Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome

Abstract

Retroviruses exploit host proteins to assemble and release virions from infected cells. Previously, most studies focused on interacting partners of retroviral Gag proteins that localize to the cytoplasm or plasma membrane. Given that several full-length Gag proteins have been found in the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings involving previously unknown host processes. Here we systematically compared nuclear factors identified in published HIV-1 proteomic studies and performed our own mass spectrometry analysis using affinity-tagged HIV-1 and RSV Gag proteins mixed with nuclear extracts. We identified 57 nuclear proteins in common between HIV-1 and RSV Gag, and a set of nuclear proteins present in our analysis and ≥ 1 of the published HIV-1 datasets. Many proteins were associated with nuclear processes which could have functional consequences for viral replication, including transcription initiation/elongation/termination, RNA processing, splicing, and chromatin remodeling. Examples include facilitating chromatin remodeling to expose the integrated provirus, promoting expression of viral genes, repressing the transcription of antagonistic cellular genes, preventing splicing of viral RNA, altering splicing of cellular RNAs, or influencing viral or host RNA folding or RNA nuclear export. Many proteins in our pulldowns common to RSV and HIV-1 Gag are critical for transcription, including PolR2B, the second largest subunit of RNA polymerase II (RNAPII), and LEO1, a PAF1C complex member that regulates transcriptional elongation, supporting the possibility that Gag influences the host transcription profile to aid the virus. Through the interaction of RSV and HIV-1 Gag with splicing-related proteins CBLL1, HNRNPH3, TRA2B, PTBP1 and U2AF1, we speculate that Gag could enhance unspliced viral RNA production for translation and packaging. To validate one putative hit, we demonstrated an interaction of RSV Gag with Mediator complex member Med26, required for RNA polymerase II-mediated transcription. Although 57 host proteins interacted with both Gag proteins, unique host proteins belonging to each interactome dataset were identified. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology.

Keywords: HIV-1; Mass spectrometry; Proteomics; Retroviruses; Rous sarcoma virus.

Fig. 1

Schematic diagram and western blot with quantitation. A RSV Gag constructs. B Subcellular fractionations were performed to separate the cytoplasm and nucleoplasm from the chromatin fractions, which were further separated using differential NaCl concentrations. The 150 mM chromatin fraction (Chr 150 mM) contains proteins associated with open chromatin (euchromatin). The 500 mM chromatin fraction (Chr 500 mM) contains proteins that are associated with condensed chromatin (heterochromatin). Wild type RSV Gag and Gag.L219A were detected in all of the fractions at different ratios. Gag.ΔNC was primarily detected in the cytoplasm with very little in the chromatin fractions. C Band densities were determined for each Gag construct for each fraction and are displayed as mean ± standard error of the mean (SEM) for three biological replicates. To assess fraction purity, cellular proteins were detected using antibodies against calnexin (cytoplasm), Med4 (nucleoplasm and euchromatin), and Histone H2B (euchromatin and heterochromatin). The position of molecular weight markers, in kilodaltons, are indicated on the right

Fig. 2

The relative representation of the IPA categories present in each protein list. A The proteins identified in our HIV-1 Gag pulldown that were also found in at least one of the previously published reports were analyzed for molecular and cellular functions. The color key on the right is the same for each pie graph. Protein functions identified in this publication (Rice) (B), Engeland 2011 [31] (C), Engeland 2014 [30] (D), Le Sage [33] (E), Li [34] (F), Ritchie [35] (G), and Jäger [32] (H)

Fig. 3

RSV Gag colocalized and co-immunoprecipitated with Med26. A Transfected RSV Gag-GFP (red) and FLAG-Med26 (green) colocalize (white) within QT6 cells. Image representative of average colocalization, as quantified in panel (B). Nuclei (blue) are outlined by a dotted white line, and regions boxed in the main images are enlarged below. White arrows are included to guide the eye. Scale bars = 2 µm. B Manders’ Overlap Coefficient values for image set represented by (A). Individual values are shown in addition mean ± SEM, n ≥ 17; ****, p < 0.0001 by unpaired two-tailed t-test. C 500 µg of RC.V8-infected QT6 nuclear lysates were incubated with an α-RSV Gag antibody (mouse α-RSV CA.A11, gift from Neil Christensen, Penn State College of Medicine), followed by antibody capture on Pierce™ Protein G Magnetic Beads. After extensive washing, proteins were eluted from beads by boiling in 1X SDS-PAGE sample buffer and run on a 10% SDS-PAGE gel, transferred to PVDF, and Western blotted first for Med26 (top) followed by RSV Gag (bottom). The position of molecular weight markers, in kilodaltons, are indicated on the left. FT, flow through; Beads, lysate only; Eluate, lysate plus antibody. Images representative of three independent experiments

Fig. 4

HIV-1 interactome pathway analysis. This diagram illustrates the HIV-1 Gag-interacting nuclear host factors discussed in this study. Factors shaded blue were uniquely identified in the present study (newly identified). Proteins shaded in purple were identified in this publication as well as at least one other published report (previously identified). Gray shading was used to show host protein complexes that are involved with Gag-interacting factors in chromatin remodeling, gene expression, nuclear export, and splicing. A HIV-1 Gag interacting factors that promote an open chromatin structure (euchromatin state) are on the left, whereas proteins involved in condensing chromatin are shown on the right. B Proteins involved in regulation of gene expression are depicted. Factors that promote transcription initiation are indicated by arrows and green plus signs. Factors that suppress or inhibit transcription are demarcated by red blocking lines. C The two nucleoporin proteins NUP98 and NUP188 were identified and are involved in trafficking between the nucleus and the cytoplasm through the nuclear pore complex. D Proteins that localize to the spliceosome and are involved in RNA splicing are shown

Fig. 5

STRING protein network map of HIV-1 Gag interacting host chromatin proteins. Protein lists were generated from mass spectrometry experiments of Gag interacting proteins. Gene lists were then categorized into gene ontology (GO) terms to identify those which were chromatin associated. This refined list was input into STRING Consortium v12.0 (https://string-db.org/) to generate a protein–protein interaction map. A total of 129 proteins were queried and the following physical protein–protein interactions were made. Among the 129 proteins queried, 117 proteins are displayed after a minimum required interaction score of 0.4 was applied. Proteins that do not have any interacting partners are shown at the bottom left corner of the map. Lines are generated based on known interactions (purple lines), predicted gene fusions (red lines), predicted gene neighborhoods (green lines), and predicted gene co-occurrences (blue lines) from the literature