Instrinsic Immunity & APOBEC3

What ARE APOBEC3 proteins?

APOBEC3 (A3) proteins are enzymes that are best known for their role in restricting retrovirus infection and retroelement transposition. Because these proteins are constitutively expressed in most human cells, and rapidly act against incoming retroviruses to prevent genomic integration, they constitute part of a specialized component of the innate immune system called intrinsic immunity.

There are seven A3 proteins in humans (A3A, A3B, A3C, A3D, A3F, A3G and A3H) that exhibit differential cell-localization and tissue expression patterns. Mice only have one A3 gene that functions similarly to A3G but is Vif-resistant. Despite having broadly overlapping innate immune functions, some A3 proteins display certain levels of specialization. A3A has a role in eliminating foreign DNA, and A3C is particularly effective in restricting the infection of the simian immunodeficiency virus (SIV), while A3D, A3F, A3G and A3H are the family members shown to mutate and potently restrict Vif-deficient HIV-1 (HIVΔVif) infection.

The ability of A3 proteins to deaminate cytidines into uridines in retroviral DNA replication intermediates represents an important mechanism responsible for its antiretroviral activity. A3G, as well as the other human A3 family members, are enzymes that act on single-stranded DNA (ssDNA) to deaminate cytidines (C) into uridines (U). The best-known target for A3 deamination is the minus- strand retroviral genomic cDNA. During the synthesis of the plus-strand retroviral DNA, deaminated cytidines (U) on the template minus strand direct the insertion of complementary adenosines (A), resulting in G-to-A substitutions (14). Extensive G-to-A mutations (also called hypermutation) in retroviral proviral DNA can potentially lead to the generation of premature termination codons and dysfunctional proteins resulting in non-infectious viral progeny. The specificity of A3 deamination is greatly influenced by the adjacent nucleotide sequences located immediately 5’ to the deaminated C. A3G will preferentially deaminate the second C in a 5’-CC-3’ DNA context, although sequences with a 5’-TC-3’ context can also be deaminated at lower frequencies. This phenomenon provides an explanation as to why tryptophan codons (UGG) are frequently mutated into UAG stop codons by A3G. Although the lethal mutagenesis is their main mechanism for inactivating retroviruses, A3 proteins have also been reported to restrict retroviral infection independently to their catalytic activity.

Antagonism of APOBEC3 proteins by VifA3 proteins present in a cell that is newly-infected by HIV are packaged into the capsids of progeny virions and exert their enzymatic and antiretroviral activities during proviral cDNA synthesis at the early stages of the infection. Packaging of A3 into HIV-1 virions is RNA-dependent and is mediated by the interaction with residues in the NC region of the retroviral structural protein Gag. HIV-1, however, has evolved specific defenses to prevent the packaging of A3 proteins. The viral infectivity factor (Vif) is an HIV-1 accessory protein whose cytosolic concentration increases over time. Vif binds to A3 proteins prior to their incorporation into virions and quickly promotes their degradation by the proteasome. HIV-1 particles that are released from infected cells expressing high levels of Vif are, therefore, devoid of A3 proteins and thus fully infectious.

In vivo, however, Vif activity against A3 proteins is imperfect and variable. Binding affinity of Vif to A3 proteins can fluctuate according to natural variations in the Vif protein sequence, thereby allowing for low levels of A3 proteins to enter the capsid. For instance, HIV-1 that expresses a Vif protein containing the K22H mutation fails to completely neutralize A3G and demonstrates abnormally high levels of A3-induced resistance mutations G16E and M36I in the viral protease. In this situation, sublethal levels of DNA deamination could actually promote HIV-1 sequence variations and help the virus accelerate adaptation to selective pressure, such as that imposed by antiretroviral drugs. Also, at the early stages of the infection, when Vif protein levels are still very low, A3 may escape destruction and mutate the first generations of progeny viruses that can then establish reservoirs of hypermutated proviruses. This escape can provide A3 proteins with the opportunity to mutate the vif gene and reduce its capacity to induce their degradation. These are some of the reasons that can explain why a large proportion of HIV-1 genomes in patients display extensive 5’-GG-to-AG and 5’- GA-to-AA mutations located at A3 deamination hotspots.