Conserved cysteine residues in Kaposi's sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

Kaposi's sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta- and gammaherpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gammaherpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the four cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components. IMPORTANCE: The initiation of late gene transcription is universally conserved across the beta- and gammaherpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the four conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production to demonstrate for the first time that these cysteines are pivotal for such functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.

Suprapterional keyhole approach for anteromedial skull base lesions: How I do it.

BACKGROUND: For a minimally invasive treatment approach to the anteromedial part of the anterior cranial fossa (ACF), a small incision and craniotomy of the posterolateral part of the ACF are preferable. METHOD: We described the concept and technique of suprapterional keyhole approach (SPKA), which uses an exoscope and endoscope to treat ACF lesions. CONCLUSION: The SPKA enables ACF observation from the lateral direction; the endoscope's extended viewing angles enable the observation of the anteromedial part of the ACF, including the bilateral olfactory groove. Facial skin and large scalp incisions are avoided, making this approach efficient for ACF lesions.

A rare case of extraluminal interstitial pregnancy treated with laparoscopic cornuotomy.

We report an extremely rare case of an extraluminal interstitial pregnancy. A 36-year-old nulliparous woman visited our hospital during the fifth week of gestation. Although no intrauterine gestational sac (GS) was identified, transabdominal ultrasonography revealed a GS-like cyst was detected in the right uterine horn. She underwent laparoscopic surgery for a suspected interstitial ectopic pregnancy. After laparoscopic cornuotomy, dye leakage was observed from the fimbria rather than the incision site. Finally, the patient was diagnosed with a right extraluminal interstitial pregnancy. Hysterosalpingography performed at three postoperative months revealed bilateral tubal passage. She conceived 7 months after surgery, with safe delivery by elective cesarean section at 38 weeks.

Analysis of the interaction between the ORF42 and ORF55 proteins encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. The tegument is a structure that is unique to herpesviruses that includes host and viral proteins, including the viral ORF42 and ORF55 proteins. Alphaherpesvirus tegument proteins have been well studied, but much is unknown regarding KSHV. Here, we report an interaction between the ORF42 and ORF55 proteins. ORF55 interacted with and recruited ORF42 from the nucleus to the cytoplasm. When ORF42 and ORF55 were expressed simultaneously in cultured cells, the expression level of these two viral proteins was higher than when either was expressed independently. ORF55, but not ORF42, was polyubiquitinated, suggesting that an unidentified regulatory mechanism may be present. A recombinant virus with an ectopic stop codon in ORF42 exhibited normal replication of genomic DNA, but fewer virus particles were released with the recombinant than with the wild-type virus. A unique R136Q mutation in ORF42, which is found in a KSHV strain that is prevalent on Miyako Island, Okinawa Prefecture, Japan, further increased the expression of ORF42 and ORF55 when these proteins were expressed simultaneously. However, the ORF42 R136Q mutation did not affect the localization pattern of ORF42 itself or of ORF55. In addition, experiments with a recombinant virus possessing the ORF42 R136Q mutation showed lower levels of production of the mutant virus than of the wild-type virus, despite similar levels of genome replication. We suggest that the R136Q mutation in ORF42 plays an important role in ORF55 protein expression and virus production.

Sofosbuvir Suppresses the Genome Replication of DENV1 in Human Hepatic Huh7 Cells.

Dengue virus (DENV) causes dengue fever and dengue hemorrhagic fever, and DENV infection kills 20,000 people annually worldwide. Therefore, the development of anti-DENV drugs is urgently needed. Sofosbuvir (SOF) is an effective drug for HCV-related diseases, and its triphosphorylated metabolite inhibits viral RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of HCV. (2'R)-2'-Deoxy-2'-fluoro-2'-methyluridine (FMeU) is the dephosphorylated metabolite produced from SOF. The effects of SOF and FMeU on DENV1 replication were analyzed using two DENV1 replicon-based methods that we previously established. First, a replicon-harboring cell assay showed that DENV1 replicon replication in human hepatic Huh7 cells was decreased by SOF but not by FMeU. Second, a transient replicon assay showed that DENV1 replicon replication in Huh7 cells was decreased by SOF; however, in hamster kidney BHK-21 cells, it was not suppressed by SOF. Additionally, the replicon replication in Huh7 and BHK-21 cells was not affected by FMeU. Moreover, we assessed the effects of SOF on infectious DENV1 production. SOF suppressed infectious DENV1 production in Huh7 cells but not in monkey kidney Vero cells. To examine the substrate recognition of the HCV and DENV1 RdRps, the complex conformation of SOF-containing DENV1 RdRp or HCV RdRp was predicted using AlphaFold 2. These results indicate that SOF may be used as a treatment for DENV1 infection.

Exo- and endoscopic lateral orbital wall approach for the medial temporal lobe glioma: how I do it.

BACKGROUND: The endoscopic transorbital approach provides a direct access to the medial temporal lobe (MTL). However, when excising a highly vascular tumour, a wider access route that enables the concurrent use of standard neurosurgical instruments with both hands is preferable. METHOD: We described the concept and technique of the lateral orbital wall approach (LOWA), which comprises orbitotomy and mini-craniotomy to treat MTL lesions using an exoscope and endoscope. CONCLUSION: The LOWA provides a safe and natural surgical corridor to the MTL and enables 2- or 3-hand surgery. Hence, LOWA can potentially improve safety and efficiency to treat MTL lesions.

Pruba de anglutinación indirecta de partículas de gelatina, un medio simple y sensible para el serodiagnóstico de la de la enfermedad de Chagas / Annual Reports 1992 / The gelatine praticle indirect agglutination test, a means of simple and sensitive serodiagnósis of Chagas' disease

An indirect agglutination test using Trypanosoma cruzi antigen-coated gelatin particles was employed to Trypanosomiasis in Paraguay. Results with this test were quite comparable to those obtained with enzyme-linked immusorbent assay (Elisa). Furthermore, nonspecific reaction to the gelatin particles alone was not found in either acute or chronic infection. This method is more convenient than the ELISA, since the antigen-conjugate particles are stable for at least 1 year at 47 ºC and the test itself is short and simple to perform and does not require specialized equipment

Caracterización de un anricuuerpo monoclonal espicífico para el Trypanosoma cruzi / Annual Reports 1992 / Characterization of a monoclonal antibody specific to Trypanosoma cruzi

Amonoclonal antibody (MoAb), designated TCY-3, against Trypanosoma cruzi was obtained from spleen cells of BALB/c mice immunized with crude soluble antigens of T. curzi trypomastigotes. This MoAb Showed no cross-reactivity with 12 other heterologous parasite antigens, including promastigotes of Leishmania amazonensis and L. panamensis. Moreover, this MoAb failed to react with various organs of mouse and rat, including brain and heart. The molecular wieght to the molecule recognized by this MoAb is approximately 53 kD. Its determinant is likely protein nature, since it is sensitive to trypsin but not to periodate. Sera from mice chronicaly infected with T. cruzi reacted with the 53 kD molecule, which suggests that TCY-3 may be useful in the purification of T. cruzi- Specific antigen