For fine mapping of ppUL35, we generated UL35 mutants by pentapeptide-scanning mutagenesis, by which 15 bp encoding 5 additional amino acids was inserted into the UL35 ORF in a transposon-mediated process. (2). The tegument proteins fulfill multiple functions during the replication cycle, such as immune modulation, experiments (14,C17) revealed the extensive influence on the cellular transport machinery by HCMV that contributes to virus maturation. Tegument proteins and posttranslational modulation of cellular proteins have been implicated in the formation of the cVAC (18, 19). Inhibition or depletion of cellular proteins Besifloxacin HCl or viral proteins involved in virus maturation and/or cVAC structure formation led to a reduction of infectious virus yield (14,C18, 20, 21). The main HCMV glycoprotein complexes, gcI (gB), gcII (gM [gpUL100] and gN [gpUL73]), and gcIII (gH [gpUL75], gL [gpUL115], and gO [gpUL74]), have important functions during attachment and entry of HCMV into the host cell, in immune evasion, and as determinants of host cell tropism (22, 23). One of the most abundant HCMV glycoproteins, gB, is posttranslationally processed in the endoplasmic reticulum and the Golgi apparatus (24, 25), transported along the secretory pathway to the plasma membrane, and internalized into the early endosome. The path of internalization is at present unclear, since inhibition of endocytosis with dominant negative dynamin I had no influence on virus production (26). Finally, gB is transported to Besifloxacin HCl cVAC vesicles, dependent on the cellular connector protein PACS-1 (27). Sorting nexins (SNXs) are evolutionarily conserved from yeast to mammals and comprise a family of proteins involved in cargo recognition and sorting during retrograde transport from the early endosome to the Golgi apparatus (28). Mammalian genomes encode 33 SNXs (29). All SNXs share the phox homology domain (PX), which binds to phosphatidylinositol phosphates, which are characteristic for the early endosomal membrane network. SNXs can be further subdivided into the SNX-BAR subfamily, whose members contain the Bin-amphiphysin-Rvs (BAR) domain, which is able to sense highly curved membranes (30). The second subfamily comprises the PX-only SNXs, and the third subfamily consists of Besifloxacin HCl SNXs with different other additional protein domains. SNX1, SNX2, SNX5, SNX6, and SNX32 form the membrane deformation subcomplex of the retromer (31,C33), while the cargo-selective subcomplex formed by VPS29, VPS26A/B, and VPS35 is responsible for cargo selection (34,C36). The main function of the retromer complex is selecting cargo proteins for retrograde transport from early endosomes to the Golgi apparatus. Depletion of one member of the SNX subcomplex results in a disturbed recycling of certain cargo proteins, such as the cation-independent mannose-6-phosphate receptor (CI-M6PR), and an altered structure of the TGN (32, 33). In this study, we show for the first time a physical interaction between the HCMV tegument protein ppUL35 and SNX5. UL35 proteins negatively regulated SNX5 function, and SNX5 overexpression reduced the virus yield. Virus with mutations in UL35 produced less progeny virus, which could be alleviated by SNX5 knockdown. These changes correlated with an altered subcellular localization of gB. RESULTS Identification of SNX5 as interaction partner of UL35. To identify host cell factors interacting with the UL35 proteins, we performed a yeast (test with the Welch correction. (B) To analyze the effects of UL35 proteins on the subcellular localization of CI-M6PR, HeLa cells were transfected with expression plasmids for EYFP-UL35 or EYFP-UL35A or for EYFP-UL82 as a control. At 24 h after transfection, cells were fixed with paraformaldehyde and stained with a specific antibody directed against CI-M6PR. At least 100 cells were scored Mouse monoclonal to PPP1A for compact or dispersed localization. Besifloxacin HCl Error bars indicate standard Besifloxacin HCl deviations. Similar results were obtained in 3 independent experiments. Significance was calculated employing a two-tailed Student’s test with the Welch correction. *, 0.05; **, 0.01. Next, we investigated the localization of the cation-independent mannose-6-phosphate receptor (CI-M6PR), which in untreated cells is concentrated in the TGN. Previously, it has been published that small interfering (siRNAs) against SNX5 and SNX6 induced a dispersed localization of CI-M6PR (32, 40). To analyze whether the UL35 proteins modulate CI-M6PR localization, we transfected HeLa cells with plasmids expressing enhanced yellow fluorescent.