Abstract Background Viral reprogramming of host cells enhances replication and is initiated by viral interaction with the cell surface. Upon human immunodeficiency virus (HIV) binding to CD4+ T cells, a signal transduction cascade is initiated that reorganizes the actin cytoskeleton, activates transcription factors, and alters mRNA splicing pathways. Methods We used a quantitative mass spectrometry-based phosphoproteomic approach to investigate signal transduction cascades initiated by CCR5-tropic HIV, which accounts for virtually all transmitted viruses and the vast majority of viruses worldwide. Results CCR5-HIV signaling induced significant reprogramming of the actin cytoskeleton and mRNA splicing pathways, as previously described. In addition, CCR5-HIV signaling induced profound changes to the mRNA transcription, processing, translation, and post-translational modifications pathways, indicating that virtually every stage of protein production is affected. Furthermore, we identified two kinases regulated by CCR5-HIV signaling—p70-S6K1 (RPS6KB1) and MK2 (MAPKAPK2)—that were also required for optimal HIV infection of CD4+ T cells. These kinases regulate protein translation and cytoskeletal architecture, respectively, reinforcing the importance of these pathways in viral replication. Additionally, we found that blockade of CCR5 signaling by maraviroc had relatively modest effects on CCR5-HIV signaling, in agreement with reports that signaling by CCR5 is dispensable for HIV infection but in contrast to the critical effects of CXCR4 on cortical actin reorganization. Conclusions These results demonstrate that CCR5-tropic HIV induces significant reprogramming of host CD4+ T cell protein production pathways and identifies two novel kinases induced upon viral binding to the cell surface that are critical for HIV replication in host cells. Electronic supplementary material The online version of this article (10.1186/s12977-018-0423-4) contains supplementary material, which is available to authorized users. Keywords: HIV-1, CCR5, Signaling, Phosphoproteomics, Transcription, Splicing, Translation, Post-translational modifications Background HIV-1 enters cells via the interactions of gp120 with its primary receptor, CD4, and a coreceptor, typically CCR5 or CXCR4 for most clinical isolates. These events lead to conformational changes in gp120 that expose the fusion peptide of gp41 and lead to host and viral membrane fusion via the formation of an energetically favorable six-helix bundle conformation (reviewed in [[35]1]). Transmission of HIV-1 is almost always associated with viruses that utilize CCR5 and these viruses predominate in the vast majority of infected individuals [[36]2–[37]7]. CXCR4-using viruses emerge in approximately 40% of patients infected with subtype B HIV [[38]8, [39]9] and are associated with accelerated CD4+ T cell loss and progression to AIDS [[40]2, [41]10, [42]11]. Emergence of CXCR4 is much less common in subtype C, which accounts for approximately half of infections worldwide [[43]12–[44]16]. CD4, CCR5, and CXCR4 are not only receptors and coreceptors for HIV-1 entry, but also have physiological roles in signal transduction in host immune cells. CD4 engagement results in the activation of the receptor tyrosine kinase p56^Lck while CCR5 and CXCR4 are G-protein coupled receptors (GPCRs) that link to Gα and Gβγ subunits, particularly Gα[i], Gα[q], and Gβγ (reviewed in [[45]17]). HIV binding to the cell surface can induce signaling through CD4 and coreceptors and has been demonstrated to trigger calcium flux [[46]18, [47]19] and to activate Pyk2 [[48]20], phosphoinositol 3-kinase (PI3K) [[49]19], Akt and Erk 1/2 [[50]21], and the small GTPase Rac1 [[51]22]. In addition, HIV can promote NF-κB, AP1, and NFAT translocation to the nucleus [[52]23–[53]25] and actin cytoskeleton rearrangement [[54]21], including activation of cofilin, which reorganizes cortical actin barriers that restrict infection in primary CD4 T cells [[55]26]. Rac1 and cofilin activation by HIV enhances viral entry into cells [[56]22, [57]26], indicating that signaling through CD4 and coreceptor can induce rapid changes in cells that promote viral replication. However, signaling by HIV also can have longer-acting effects on the cellular environment: PI3 K activation by HIV was found to enhance infection via a post-entry step prior to integration [[58]19] and, more recently, a phosphoproteomic screen demonstrated that CXCR4-tropic HIV causes extensive alterations to the cellular microenvironment including to the splicing factor SRm300 (SRRM2) that enhances viral production by regulating alternative splicing of HIV mRNAs [[59]27]. Quantitative mass spectrometry-based phosphoproteomics is