# Visualization of Influenza Viral RNA Assembly

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2020 · $382,143

## Abstract

Influenza  A  viruses  (IAV)  pose  a  major  public  health  threat  through  both  seasonal epidemics and sporadic pandemics. The segmented nature of the viral genome promotes reassortment,  a  process  where  the  genetic  material  between  viruses  is  exchanged  in  a co-­infected cell. In nature, reassortment leads to increased viral diversity and emergence of  pandemic  influenza  viruses.  For  example,  the  2009  influenza  H1N1  (‘swine  flu’) pandemic virus, emerged from reassortment of two circulating swine viruses. Prediction of future pandemic influenza viruses from circulating zoonotic virus populations is difficult because  very  little  is  known  about  the  mechanism  of  reassortment  within  a  single co-infected cell. To accurately define the process of reassortment, we must first understand the  dynamics  of  intracellular  viral  RNA  (vRNA)  assembly.  Influenza  vRNA  replicates  in the  nucleus  and  is  transported  to  the  plasma  membrane  for  packaging,  which  requires one  copy  of  all  eight  segments  to  assemble  within  a  single  virion  to  produce  a  fully infectious  virus.  In  this  proposal,  we  will  build  upon  our  previous  data  on  influenza assembly  and  define  1)  the  assembly  dynamics  in  physiologically  relevant  human  and swine cell types, 2) the cellular proteins modulating vRNA transport, and 3) the location of reassortment within a co-­infected cell. Our central hypothesis is that vRNA assembly occurs  in  a  cell-­type  specific  manner  that  correlates  with  IAV  reassortment  in  different host  species.  The  Specific  Aims  of  this  application  will  use  a  variety  of  sophisticated microscopy  tools,  including  live  cell  imaging  with  a  custom  light-­sheet  microscope,  to determine  the  assembly  mechanism  in  various  cell  culture  models.  Aim  1  will  utilize multicolor fluorescent in situ hybridization and live cell imaging techniques to explore the dynamics of influenza vRNA assembly in human and swine differentiated airway epithelial cells.  Aim  2  will  uncover  the  identity  and  roles  of  cellular  cytoskeletal  proteins  and membranous  organelles  utilized  during  influenza  vRNA  assembly  using  biochemical approaches  like  proximity-­dependent  biotinylation.  Aim  3  will  combine  imaging  and genomic  approaches  to  characterize  the  cellular  location  of  vRNA  intermingling  during co-infection  with  two  heterologous  viruses  in  differentiated  airway  epithelial  cells.  The proposed  work  will  address  many  outstanding  questions  in  influenza  biology  regarding reassortment  that  have  remained  unanswered  due  to  a  lack  of  tools  to  track  vRNA movement in live cells during a productive infection. In addition, these studies will identify novel  host  factors  involved  in  vRNA  packaging  that  can  be  pursued  as  potential therapeutic targets.

## Key facts

- **NIH application ID:** 9973079
- **Project number:** 5R01AI139063-03
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Seema S. Lakdawala
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $382,143
- **Award type:** 5
- **Project period:** 2018-08-16 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9973079

## Citation

> US National Institutes of Health, RePORTER application 9973079, Visualization of Influenza Viral RNA Assembly (5R01AI139063-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9973079. Licensed CC0.

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