Assignment of the Lassa virus transmembrane domain in the prefusion and postfusion states in detergent micelles.

Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa, and recently isolated cases worldwide, there are no current therapeutics or vaccines. As such, LASV poses a significant global public health threat. One of the key steps in LASV infection is delivering its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Previously, we showed that the TM undergoes pH-dependent structural changes that result in a helical extension. Here, we provide the 1H, 15N, and 13C assignment of the LASV TM backbone in the prefusion and postfusion states. We also provide the 1H, 15N, and 13C assignment of two mutants, G429P and D432P, which prevent this helical extension. These results will help understand the role the TM plays in membrane fusion and can lead to the design of therapeutics against LASV infection.

Purification and characterization of the Lassa virus transmembrane domain.

Lassa virus (LASV) is the most prevalent arenavirus afflicting humans and has high potential to become a threat to global public health. The transmembrane domain (TM) of the LASV glycoprotein complex forms critical interactions with the LASV stable signal peptide that are important for the maturation and fusion activity of the virus. A further study of the structure-based molecular mechanisms is required to understand the role of the TM in the lifecycle of LASV in greater detail. However, it is challenging to obtain the TM in high quantity and purity due to its hydrophobic nature which results in solubility issues that makes it prone to aggregation in typical buffer systems. Here, we designed a purification and detergent screen protocol for the highly insoluble TM to enhance the yield and purity for structural studies. Based on the detergents tested, the TM had the highest incorporation in LMPG. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were utilized to confirm the best detergent system for structural studies. Through CD spectroscopy, we were able to characterize the secondary structure of the TM as largely alpha-helical, while NMR spectroscopy showed a well-structured and stable TM in LMPG. From these results, LMPG was determined to be the optimal detergent for further structural studies.

Physeal-sparing technique for femoral tunnel drilling in pediatric anterior cruciate ligament reconstruction using a posteromedial portal.

Pediatric anterior cruciate ligament (ACL) tears present a technical dilemma for orthopaedic surgeons. Multiple surgical techniques have been described to protect the distal femoral and proximal tibial physes. We present an ACL reconstruction technique performed on a 12-year-old girl with open physes who sustained an ACL tear after a noncontact twisting injury while playing soccer. A hamstring autograft reconstruction was performed by use of a posteromedial portal to drill the femoral tunnel in an all-epiphyseal fashion at the anatomic footprint of the native ACL. This case provides a new surgical technique to achieve anatomic fixation for ACL reconstruction in a skeletally immature individual using a posteromedial portal to drill a physeal-sparing lateral femoral tunnel for anatomic ACL reconstruction. This advancement may make drilling the femoral tunnel less technically challenging compared with other proposed methods while maintaining the lateral wall of the distal femur.