Interactome and Ubiquitinome Analyses Identify Functional Targets of Herpes Simplex Virus 1 Infected Cell Protein 0.

Herpes simplex virus 1 (HSV-1) can productively infect multiple cell types and establish latent infection in neurons. Infected cell protein 0 (ICP0) is an HSV-1 E3 ubiquitin ligase crucial for productive infection and reactivation from latency. However, our knowledge about its targets especially in neuronal cells is limited. We confirmed that, like in non-neuronal cells, ICP0-null virus exhibited major replication defects in primary mouse neurons and Neuro-2a cells. We identified many ICP0-interacting proteins in Neuro-2a cells, 293T cells, and human foreskin fibroblasts by mass spectrometry-based interactome analysis. Co-immunoprecipitation assays validated ICP0 interactions with acyl-coenzyme A thioesterase 8 (ACOT8), complement C1q binding protein (C1QBP), ovarian tumour domain-containing protein 4 (OTUD4), sorting nexin 9 (SNX9), and vimentin (VIM) in both Neuro-2a and 293T cells. Overexpression and knockdown experiments showed that SNX9 restricted replication of an ICP0-null but not wild-type virus in Neuro-2a cells. Ubiquitinome analysis by immunoprecipitating the trypsin-digested ubiquitin reminant followed by mass spectrometry identified numerous candidate ubiquitination substrates of ICP0 in infected Neuro-2a cells, among which OTUD4 and VIM were novel substrates confirmed to be ubiquitinated by transfected ICP0 in Neuro-2a cells despite no evidence of their degradation by ICP0. Expression of OTUD4 was induced independently of ICP0 during HSV-1 infection. Overexpressed OTUD4 enhanced type I interferon expression during infection with the ICP0-null but not wild-type virus. In summary, by combining two proteomic approaches followed by confirmatory and functional experiments, we identified and validated multiple novel targets of ICP0 and revealed potential restrictive activities of SNX9 and OTUD4 in neuronal cells.

Host MOV10 is induced to restrict herpes simplex virus 1 lytic infection by promoting type I interferon response.

Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.

Peptide Presentations of Marsupial MHC Class I Visualize Immune Features of Lower Mammals Paralleled with Bats.

Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus-host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex. Notably, we found the two brushtail possum-specific insertions, the 3-aa Ile52Glu53Arg54 and 1-aa Arg154 insertions are located in the Trvu-UB*01:01 peptide binding groove (PBG). The 3-aa insertion plays a pivotal role in maintaining the stability of the N terminus of Trvu-UB*01:01 PBG. This aspect of marsupial PBG is unexpectedly similar to the bat MHC I Ptal-N*01:01 and is shared with lower vertebrates from elasmobranch to monotreme, indicating an evolution hotspot that may have emerged from the pathogen-host interactions. Residue Arg154 insertion, located in the α2 helix, is available for TCR recognition, and it has a particular influence on promoting the anchoring of peptide WPDV-12. These findings add significantly to our understanding of adaptive immunity in marsupials and its evolution in vertebrates. Our findings have the potential to impact the conservation of the protected species brushtail possum and other marsupial species.

Reconstruction of skull and dural defects using anterolateral thigh flaps: A STROBE-compliant article.

It is difficult to repair large skull and dural defects. We observed the therapeutic effects of anterolateral thigh flaps with vascular fascia lata for repairing large skull and dural defects.From December 2008 to June 2019, we repaired large skull and dural defects for 28 cases including 12 cases with scalp malignant tumor and 16 cases requiring removal of titanium mesh which had been once placed due to craniocerebral trauma. The scalp malignant tumor invaded full-thickness skull in 12 cases; and invaded cervical lymph nodes, dura mater or brain tissue in 3 cases. In the 12 cases with scalp malignant tumor, the scalp defects of 12 cm × 9 cm to 22 cm × 18 cm and skull defects of 9 cm × 7 cm to 15 cm × 12 cm after radical tumor resection were repaired using anterolateral thigh flaps of 14 cm × 11 cm to 23 cm × 19 cm with fascia lata of 10 cm × 8 cm to 16 cm × 12 cm. Postoperative radiotherapy and chemotherapy were also performed in the 3 cases with tumor metastasis. In the 16 cases requiring removal of titanium mesh, the skull and dural defects of 8 cm × 7 cm to 15 cm × 11 cm after removal of titanium mesh were repaired using anterolateral thigh flaps of 10 cm × 8 cm to 16 cm × 12 cm.In all cases, the transplanted anterolateral thigh flap with fascia lata survived after surgery and no vascular crisis occurred. During the followup of 8 months to 9 years, the flap appearance in the head-repaired area was fine, no external hernia of brain tissue occurred, the appearance of the femoral donor site was acceptable, and femoral muscle strength and movements were normal in all cases. The 12 cases with scalp malignant tumor had no local recurrence or distant metastasis.Repairing the skull and dural defects caused by radical surgery for scalp malignant tumor or removal of titanium mesh using anterolateral thigh flaps with vascular fascia lata, is effective. The appearance in the head-repaired area is fine without external hernia of brain tissue.

MicroRNA-25-3p regulates osteoclasts through nuclear factor I X.

Osteoporosis is a bone metabolic disease, characterized by loss of bone density leading to fractures. Its incidence increases with age and affects patient quality of life. Although osteoclasts play a significant role in osteoporosis, their underlying regulatory mechanisms remain unclear. In this study, we found that microRNA (miR)-25-3p negatively regulates osteoclast function through nuclear factor I X (NFIX). Overexpression of NFIX promoted osteoclast proliferation and increased the expression of the osteoclast differentiation and activity markers tartrate-resistant acid phosphatase and cathepsin K. MiR-25-3p transfection inhibited NFIX expression, which in turn inhibited osteoclast proliferation. Collectively, our results suggest that miR-25-3p promotes osteoclast activity by regulating the expression of NFIX. Therefore, targeting miR-25-3p in osteoclasts could be a promising strategy for treating skeletal disorders involving reduced bone formation.

Repair of knee deep burn wound with descending genicular artery-saphenous artery perforator flaps in elderly patients (a STROBE-compliant article).

It is difficult to repair knee deep burn wounds in elderly patients. In this study, we observed the therapeutic effects of descending genicular artery-saphenous artery perforator flaps on knee deep burn wounds in elderly patients.Between December 2013 and February 2018, we repaired knee third-degree burn wounds using descending genicular artery-saphenous artery perforator flaps of 20 × 12 cm to 23 × 13 cm in 56 elderly patients. For the patella and patellar ligament with complete necrosis, the patella and patellar ligament were completely removed, whereas for the patella and patellar ligament with partial necrosis, necrotic parts were removed first. The donor area was repaired using intermediate thickness free skin graft. The 56 patients were 76- to 85 years' old and all had unilateral knee burn.All flaps survived in the 56 patients. After the follow-up of 2 to 36 months, the flaps were excellent in texture and appearance, and exhibited sensory recovery. In the 8 patients with completely necrotic patella and patellar ligament as well as open knee joint, the weight-bearing function of knee joint was retained, which met patients' requirements of limb salvage and weight-bearing function. In the other 48 patients with partially necrotic patella and patellar ligament as well as open joint capsule, the postoperative flexion and extension of the knee joint were good.In elderly patients, it is an effective method to repair knee deep burn wounds using the descending genicular artery-saphenous artery perforator flaps.