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

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.

Cloning and expression in Pichia pastoris of a genetically engineered single chain antibody against the rat transferrin receptor.

The present investigation describes the construction of a genetically engineered single chain antibody (scFv) against the rat transferrin receptor (OX26), and demonstrates that this scFv antibody can be fully processed and expressed as a soluble secreted molecule in the methylotrophic yeast Pichia pastoris. Restriction endonuclease sites located at both 5'- and 3'-flanking regions of OX26 coding region in the prokaryote pOPE-OX26 vector were engineered to incorporate yeast compatible restriction endonuclease sites (i.e. EcoRI and SmaI or AvrII). The modified OX26 cDNA was subcloned into the Pichia expression vectors pPIC9 and pHIL-S1. An OX26 scFv high producer clone [GS115 His+ Mut+ (pPIC-OX26 SacI)] was isolated and used for large-scale production and characterization. Because the engineered scFv contains both a c-myc tag and a (His)5 tail, the OX26 scFv was purified to homogeneity by immobilized metal affinity chromatography. The identity of the OX26 scFv was confirmed by Western blot analyses with both anti c-myc and anti poly-His antibodies. Minor immunoreactive bands corresponding to hyperglycosylated and partially processed alpha-factor leader prosequence were also detected in the purified OX26 scFv, and these contaminants were markedly reduced when the expression of the OX26 scFv was performed in minimal methanol medium buffered with phosphate at pH = 7. The present investigation suggests that this expression system may be useful for the production of anti-receptor single chain antibodies that can be used as brain drug delivery vectors.