Cryo-EM structure of DNA polymerase of African swine fever virus.

African swine fever virus (ASFV) is one of the most important causative agents of animal diseases and can cause highly fatal diseases in swine. ASFV DNA polymerase (DNAPol) is responsible for genome replication and highly conserved in all viral genotypes showing an ideal target for drug development. Here, we systematically determined the structures of ASFV DNAPol in apo, replicating and editing states. Structural analysis revealed that ASFV DNAPol had a classical right-handed structure and showed the highest similarity to the structure of human polymerase delta. Intriguingly, ASFV DNAPol has a much longer fingers subdomain, and the thumb and palm subdomain form a unique interaction that has never been seen. Mutagenesis work revealed that the loss of this unique interaction decreased the enzymatic activity. We also found that the ß-hairpin of ASFV DNAPol is located below the template strand in the editing state, which is different from the editing structures of other known B family DNAPols with the ß-hairpin above the template strand. It suggests that B family DNAPols have evolved two ways to facilitate the dsDNA unwinding during the transition from replicating into editing state. These findings figured out the working mechanism of ASFV DNAPol and will provide a critical structural basis for the development of antiviral drugs.

NS2 induces an influenza A RNA polymerase hexamer and acts as a transcription to replication switch.

Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals.

Structure of monkeypox virus DNA polymerase holoenzyme.

The World Health Organization declared mpox (or monkeypox) a public health emergency of international concern in July 2022, and prophylactic and therapeutic measures are in urgent need. The monkeypox virus (MPXV) has its own DNA polymerase F8, together with the processive cofactors A22 and E4, constituting the polymerase holoenzyme for genome replication. Here, we determined the holoenzyme structure in complex with DNA using cryo-electron microscopy at the global resolution of ~2.8 angstroms. The holoenzyme possesses an architecture that suggests a "forward sliding clamp" processivity mechanism for viral DNA replication. MPXV polymerase has a DNA binding mode similar to that of other B-family DNA polymerases from different species. These findings reveal the mechanism of the MPXV genome replication and may guide the development of anti-poxvirus drugs.

Predicting the factors of lateral lymph node metastasis in papillary microcarcinoma of the thyroid in eastern China.

INTRODUCTION: Lateral lymph node metastasis is common in papillary thyroid microcarcinoma (PTMC). The present study evaluated the clinicopathologic characteristics and ultrasonographic (US) findings in predicting lateral LNM from PTMC in eastern China. MATERIALS AND METHODS: A total of 176 patients with confirmed PTMC by final histological examination who underwent central lymph node dissection (LND) and lateral LND were enrolled in our study. The clinicopathological and US data from the cases were analyzed retrospectively to determine the independent predictive factors for lateral LNM. Then, a scoring system was developed on the basis of independent factors. The sum of the points for individuals was evaluated for the value in predicting lateral LNM. RESULTS: Central LNM, underlying Hashimoto's thyroiditis, upper pole location, no well-defined margin and presence of calcifications were independent predictive factors for lateral LNM on multivariate analysis. Clinicopathological and US index points were statistically significant, with ≤ 2 favoring lateral LNM negativity with a sensitivity of 83.3 %, positive predictive value of 89.6 % and negative predictive value of 72.9 %. CONCLUSIONS: When the evaluation for lateral lymph nodes from a preoperative approach is inadequate or not obvious, our scoring system for prediction of lateral LNM can be another choice. Patients with clinicopathological and US index points ≤ 2 could be considered as lateral LNM negative, so more diagnostic approach is recommended for patients with clinicopathological and US index points >2.

The BRAF mutation is predictive of aggressive clinicopathological characteristics in papillary thyroid microcarcinoma.

BACKGROUND: This study analyzed the utility of BRAF mutation screening of ultrasonography-guided fine-needle aspiration biopsy (FNAB) specimens for predicting aggressive clinicopathological characteristics of papillary thyroid microcarcinoma (PTMC). METHODS: We assessed the T1799A BRAF mutation status in FNAB specimens obtained from 61 PTMC patients before undergoing operations for PTMC. We examined whether the BRAF mutation was associated with clinicopathologic characteristics in PTMC. Additionally, we reviewed the BRAF mutation status, and clinical, ultrasound (US), hematological, and pathology records of the patients and analyzed the associations between these characteristics and lateral lymph node metastasis (LNM). RESULTS: Analysis of the preoperative FNABs accurately reflected the BRAF status of the resected tissues in 19 of the 20 paired samples (95% concordance). We observed that the BRAF mutation was statistically significantly associated with multifocality, extrathyroidal invasion, lateral LNM, and advanced tumor stages III and IV. The BRAF mutation, pathologic features (central LNM), and US features (upper pole location) were independent predictive factors for lateral LNM in a multivariate analysis with odds ratios of 18.144 (95% confidence interval [95% CI], 1.999-164.664; P = 0.01), 8.582 (95% CI, 1.014-76.662; P = 0.049) and 9.576 (95% CI, 1.374-66.728; P = 0.023), respectively. CONCLUSIONS: BRAF mutation-positive PTMCs were more likely to manifest aggressive characteristics (extrathyroidal extension and LNM). The BRAF mutation screening of FNAB specimens can be used to predict aggressive clinicopathological characteristics of PTMC. Lateral neck nodes should be meticulously analyzed for cases of PTMC demonstrating the following three characteristics: BRAF mutation, central LNM, and US features in the upper pole location.