a powerful technique to broadly monitor phosphorylation changes in cells following exposure to a variety of stimuli. Here, we sought to build upon the study by Wojcechowskyj and colleagues [[60]27] by performing a similar investigation but using CCR5-tropic HIV, which accounts for nearly all transmission events and the vast majority of infections. To gain additional insights, we examined phosphorylation changes at 1, 15, and 60 min after exposure to HIV and included conditions with the CCR5 antagonist maraviroc that blocks CCR5 signaling [[61]28]. Together with an experimental setup closely related to that used by Wojchechowskyj, this design enabled comparisons between CCR5 and CXCR4-tropic HIV signaling, the transience or durability of phosphorylation changes, and how CCR5 signaling contributes to the overall reprogramming of host cells. Here, we report that CCR5-tropic HIV signaling reprogrammed not only cellular mRNA splicing pathways, but also transcription, translation, and post-translational modification pathways. Signaling by HIV also induced rearrangement in phosphoproteins regulating cytoskeletal transport, endocytosis and movement of cargo along cytoskeletal networks. Surprisingly, many of these pathways were stimulated regardless of the presence of maraviroc, suggesting that the effects of HIV particles on cells are primarily due to CD4 signaling or from other host molecules incorporated into viruses rather than on the coreceptors themselves. These findings provide new insights into how HIV-1 actively manipulates the target cell environment to enhance its replication. Results Phosphoproteomics of CCR5-tropic HIV signaling in primary memory CD4+ T cells HIV-1 binding to the cell surface induces signals within CD4+ T cells that have been characterized primarily using biochemical approaches [[62]18–[63]26]. More recently, Wojcechowskyj and colleagues used a mass spectrometry-based proteomics approach to identify proteins that were differentially phosphorylated in primary resting CD4+ T cells following a 1-min exposure to CXCR4-tropic HIV [[64]27]. CXCR4 is expressed on the majority of CD4+ T cells [[65]29]; however, CXCR4-tropic HIV-1 variants account for only a fraction of viruses worldwide and almost none that are involved in transmission and early infection [[66]2–[67]7]. To gain insight into phosphorylation changes induced by CCR5-tropic HIV-1 binding to cell surface receptors, we collected leukapheresis samples from several donors and removed naïve CD4+ T cells—which express little CCR5 and are not infected to an appreciable extent by R5-tropic HIV—by rosette depletion to yield pooled, unstimulated memory CD4+ T cells. 150 × 10^6 memory CD4+ T cells were exposed to 20 μg/ml p24 equivalent AT2-inactivated HIV-1 THRO or an equivalent protein concentration of non-viral extracellular vesicles. The high concentration of HIV was chosen to synchronize signaling events within CD4+ T cells as well as to maintain consistency with the CXCR4-tropic HIV phosphoproteomic study [[68]27] to facilitate comparisons between R5- and X4-tropic HIV signaling. To further improve understanding of HIV-1 signaling events, we also exposed cells to R5-tropic HIV in the presence of 100 μM of the CCR5 antagonist maraviroc to block co-receptor signaling and included 1, 15, and 60 min stimulations for all experimental conditions to assess the durability of phosphorylation changes. A schematic of the experimental design is shown in Fig. [69]1a. Importantly, 100 μm maraviroc completely blocked entry of HIV-1 THRO into primary CD4+ T cells (Fig. [70]1b). Fig. 1. [71]Fig. 1 [72]Open in a new tab Phosphoproteomics of CCR5-tropic HIV signaling in primary memory CD4+ T cells. a Schematic of experimental design. Purified, unstimulated memory CD4+ T cells were exposed to extracellular vesicles (EVs), HIV, or HIV in the presence of 100 μm maraviroc (MVC) for 1, 15, or 60 min. A fraction of the experimental samples were pooled and run in triplicate for technical replicates. b Entry of HIV-1 THRO into primary CD4+ T cells is inhibited by treatment with 100 μM maraviroc. c Peptides demonstrating coefficients of variation (CVs) of > 50% in the technical replicate samples were excluded from further analysis. d Table of summary counts from the phosphoproteomics experiment; FDR-filtered results are selected if meeting the FDR = 0.001 cutoff. e Phosphopeptide-level MA plots across different treatment conditions and time points. Fold change (FC) calculations were in reference to extracellular vesicle control. Mean peptide intensity was calculated between the control and designated treatment condition. Grey circles indicate peptides that do not meet the log2(FC) cutoff After applying filters to remove phosphopeptides demonstrating unreliable detection in three technical replicate controls (Fig. [73]1c), we removed peptides with non-phosphorylation post-translational modifications such as alkylation of cysteine residues. The final list contained 2816 phosphorylation sites from 1363 unique proteins using label-free proteomics. 