Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.

Moloney leukemia virus type 10 protein (MOV10) is an RNA helicase that is induced by type I interferon. It inhibits HIV replication at several steps of its replicative cycle. Of interest, MOV10 is a component of mRNA processing (P) bodies, which inhibit retrotransposition (RTP) of intracisternal A particles (IAP). In this report, we studied the effects of MOV10 on IAP RTP and its dependence on P bodies. Indeed, MOV10 inhibited IAP RTP. It decreased significantly not only the products of reverse transcriptase but also its endogenous activity. MOV10 also associated with IAP RNA. Furthermore, although it was found in IAP virus-like particles, it did not affect their incorporation of IAP RNA, primer tRNAPhe (phenylalanine tRNA), or IAP Gag. Concerning P bodies, the exogenously expressed MOV10 had no effect on their size and number, and the inhibition of IAP RTP persisted despite the depletion of their RCK subunit. Thus, by interfering with reverse transcription, MOV10 inhibits IAP RTP, and this inhibition is independent of P bodies.

Transcriptional cyclin-dependent kinases play important roles in eukaryotic gene expression. CDK7, CDK9 (P-TEFb), and CDK13 are also critical for HIV replication. However, the function of CDK11 remained enigmatic. In this report, we determined that CDK11 regulates the cleavage and polyadenylation (CPA) of all viral transcripts. CDK11 was found associated with the TREX/THOC, which recruited this kinase to DNA. Once at the viral genome, CDK11 phosphorylated serines at position 2 in the CTD of RNAPII, which increased levels of CPA factors at the HIV 3' end. In its absence, cleavage of viral transcripts was greatly attenuated. In contrast, higher levels of CDK11 increased the length of HIV poly(A) tails and the stability of mature viral transcripts. We conclude that CDK11 plays a critical role for the cotranscriptional processing of all HIV mRNA species.

Transcriptional cyclin-dependent kinases (CDKs) regulate RNA polymerase II initiation and elongation as well as cotranscriptional mRNA processing. In this report, we describe an important role for CDK12 in the epidermal growth factor (EGF)-induced c-FOS proto-oncogene expression in mammalian cells. This kinase was found in the exon junction complexes (EJC) together with SR proteins and was thus recruited to RNA polymerase II. In cells depleted of CDK12 or eukaryotic translation initiation factor 4A3 (eIF4A3) from the EJC, EGF induced fewer c-FOS transcripts. In these cells, phosphorylation of serines at position 2 in the C-terminal domain (CTD) of RNA polymerase II, as well as levels of cleavage-stimulating factor 64 (Cstf64) and 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF73), was reduced at the c-FOS gene. These effects impaired 3' end processing of c-FOS transcripts. Mutant CDK12 proteins lacking their Arg-Ser-rich (RS) domain or just the RS domain alone acted as dominant negative proteins. Thus, CDK12 plays an important role in cotranscriptional processing of c-FOS transcripts.

Mapping host-pathogen interactions has proven instrumental for understanding how viruses manipulate host machinery and how numerous cellular processes are regulated. DNA viruses such as herpesviruses have relatively large coding capacity and thus can target an extensive network of cellular proteins. To identify the host proteins hijacked by this pathogen, we systematically affinity tagged and purified all 89 proteins of Kaposi's sarcoma-associated herpesvirus (KSHV) from human cells. Mass spectrometry of this material identified over 500 virus-host interactions. KSHV causes AIDS-associated cancers, and its interaction network is enriched for proteins linked to cancer and overlaps with proteins that are also targeted by HIV-1. We found that the conserved KSHV protein ORF24 binds to RNA polymerase II and brings it to viral late promoters by mimicking and replacing cellular TATA-box-binding protein (TBP). This is required for herpesviral late gene expression, a complex and poorly understood phase of the viral lifecycle.

Abstract Background The increasing availability of HIV-host interaction datasets, including both physical and genetic interactions, has created a need for software tools to integrate and visualize the data. Because these host-pathogen interactions are extensive and interactions between human proteins are found within many different databases, it is difficult to generate integrated HIV-human interaction networks. Results We have developed a web-based platform, termed GPS-Prot http://www.gpsprot.org, that allows for facile integration of different HIV interaction data types as well as inclusion of interactions between human proteins derived from publicly-available databases, including MINT, BioGRID and HPRD. The software has the ability to group proteins into functional modules or protein complexes, generating more intuitive network representations and also allows for the uploading of user-generated data. Conclusions GPS-Prot is a software tool that allows users to easily create comprehensive and integrated HIV-host networks. A major advantage of this platform compared to other visualization tools is its web-based format, which requires no software installation or data downloads. GPS-Prot allows novice users to quickly generate networks that combine both genetic and protein-protein interactions between HIV and its human host into a single representation. Ultimately, the platform is extendable to other host-pathogen systems.

Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.

The biogenesis of the multi-subunit vacuolar-type H⁺-ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that ATP6AP2 deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into Drosophila led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both ATP6AP2 mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in ATP6AP2 lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy.

Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl^-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.

Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane-bound compartment—the inclusion—and secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydia's intracellular life cycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, which relocalizes SNX5/6 to the inclusion membrane and augments inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.