Assessing the importance of ubiquitination in regulation of nuclear transit of HPIV3 matrix protein

Abstract

Human Parainfluenza Viruses (HPIV) cause the common cold. One subtype, HPIV3, infects most people by 2 years old and is more likely than other subtypes to contribute to lower respiratory disease. Because of the high rates of infection, and high likelihood for complication, new interventions for HPIV3 would be of great benefit to decrease the burden of disease. A protein within HPIV3 that is a strong candidate for future intervention is the matrix (M) protein which gathers all other viral components and results in release of the virus particles from infected cells. Two amino acid motifs, the nuclear localization sequence which stretches from AA 240-265 (with K258 as the targeted Lysine) and the nuclear export sequence AA 101-121 (with 106/107 as the identified target to alter), within the M protein have been identified as important in trafficking the M protein both in and out of the nucleus. Disruption of similar regions in related paramyxoviruses show altered nuclear localization, resulting in decreased virus particle release. To bridge this knowledge gap, it is important to determine whether amino acid K258 within the NLS is a key site of ubiquitination within the HPIV3 matrix protein. The NLS and nuclear export sequence (NES) mutations have not yet been moved to the infectious clone to see whether the virus was still replication competent. To confirm this, the NES and NLS mutations should be transferred into the infectious clone. Interestingly, changing K258 in HPIV3 M did not show a clear change in ubiquitination by Western blot as seen in other viruses. Further lysine sites(there are 31 total in HPIV3 M) should be examined moving forward to determine whether ubiquitination (and the impact on nuclear transit) can be impacted as dramatically as in other paramyxoviruses.