| dc.description.abstract | The human apolipoprotein B mRNA editing enzyme catalytic peptide-like 3 (APOBEC3; A3) family of proteins are a family of seven cytidine deaminases (A3A, A3B, A3C, A3D, A3F, A3G and A3H) that restrict certain viral infections. These innate defense factors are best known for their ability to restrict the replication of human immunodeficiency virus type 1 (HIV-1) lacking a functional Vif protein (HIV-1Δvif) through the deamination of cytidine residues to uridines during reverse transcription, ultimately leading to lethal G-to-A changes in the viral genome. The best studied of the A3 proteins has been A3G because of its potent activity against HIV-1Δvif. However, one member of this family, A3A, has biological properties that make it unique among the A3 proteins. Despite potent restriction activity when expressed in myeloid cells, human A3A (hA3A) is generally known lacking inhibitory function towards HIV-1Δvif. Previous studies in our laboratory have shown that rhesus macaque A3A (rhA3A) is capable of restricting both simian–human immunodeficiency virus lacking vif gene (SHIVΔvif) and human immunodeficiency virus lacking vif gene (HIV-1Δvif) to a greater extent than hA3A. Chimeric studies by substituting amino acids 25–33 (AC loop 1 region) from rhA3A into hA3A showed that this region is critical in determining the restriction pattern against lentiviruses. Further studies identified three Old World Monkey A3As that were able to restrict HIV-1 replication. Restriction assays with various A3As from hominids, New World monkeys, and Old World monkeys found that agmA3A and colA3A restricted the replication of SHIVΔvif and that all three Old World monkey A3A proteins (agmA3A, colA3A, and rhA3A) restricted HIV-1Δvif to some extent. More impressively, it is also showed that the A3A protein from the black and white colobus monkey (mantled guereza; Colobus guereza; colA3A) potently inhibited not only HIV-1Δvif replication but also wild-type HIV- 1 in producer cells. Since previous studies indicated the importance of AC loop 1 region in determining the restriction ability and our sequence alignment analysis showed that A3A proteins from Mandrillus sphinx (mndA3A) and Cercopithecus neglectus (debA3A) had an AC-loop1 region that was almost identical to colA3A, we tested the restriction ability of mndA3A and debA3A. The restriction assay showed that both debA3A and mndA3A restricted HIV-1 similar as colA3A. Next, we used chimeric protein and amino acid deletion studies to show that AC loop 1 region, especially the 27WVS29 motif, is the key determinant for HIV-1 and AAV-2 restriction. First, by fusing the first 100 amino acids of colA3A and the C-terminal region of hA3A (h1–100 colA3A), or the first 100 amino acids of hA3A and the C-terminal region of colA3A (col1–100hA3A), we found that the N-terminal domain of colA3A contains the determinants for HIV-1 restriction. To further confirm that, we truncated the C-terminal off and constructed the mutant proteins that expressed only the N-terminal 100 amino acids (colA3A1–100, mndA3A1–100, debA3A1–100). The truncated colA3A, mndA3A and debA3A proteins were all able to restrict HIV-1 replication. We then narrowed the region down by examining chimeric proteins with the N-terminal 75, 50 and 33 amino acids of colA3A fused to hA3A (col1–75hA3A, col1–50hA3A, and col1–33hA3A). The results indicated that the first 33 amino acids harbor the critical determinants for HIV-1 restriction. As previous experiments indicate the importance of the AC loop 1 region, a chimeric hA3A containing the AC Loop1 region of mndA3A (mnd25–33hA3A) was made. Restriction assays revealed that the mnd25–33hA3A chimera was able to restrict HIV-1, indicating that the AC Loop1 region of mndA3A was critical to its restriction properties. As the hominids have a three amino acid deletion in this region of A3A, we constructed a chimeric hA3A with the WVS motif and found that this insertion completely restored the antiviral activity of hA3A. In addition, insertion of “WVS” or AC Loop-1 region into hA3A also changed the restriction pattern of adeno-associated virus 2 (AAV-2). Therefore, we conclude that this WVS motif is the key for both retrovirus and parvovirus restriction. As we found that the WVS motif in the AC loop 1 region plays a key role in HIV-1 restriction, we determined the role of each of these three amino acids in inhibiting HIV-1 replication. We constructed a series of single amino acid substitutions and single/double deletion mutants in this region and performed restriction assays on these mutants. The results suggested that the nature (charge and hydrophobicity) of the three amino acids was not as important as the length for restriction of HIV-1. Finally, we confirmed this by substituting the WVS motif with either AAA or GGG, and found that both hA3A[27AAA29] and hA3A[27GGG29] were able to inhibit HIV to the level similar as hA3A[27WVS29]. Finally, we explored the mechanism of hA3A[27WVS29] restriction of HIV-1 replication. First, we examined the viral replication in producer cells. We determined the viral protein synthesis in producer cells, using p24 and pulse-chase analysis, were similar in controls and cells expressing hA3A[27WVS29]. Next, we excluded the possibility of subcellular localization contributing to defective viruses production as hA3A and hA3A[27WVS29] were both expressed in cytoplasm and the nucleus at similar level in producer cells. Further, we examined the viral RNA packaging and found that viral RNA was packaged at similar levels in in progeny viruses from both control and hA3A[27WVS29] expressing cells. In addition, we showed that the viral reverse transcriptase enzyme activity of the progeny viruses was not affected by the presence of hA3A[27WVS29]. We next examined restriction in target cells. We first confirmed that the viral replication was inhibited in target cells by showing that the viral protein biosynthesis was significantly lower in target cells using pulse-chase experiments. We also excluded that canonical hypermutation contributed to the restriction. We found the levels of early and late reverse transcription products were decreased approximately 10-fold in the presence of hA3A[27WVS29]. This was also reflected in decreased viral integration and viral transcripts in the target cell. Thus, we concluded that the restriction of HIV-1 by hA3A[27WVS29] occurred at the level of reverse transcription in the target cells. In summary, we were able to restore the ability of hA3A to restrict HIV-1 and abolish the ability of hA3A to restrict AAV-2 by inserting only three amino acids in the AC loop 1 region. This reveals that the AC loop 1 region harbors key determinants for both retrovirus and parvovirus restriction, and provides promising preliminary data for further translational application such as gene therapy. | |