1678 phosphorylation sites from 931 unique proteins were found to be HIV-responsive using an FDR cutoff of 0.001 (0.1%) compared to the respective extracellular vesicle control for at least one R5-tropic HIV-1 condition (Fig. [74]1d). The majority of peptides demonstrated modest fold-change variation (Fig. [75]1e); however a small number of peptides, particularly in the 1- and 15-min time points, showed much greater fold-change values Additional file [76]1: Table S1). We analyzed phosphopeptides with large fold-changes at both the 1- and 15-min time points and found nearly all were from proteins with roles in either (1) actin cytoskeletal architecture, endocytosis, and movement of vesicles along actin and tubulin networks, or (2) transcription, mRNA splicing and capping, translation, or acetylation of methionine residues on newly produced proteins. These data support previous studies demonstrating that these pathways are actively modulated by signaling upon HIV binding to the cell surface [[77]22, [78]26, [79]27, [80]30]. Signaling by CCR5-tropic HIV results in phosphorylation patterns with both significant overlap and differences compared to CXCR4-tropic HIV HIV-1 binding to the cell surface can signal through several different pathways. First, specific interactions between gp120 and the host receptors CD4 and CCR5/CXCR4 can trigger responses in their respective signal transduction cascades. CD4 activates the receptor tyrosine kinase p56^Lck while CCR5 and CXCR4 signal via Gα and Gβγ subunits, particularly Gα[i], Gα[q], and Gβγ (reviewed in [[81]17]). Second, signaling from gp120 interacting with non-receptor molecules has also been reported, such as the induction of cdc42 in dendritic cells via engagement of DC-SIGN [[82]31]. It is possible that Env proteins could signal through other, currently unidentified cellular receptors as well. Third, HIV-1 incorporates host proteins into virions including HLA-DR, LFA-1, and ICAM-1 [[83]32–[84]34], which could also potentially induce signals in target cells. We reasoned that CCR5-tropic and CXCR4-tropic HIV would share considerable overlap in induced signaling pathways but could also vary due to potential signaling differences following CCR5 or CXCR4 engagement or due to the exclusion of naïve CD4+ T cells that are not readily infected by most HIV-1 isolates. Indeed, we found a considerable overlap between HIV-responsive proteins following CCR5- and CXCR4-HIV signaling (Fig. [85]2a), with 111 shared proteins between the two studies. Our study identified 63.4% (111/175, p < 1.0 × 10^−15) of the HIV-responsive proteins identified by Wojecechowskyj and colleagues and an additional 820 HIV-responsive proteins not identified by their study. The larger number of HIV-responsive proteins found here is likely due to a combination of factors, including a larger number of total phosphopeptides detected, indicating more robust coverage of the CD4+ T cell proteome and a larger number of experimental conditions including 1, 15, and 60 min time points and CCR5-signaling blockade with maraviroc. Fig. 2. [86]Fig. 2 [87]Open in a new tab Venn diagram displaying overlap across three HIV datasets. CCR5-HIV signaling refers to HIV-responsive phosphoproteins (responsive in either HIV or HIV + MVC groups compared to MV controls) described in this study. CXCR4-HIV are HIV-responsive peptides reported by Wojcechowskyj and colleagues and HHPID is the HIV-1 Human Protein Interaction Database. There is statistically significant overlap of members between a the CCR5-HIV dataset vs. CXCR4-HIV (p < 1 × 10^−15, hypergeometric test) and b between CCR5-HIV and the HHPID (p < 1 × 10^−15, hypergeometric test). Both tests were calculated against a background of 21,764 protein-coding genes from the GeneCards database, as of March 2017. c Overlap of proteins in the CCR5-HIV, CXCR4-HIV, and HHPID datasets To gain further insight into the proteins identified as HIV-responsive following CCR5-HIV signaling, we compared our results with a manually curated list of human proteins shown to interact with HIV—the HIV-1 human protein interaction database (HHPID). This comparison was of particular interest as signaling has previously been demonstrated to influence host proteins levels that subsequently affect viral replication within host cells [[88]19, [89]22, [90]26, [91]27]. Again, we found a highly significant overlap (Fig. [92]2b), with 