# Topological Heterogeneity as an Evolved Source of Structural Diversity in Viral Polyproteins

> **NIH NIH R21** · TRUSTEES OF INDIANA UNIVERSITY · 2020 · $186,959

## Abstract

ABSTRACT
Viruses rely on the host cellular machinery for the production of their proteins and, ultimately, for the assembly
of infectious virions. In most cases, viral proteins are recognized and processed in the same manner as host
proteins. However, some viral sequence elements have evolved to distort the activity of the host machinery in a
manner that increases the structural and/ or functional diversity among translation products. Like many other
RNA viruses, alphaviruses leverage ribosomal errors to facilitate the production of multiple proteins from a single
open reading frame. This process, which is known as -1 programmed ribosomal frameshifting (-1PRF), is often
mediated by structural elements within the polyprotein mRNA. However, it was recently found that the protein
downstream of the -1PRF site exhibits a distinct topology with respect to the endoplasmic reticulum (ER)
membrane. This finding suggests the formation of distinct topological states in the nascent polypeptide is coupled
to ribosomal frameshifting. In this proposal, we seek to explore the mechanistic basis of this phenomenon and
to determine whether sequence elements in the viral polyprotein have evolved to exploit topological errors for
functional gain. Sequence-based topology predictors reveal several ambiguous topological signals within the
alphavirus polyprotein, which could potentially facilitate the formation of a distinct topology in the frameshifted
translation product. Notably, we have identified a TM domain in a position to partition into the membrane and
impose a tension on the nascent chain at the precise moment the frameshift site passes through the ribosome.
Given that mechanical tension on the nascent chain is a hallmark of-1PRF, this observation is suggestive of a
novel mechanistic link between the translocon and -1PRF. Moreover, this TM domain is somewhat polar and
partitions into the membrane with a marginal efficiency that is comparable to the frequency of -1PRF. Based on
this preliminary evidence, we hypothesize that the membrane integration of this TM domain serves as a trigger
for -1PRF. In the following, we propose a series of experiments aimed at testing this hypothesis and elucidating
the topological determinants within the polyprotein. More generally, we also seek to determine whether the
ambiguous signals in the polyprotein have evolved to promote the formation of multiple topologies during
translation. Using energetic predictors and multiple sequence alignments, we outline a novel protein engineering
approach to trap the nascent polyprotein within discrete topologies during translation. We will then evaluate the
manner in which these modifications to the topological energetics influence viral fitness in the context of both
host and vector cells. Finally, to determine whether topological heterogeneity represents a selected trait, we will
screen for revertant strains of these engineered viruses, and assess whether their sequences regenerate the
topo...

## Key facts

- **NIH application ID:** 9827536
- **Project number:** 5R21AI142383-02
- **Recipient organization:** TRUSTEES OF INDIANA UNIVERSITY
- **Principal Investigator:** Jonathan Patrick Schlebach
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $186,959
- **Award type:** 5
- **Project period:** 2018-11-20 → 2021-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9827536

## Citation

> US National Institutes of Health, RePORTER application 9827536, Topological Heterogeneity as an Evolved Source of Structural Diversity in Viral Polyproteins (5R21AI142383-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9827536. Licensed CC0.

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