Polymerase-tagged respiratory syncytial virus reveals a dynamic rearrangement of the ribonucleocapsid complex during infection
Authors: Blanchard, Emmeline L.; Braun, Molly R.; Lifland, Aaron W.; Ludeke, Barbara; Noton, Sarah L.; Vanover, Daryll; Zurla, Chiara; Fearns, Rachel; Santangelo, Philip J.
Author summary Respiratory syncytial virus is a common respiratory disease among children under the age of five. Yearly, approximately three million children infected require hospitalization for treatment. However, no vaccine or effective antiviral treatment for respiratory syncytial virus yet exists. Understanding how respiratory syncytial virus replicates in cells will provide key knowledge for the development of more effective countermeasures. Here, we investigated the interactions between the large protein subunit of the respiratory syncytial virus polymerase, which is responsible for viral mRNA synthesis and replication, and other viral proteins during infection, as well as the localization of the large protein subunit over time in key viral structures. Our study provides new insights into the localization and activity of the respiratory syncytial virus polymerase, and suggests ribonucleocapsid complex rearrangement plays a key role in the function of the respiratory syncytial virus polymerase in replication of the virus, leading to a better understanding of respiratory syncytial virus biology. The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 fromin vitrotranscribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.
Ubiquitin-Conjugating Enzyme E2 E Inhibits the Accumulation of Rice Stripe Virus inLaodelphax striatellus(Fallen)
Authors: Li, Yao; Zhou, Ze; Shen, Mi; Ge, Linquan; Liu, Fang
The ubiquitin-proteasome system (UPS) is an essential protagonist in host-pathogen interactions. Among the three classes of enzymes in the UPS, ubiquitin-conjugating enzyme E2 plays a dual role in viral pathogenesis; however, the role of insect E2s in interactions with plant viruses is unclear. Twenty E2-encoding genes inLaodelphax striatellus, the small brown planthopper, were identified and classified into 17 groups by transcriptomic and phylogenetic analysis. Full-length cDNAs of fourLstrE2s(LstrE2 A/E/G2/H) were obtained by rapid-amplification of cDNA ends (RACE-PCR) analysis. Expression of the fourLstrE2sshowed tissue- and development-specific patterns. RT-qPCR analyses revealed that Rice stripe viruse (RSV) infection increased the level ofLstrE2 A/E/G2/H. Further study indicated that repression ofLstrE2 Evia RNAi caused significant increases in the expression of RSV coat protein mRNA and protein levels. These findings suggest that LstrE2 E inhibits RSV accumulation in the planthopper body. Understanding the function of LstrE2 E in RSV accumulation may ultimately result in the development of novel antiviral strategies.