43.1% (401/931, p < 1.0 × 10^−15) of the HIV-responsive proteins identified here being part of the HHPID. The overlap between the HHPID, CCR5- and CXCR4-HIV signaling is shown in Fig. [93]2c. Shared proteins between the current study, CXCR4-HIV signaling, and the HHPID are listed in Additional file [94]1: Table S2. CCR5-tropic HIV signaling extensively reprograms cellular pathways involved in protein production and cytoskeletal regulation Following integration into the host chromosome, HIV is transcribed into mRNA by the cellular RNA polymerase II complex. HIV contains at least four splice donors (D1–D4) and eight splice acceptors (A1–A8) that result in over 40 varieties of spliced mRNA transcripts. Splicing is regulated in a temporal fashion, with fully spliced 1- to 2-kb transcripts appearing first, followed by intermediate 4- to 5-kb transcripts, and finally by full-length 9-kb mRNAs that encode the Gag-Pol proteins and comprise the genome of budding viruses. The phosphoproteomic analysis of CXCR4-tropic HIV signaling performed by Wojcechowskyj and colleagues revealed a reprogramming of the cellular mRNA splicing pathways and identified SRRM2 as an HIV responsive protein that was also required for optimal HIV replication. Five additional SR proteins involved in mRNA splicing were identified as HIV responsive, including SRRM1, ACIN1, PNN, PPIG, and TRA1. Our results with CCR5-tropic signaling strongly support the finding that HIV actively modulates splicing pathways: 5 of 6 of the SR proteins identified as CXCR4-HIV responsive were also CCR5-HIV responsive, the lone exception being PPIG. In addition, we identified a large number of additional splicing regulators including serine/arginine-rich splicing factors (SRSF) 1, 2, 5, 6, 7, 9, 10 and 11 and the upstream SRSF kinase 1. In particular, the SRSF1, 2, 5, and 6 proteins have previously been implicated in binding to the splice-donor region of the major 5′ splice site of HIV [[95]35]. HIV reprogramming of mRNA splicing is also apparent on a more global scale: there was a highly significant enrichment of HIV-responsive proteins that were members of mRNA splicing and major mRNA splicing pathways in the REACTOME database (Additional file [96]1: Table S3). In addition to the splicing pathways, bioinformatic analysis of the REACTOME databases revealed significant enrichment of HIV-responsive proteins in several other pathways relating to mRNA and protein production pathways: transcriptional regulation, processing of capped intron-containing pre-mRNA, metabolism of proteins, and post-translational protein modifications. These results suggest that HIV signaling induces profound changes at multiple levels of protein production, from mRNA transcription to post-translational modifications. In particular, the eukaryotic translation initiation (EIF) proteins, S ribosomal proteins (RPS), secretory (SEC) proteins involved in ER to Golgi transport, and the ubiquitin specific peptidase (USP) families all demonstrated multiple members that were responsive to HIV signaling. Signaling by Rho GTPases and Rho GTPase cycle pathways were also highly enriched for proteins responsive to HIV signaling. Rho GTPases regulate a variety of cellular processes including cytoskeletal dynamics, transcription, endosomal trafficking, cell cycle, cell adhesion and cytokinesis. The observation that HIV signaling influences Rho GTPase signaling pathways has been explored by our lab recently, in combination with a small molecule screen that implicated Rho GTPases in regulation of HIV infection [[97]30]. Briefly, CCR5-tropic signaling was found to dramatically alter the cellular Rho GTPase landscape, regulating not only cdc42 and Rac1 GTPases but also at least 25 guanosine dissociation inhibitors (GDIs), guanosine exchange factors (GEFs) and GTPase activating proteins (GAPs) that influence GTPase signaling. Furthermore, inhibition of cdc42, RhoA or Rho-associated protein kinase (ROCK) inhibited viral infection, demonstrating that the appropriate function of Rho family GTPases and their substrates is essential to optimal infection of CD4+ T cells. These results add to previous studies demonstrating that HIV-1 signaling actively regulates cytoskeletal dynamics and that inhibition of these pathways negatively affects viral replication [[98]22, [99]26, [100]36]. Identification of kinases activated or inhibited by CCR5-tropic HIV One of primary advantages of phosphoproteomics is that it enables collection of information on thousands of differentially regulated phosphorylation sites that are the result of changes in the activity of proximal kinases and phosphatases acting upon their substrates. Since different kinases have varying preferences for consensus substrate