# Identification and characterization of host proteins involved in the alphavirus exit pathway

> **NIH NIH R21** · ALBERT EINSTEIN COLLEGE OF MEDICINE · 2022 · $210,000

## Abstract

Alphaviruses include important and emerging human pathogens such as encephalitic viruses and the
arthritogenic Chikungunya, Ross River, and Mayaro viruses. There are currently no licensed antiviral therapies
or vaccines, and new approaches and information are needed to develop antiviral strategies.
 Alphaviruses are small enveloped RNA viruses with highly ordered structures. The nucleocapsid core is
composed of the plus-sense RNA genome enclosed in a capsid protein shell, and is surrounded by the virus
envelope, a membrane containing an organized lattice of the E2 and E1 transmembrane proteins. There is
increasing understanding of alphavirus structure, entry, and replication, and the functions of host proteins in
these processes. In contrast, much less is known about the roles of host proteins in the late steps of the
alphavirus exit pathway, including nucleocapsid assembly/transport and virus budding at the plasma
membrane. This grant aims to use proteomics to identify host proteins that interact with the virus capsid
protein, and determine their roles in virus exit. Such an approach has been difficult because capsid protein is
highly basic in nature and interacts non-specifically with host proteins during retrieval. Tagging the capsid
protein with relatively large tags such as fluorescent proteins leads to major distortions in the organized capsid
lattice and the viral particle. Instead, we have developed an approach based on insertion of a 13 residue biotin
acceptor peptide into a permissive site on capsid. Infection of cells expressing the BirA biotinylation enzyme
leads to efficient biotin labeling of capsid and nucleocapsid in cells and virus particles. Our preliminary data
have established efficient and specific retrieval of biotinylated capsid using Streptavidin magnetic beads. We
will use this system to identify capsid-associated cellular proteins in infected cells. The specific aims are:
 1. Use a novel biotin-based capsid pull-down strategy to identify host proteins involved in
alphavirus exit. We will use our optimized methods to perform chemical crosslinking, specific biotin-based
retrieval, and elution of capsid-associated cellular proteins. Proteins will be identified by state of the art mass
spectrometry methods and prioritized for further study based on statistical and bioinformatics analyses of
samples and controls, and on protein cellular locations, pathways, and functions.
 2. Define the role of host proteins in alphavirus exit. We will test a prioritized set of candidate host
proteins for roles in the alphavirus exit pathway. Host proteins will be depleted by siRNA targeting and effects
on alphavirus primary infection and virus production determined. Proteins showing the most specific effects on
alphavirus exit will be further characterized in knock-out and overexpressing cells using our established battery
of assays for defined steps in virus exit.
 The results of these studies will provide fundamental information on host pr...

## Key facts

- **NIH application ID:** 10495264
- **Project number:** 5R21AI166194-02
- **Recipient organization:** ALBERT EINSTEIN COLLEGE OF MEDICINE
- **Principal Investigator:** MARGARET KIELIAN
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $210,000
- **Award type:** 5
- **Project period:** 2021-09-24 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10495264

## Citation

> US National Institutes of Health, RePORTER application 10495264, Identification and characterization of host proteins involved in the alphavirus exit pathway (5R21AI166194-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10495264. Licensed CC0.

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