif proteins have multiple interactions with a cluster of proteins that function in the ubiquitin-proteasome pathway (Figure 3B), indicating that these viral factors may play a structural role in the HIV RTC to recruit the proteosomal machinery and facilitate uncoating or reverse transcription.
Posttranslational Modifications in Early HIV Replication
Previously, the cAMP-dependent protein kinase (PKA) was implicated in regulating early steps of HIV-1 replication (Cartier et al., 2003). AKAP13 is a PKA scaffold protein (Table 1) and as such may be involved in mediating PKA-dependent regulation of HIV-1 reverse transcription. Protein dephosphorylation events may also act to regulate HIV-1 reverse transcription, as indicated by the importance of protein phosphatase 1 regulatory subunit 14D (PPP1R14D), a negative regulator of the catalytic subunit of the serine/threonine protein phosphatase 1 (Table 1).
SUMOylation events have been proposed to be important in the early steps of MuLV infection (Yueh et al., 2006). We have found that SUMO-2, one of three small, ubiquitin-related modifier proteins, is important during the late stage of HIV-1 (and MuLV) reverse transcription (Table 1). In addition, RANBP2, a SUMO-1 E3 ligase that is a component of the cytoplasmic filaments of the nuclear pore complex, was required for nuclear import of the HIV-1 DNA (Table 1), perhaps through the sumoylation of viral proteins in the PIC or host factors required in the PIC. Since RANBP2 influenced HIV, but not MuLV, infection (Figure 4B), different SUMO conjugating systems may be important for each of these two viruses.
Host Factors Required for Nuclear Import of the Viral Preintegration Complex
The mechanism of HIV nuclear import is controversial, with multiple proteins and nucleic acids proposed to play a role (Suzuki et al., 2007). Our studies, combined with those of Brass et al., 2008, indicate the involvement of NUP153, RANBP2, and TNPO3 as factors involved in HIV-1 PIC import (Table 1). We have also uncovered roles for NUP214; the nascent polypeptide-associated complex alpha subunit 2, NACA2; and prostaglandin E synthase, PTGES3 (Table 1 and Figure 5). The potential role of prostaglandins in HIV-1 nuclear import is particularly intriguing, because these factors are already known to regulate the import of other types of nuclear cargo (Gomez et al., 2005, Malki et al., 2005), and they may represent a new therapeutic target for HIV-1 infection.
Host Proteins and Viral DNA Integration
Our studies have also revealed several cellular factors important for HIV-1 DNA integration. The first is ANAPC2, a component of the anaphase-promoting complex that promotes the metaphase-anaphase transition. ANAPC2 is a cullin protein with a role in ubiquitin ligase activity (Table 1), suggesting that ubiquitin modification and/or proteolysis of virus PIC components may play a regulatory role during the integration stage of viral DNA replication. Recent reports have suggested that passage through mitosis may promote HIV as well as MLV DNA integration (Mannioui et al., 2004, Roe et al., 1993), providing a possible role for ANAPC2. The second factor, SNW1, is a transcriptional coactivator that associates with a cyclophilin-like protein, peptidyl-prolyl isomerase-like 1 (PPIL1) (Table 1). The structure of PPIL1 resembles that of other members of the cyclophilin family, in particular Cyclophilin A (Xu et al., 2006), suggesting that cyclophilin proteins might be involved in regulating both late (integration) and early (uncoating) (Luban, 2007) replication steps. A third factor, aquarius or AQR, also regulates HIV-1 DNA integration (Figure 5A). This factor associates with XAB2, a protein involved in transcription-coupled repair (Kuraoka et al., 2008), suggesting that this DNA repair pathway might also play a role during viral DNA integration.
A Model for Import-Coupled Integration
Protein networks that are implicated in nuclear import and integration of the HIV PIC are shown in Figure 5C. This network was constructed based upon known or experimentally determined interactions between host factors and HIV-encoded proteins, and proteins are organized and oriented on the basis of the quantitative PCR mapping data (Figure 5A). Based upon these data, we propose a model wherein nuclear import of the PIC and proviral DNA integration are molecularly coupled events mediated by nucleoporins, karyopherin, and putative tethering factors (Figure 5D). Specifically, we hypothesize that nuclear transport of the viral PIC through the nuclear pore complex is mediated by soluble transport receptors, such as TNPO3, and nuclear pore components (Stewart, 2007). Next, the PIC cargo is transferred consecutively to phenylalanine-glycine (FG) repeat domains of variant nucleoporins, for example NUP358/RANBP2. Subsequently, in this model, the cargo is delivered to the FG repeats of NUP214 (anchored to the cytoplasmic ring) and NUP153 (distal ring). The FG repeats of both NUP214 and NUP153 are highly flexible and can either reach down to the nuclear basket (NUP214) or toward the cytoplasmic periphery of the central pore (NUP153) (Paulillo et al., 2005), thereby possibly acting to accelerate PIC translocation. As an alternative, we cannot rule out that RNAi silencing of nuclear import machinery components may be acting indirectly by altering the localization of protein(s) required for HIV nuclear import.
Upon traversing the nuclear pore, we hypothesize that the PIC is then released into the nucleus through interaction with the FG-containing NUP98 located near the nuclear basket, which dynamically associates with and dissociates from the nuclear pore (Griffis et al., 2004). Our data indicate that NUP98 is essential for viral integration, suggesting that NUP98 likely directs the viral PIC from the nuclear pore to the proximity of the chromatin. The intranuclear mobility of NUP98 has previously been linked to active transcription sites, possibly through direct interactions with the transcriptional machinery or with newly produced transcripts and RNP complexes (Griffis et al., 2004). Additionally, the PIC is anchored to chromatin through potential "tethering" factors. Targeted integration into active transcription units is mediated by PSIP1/LEDGF/p75 (Ciuffi et al., 2006) (and references therein). Additional factors may help direct integration to transcriptionally active regions, as suggested by the finding that histone posttranslational modifications associated with expression positively correlate with HIV integration frequency (Wang et al., 2007). We have identified several proteins that are required for viral integration and are linked to transcription, signaling, and splicing (ANAPC2, MT1X, SNW1, IK, PRPF38A, and AQR). We hypothesize that these proteins might act to target the PIC to the chromatin and/or act as enzymatic cofactors for DNA integration. Our data indicate that karyopherin KPNB1 is also required for viral integration, suggesting that it may either cooperate with NUP98 to direct the PIC to transcriptionally active chromatin or, alternatively, could serve as an intermediate to couple the viral PIC to the tethering factors. These data support a model in which components of nuclear import machinery facilitate both HIV nuclear entry and integration, and they suggest that the coupling of these steps is required for the establishment of the HIV provirus.
Taken together, this integrative approach toward genome-wide host-pathogen interaction analysis has revealed cellular factors that coordinately regulate the early steps of HIV viral replication, including those required for reverse transcription and proviral establishment. To our knowledge, altogether new pathways, such as the possible involvement of Notch signaling in reverse transcription and prostaglandins in nuclear import, are suggested for the first time. An understanding of their role in cell types targeted by HIV during infection, such as helper T cells, macrophages, and dendritic and microglial cells, will be critical for characterizing the contribution of these factors toward disease progression. The development of small molecules that modulate the activity of these proteins may provide novel strategies for the treatment of HIV/AIDS, particularly since the inhibition of early stages in the viral life cycle has already proven to be therapeutically effective.
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