The APOBEC3 (A3) gene family encodes proteins that can mutate viral genomes by changing cytosines to uracils in single-stranded DNA through a deamination reaction that results in potentially lethal G-to-A mutations. The antiviral activity of A3 proteins involves a producer cell as well as a target cell. In the HIV-infected producer cell, A3 proteins get packaged into nascent HIV-1 virions that later go on to infect a target cell in which A3 is released to deaminate viral genomes during reverse transcription. To counteract A3, HIV-1 encodes for the accessory protein Vif (viral infectivity factor) that recruits cellular machinery in the producer cell to target A3 for degradation and preventing its incorporation into virions.
Throughout evolutionary history, the APOBEC3 gene family has undergone duplication, recombination, and deletion events that resulted in seven APOBEC3 genes in primates: A3A, A3B, A3C, A3D, A3F, A3G, and A3H. These seven A3 members are diverse in function but structurally can be categorized by their type and quantity of deaminase domains. In general, the most active A3 proteins have double deaminase domains. For instance, in humans, double-deaminase-domain A3D, A3F, A3G have antiviral activity against HIV-1, whereas the single domain A3C is a weak HIV-1 restrictor. However, an A3C polymorphism found at low frequency in humans that encodes for an isoleucine instead of a serine at position 188 has increased antiviral activity against HIV-1. This increased antiviral activity correlates with increased propensity to form dimers. For her thesis project, Mollie McDonnell, an MCB graduate student in the Emerman Lab (Human Biology), investigated if the antiviral activity of A3C could be improved by linking two A3C sequences in a synthetic tandem domain protein. The group recently published their findings in the journal mBio.
To investigate if the antiviral activity of A3C could be improved by duplicating A3C sequences, the researchers created two versions of the A3C double domain to account for polymorphisms found in humans at position 188. First, the investigators demonstrated that the double domain proteins A3CS188-A3CS188 and A3C I188-A3CI188 could be stably expressed and successfully packaged into HIV-1 virions in the absence of Vif. Notably, both double domains were better packaged into virions than the A3C single domain, and this increase in packaging correlated with increased antiviral activity.
Next, the investigators tested if this increased activity was due to the combined effect of two deaminase domains. To test this, they introduced inactivating point mutations at the N-terminal and C-terminal catalytic sites in the A3CS188-A3CS188 double domain, individually and in combination. Surprisingly, the inactivation of the C-terminal domain resulted in an even more potent HIV-1 restriction than the original A3CS188-A3CS188 double domain. In this case, the investigators also observed that the increase in antiviral activity correlated with increased packaging into virions. When the investigators mutated both the N-terminal and the C-terminal sites or only the N-terminal site, they found that these mutants restricted HIV-1 to similar levels as the original the A3CS188-A3CS188 double domain, showing that the synthetic tandem domain protein functions as an antiviral protein in a cytidine deaminase-independent manner.
The investigators then hypothesized that the increased antiviral activity in the A3C tandem domains might be due to a decrease in the levels of reverse transcriptase products. To test this hypothesis, they measured the copies of reverse transcriptase products in cells expressing the A3C tandem domains or the A3C single domain. They found that in the cells with A3C tandem domains contained less reverse transcription products compared to the A3C single domain. Finally, the investigators evaluated the activity of the A3C tandem domain proteins in the presence of HIV-1 Vif. The results indicated that in contrast to the A3C single domain, the A3C tandem domains were resistant to Vif degradation.
Mollie McDonnell summarized the findings of the study: “Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain, and furthermore, that these novel proteins are relatively resistant to the viral antagonist, Vif. We call this type of protein a super restriction factor because it is an evolution guided variant of a naturally occurring protein with improved antiviral activity and resistant to viral antagonism. Together, these data give insights about how nature has evolved a defense against viral pathogens like HIV.”
This research was supported by a grant from the University of Washington CMB Training Program, an NSF predoctoral fellowship, and grants from the National Institutes of Health.
UW/Fred Hutch Cancer Consortium member Dr. Michel Emerman contributed to this work.
McDonnell, M. M., Crawford, K. H. D., Dingens, A. S., Bloom, J. D., & Emerman, M. (2020). APOBEC3C Tandem Domain Proteins Create Super Restriction Factors against HIV-1. mBio, 11(2). http://doi.org/10.1128/mBio.00737-20