Viruses like HIV and influenza are constantly evolving and, because of this, they are especially good at evading the host immune system as well as anti-viral drugs. But what if a virus had an Achilles' heel—a place that didn’t tolerate mutations where an antibody or a drug could always bind?
Essential proteins have regions that are constrained by their structural and functional requirements. That is, those parts of the protein do not tolerate change very well because change would prevent the virus from replicating. It is commonly assumed that those regions of viral proteins that do not appear to change much over time are under a large amount of functional constraint. “However, this assumption has never been rigorously tested,” said Hugh Haddox, MCB graduate student in the Bloom Laboratory (Basic Sciences Division).
In a recent publication in PLOS Pathogens, scientists in the Bloom Laboratory (Basic Sciences Division) present their results that largely support this hypothesis. Their study focused on the HIV gene env, which codes for a surface protein that attaches the virus particle to receptors on the host cell, stimulating viral entry. Without a functional Env protein, short for envelope, HIV cannot infect or replicate within the host.
"The protein we're studying is HIV's most rapidly evolving protein. It's ability to change so rapidly makes it difficult for the immune system to mount an effective and long-lasting response against it,” said Haddox. So are there actually parts of Env that can’t change?
Using a technique called deep mutational scanning, Haddox generated a library of HIV virions with random codon mutations in the region of the env gene that codes for the part of the protein that is presented on the outside of the virion. Each virion had an average of ~1 codon mutation per env gene. By making a library of ~1 million virions, the authors estimate that nearly all single amino-acid mutations are represented in their library. Haddox and his colleagues generated three identical but independently synthesized libraries of HIV env. They deep sequenced each library (using molecular barcodes to reduce error rates) to estimate the starting frequency of each mutation.
To measure which amino-acid changes produced an infectious virus, they tested each strain’s ability to infect and replicate in cells cultured in a petri dish. For each round of the experiment with one of the three HIV libraries, they generated infectious viruses by transfecting 293T cells with HIV genomic plasmids encoding the mutant env genes. They then collected the virus-laden media from the initial dish of cells and moved it through two subsequent passages in a human T-cell line(see figure). In each round they also passaged wild-type (specifically, the LAI strain) virus in order to estimate and correct for the background rate of mutation that occurs during replication. Importantly, these host cells do not express detectable levels of a protein known to hypermutate HIV called APOBEC3G.
Many of their results match existing data concerning which parts of Env are most variable and which evolve less rapidly. For example a residue found to be important for receptor binding, site 457, strongly prefers to be an aspartic acid and changes are not tolerated2. Their results also estimate the mutational tolerance of a large number of sites that have never before been functionally characterized. This allowed the scientists to comprehensively examine Env’s mutational tolerance across the entire protein. When examining a set of the conserved antibody epitopes being targeted in vaccine design, they found that residues in these epitopes indeed tended to be less tolerant of mutations than other sites in Env. This finding provides rigorous support for a long-standing hypothesis – that these regions are conserved, in part because they are intolerant of mutations. Such sites would therefore be excellent targets for therapeutics.
The results are also illuminating where they differ with existing data estimating mutational tolerance from natural isolates of HIV. For example, the correlation between the mutational tolerance estimated by their results and the tolerance estimated from sequences from natural isolates is stronger for core than surface-exposed amino acids. This likely reflects the fact that core amino acids, being the biochemical and biophysical core of the protein, are required for viral replication both in nature and in the lab while many surface residues make contacts with host proteins, some of which are only present in nature (e.g., antibodies). Because their experiments were done in a laboratory setting free of the tight selective pressure of the host immune system, it makes sense that the surface residues could be more free to change in the laboratory than in a host organism.
“The results allow us to see which mutations are expected under defined selection pressures,” said principal investigator Dr. Jesse Bloom. “We can then compare these results to evolution in nature to better understand the selection pressures shaping HIV evolution in nature."
"We are also very excited about extending this technique to study other aspects of HIV biology,” continues Haddox. “For example, another graduate student in the lab is using this technique to measure the effects of all amino-acid mutations on antibody escape."
Haddox HK, Dingens AS, Bloom JD. 2016. “Experimental Estimation of the Effects of All Amino-Acid Mutations to HIV’s Envelope Protein on Viral Replication in Cell Culture.” PLOS Pathogens.
2. Olshevsky U, Helseth E, Furman C, Li J, Haseltine W, Sodroski J. 1990. "Identification of individual human immunodeficiency virus type 1 gp120 amino acids important for CD4 receptor binding." 64(12):5701-5707. PMID:2243375
See also the Fred Hutch News Article covering this work.
This research was supported by funding from the National Institutes of Health, the National Science Foundation, and the Burroughs Wellcome Fund.