APOBEC3C Tandem Domain Proteins Create Super Restriction Factors against HIV-1

Abstract

Humans encode proteins, called restriction factors, that inhibit replication of viruses such as HIV-1. The members of one family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; shortened here to A3), act by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A to A3H. These genes have either one or two cytidine deaminase domains, and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a “super restriction factor” that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein resulted in an even greater increase in the antiviral activity—recapitulating a similar evolution seen in A3F and A3G (double domain A3s that use only a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity but instead inhibit formation of reverse transcription products, which correlates with their ability to form large higher-order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein Vif. IMPORTANCE As a part of the innate immune system, humans encode proteins that inhibit viruses such as HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. 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 the proteins thus generated are relatively resistant to the viral antagonist Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens such as HIV.

Insights into the Structures and Multimeric Status of APOBEC Proteins Involved in Viral Restriction and Other Cellular Functions

“…This higher anti-HIV activity of S188I was attributed to the gained ability to form dimer, which is supported by the evidence from size exclusion chromatography (SEC) and cross-linking experiments [ 116 , 132 ]. This notion is further strengthened by the similarly enhanced anti-HIV activity of a forced tandem dimer through linking two A3C or two S188I variants together [ 116 , 132 , 134 ]. These results indicate that forced dimerization is sufficient to significantly increase the anti-HIV activity regardless of the A3C S188 or the I188 variant at position 188 [ 116 , 132 , 134 ].…”

“…This notion is further strengthened by the similarly enhanced anti-HIV activity of a forced tandem dimer through linking two A3C or two S188I variants together [ 116 , 132 , 134 ]. These results indicate that forced dimerization is sufficient to significantly increase the anti-HIV activity regardless of the A3C S188 or the I188 variant at position 188 [ 116 , 132 , 134 ]. This significantly enhanced anti-HIV activity cannot be accounted for by the slight increase of deaminase activity and the virion package level, which highlights the importance of dimerization of A3C in restricting HIV.…”

Potential Utilization of APOBEC3-Mediated Mutagenesis for an HIV-1 Functional Cure

“…Another interesting challenge is the creation of a “super restriction factor” to function as an inhibitor more potent than the original protein ( McDonnell et al, 2020 ). Single-domain deaminase A3C proteins have weak HIV-1 restriction activity compared with that of double-domain deaminases, e.g., A3D, A3F, and A3G proteins ( Bishop et al, 2004 ; Hultquist et al, 2011 ; Wittkopp et al, 2016 ; Anderson et al, 2018 ).…”

“…Single-domain deaminase A3C proteins have weak HIV-1 restriction activity compared with that of double-domain deaminases, e.g., A3D, A3F, and A3G proteins ( Bishop et al, 2004 ; Hultquist et al, 2011 ; Wittkopp et al, 2016 ; Anderson et al, 2018 ). Synthetic tandem double deaminase A3C proteins represent one attempt to create HIV-1 restrictive A3C proteins that are more potent than the native proteins ( McDonnell et al, 2020 ). The novel double-domain A3C proteins showed improved HIV-1 restriction activity and were largely resistant to Vif-mediated degradation.…”

Highly-potent, synthetic APOBEC3s restrict HIV-1 through deamination-independent mechanisms

“…For example, not all of the double Z domain combinations have been sampled in primates, and many combinations of A3 double domains with polymorphisms are unsampled [12]. We predicted that novel double domain combinations may prove to be more effective inhibitors of HIV-1 and we refer to these kinds of evolutionary-based variants of natural antiviral proteins with improved potency and/or escape from antagonism as "super restriction factors" [21][22][23]. Our previous study showed that duplicating the single A3Z2 domain protein A3C created an A3C-A3C tandem domain protein with increased antiviral activity relative to its single domain counterpart that was also largely resistant to degradation by HIV-1 Vif [22].…”