Structural Biochemistry/Enzyme/APOBEC3G
Introduction[edit | edit source]
APOBEC3G, commonly abbreviated as A3G, is a human enzyme which acts as an effective cellular host defense factor which prohibits the expression of the functional form of the HIV-encoded protein Vif. Because the wild type of Vif targets the A3G enzyme, when the enzyme is attacked, any sort of host defense advantage that A3G may provide is severely deminished or lost. Because of this, recent studies provide clues that suggest that if compromised, the A3G enzyme may be actually promoting the emergence of more virulent strains of the HIV virus[1].
A3G is a double agent in defense, meaning that it can act as an antiviral factor, helping to cause the destruction of virus, or it can help diversify viral genomes through gene mutation, leading to the virus being more resistant to drugs. A3G needs to be incorporated into the virus for it to be effective. Vif is the HIV encoded protein that cause the destruction or degradation of A3G. Degradation of the A3G occured in the 26S proteosome. The presence of Vif can enhance the ability of a virus to infect a cell. [2].
The A3G, which has the full name of APOlipoprotein B mRNA-editing, Enzyme-Catalytic, polypeptide-like 3G, is a human enzyme encoded by the APOBEC3G gene. The enzyme belongs to the family of APOBEC superfamily of proteins. This family of proteins have important roles in innate anti-viral immunity.
Relevant Key Terms[edit | edit source]
In order for the concept of the A3G enzyme to be well understood, the following terms need to be first defined.
APOBEC[edit | edit source]
APOBEC is a family of proteins that contains a zinc-dependent deaminase motif and is named after the first enzyme that was discovered in the family (apolipoprotein B editing catalytic subunit 1). The family consists of activation-induced deaminase (AID), A1, A2, A3A-A3H, and A4. A3G and A3F are a bit different than the rest of the family of proteins because they have different nearest neighbor preferences in single-stranded DNA for deamination and are also different in interacting with Vif. A3G is more likely to the process of Vif-dependent degradation. On the other hand, A3G protein is expressed at a higher rate than the A3F protein[3].
HMM and LMM complexes[edit | edit source]
HMM stands for high molecular mass and LMM stands for low molecular mass. These two complexes are defined by the biochemical sizing methods of A3G in cell extracts. Their sizes vary, but generally, HMM are much bigger than LMM complexes. A3G is able to bind nonselectively to cellular RNA's and are also associated with a plethora of cellular proteins. On the other hand, A3G LMM has barely any RNA associations with A3G subunits.
Permissive and nonpermissive cells[edit | edit source]
This term describes whether or not a cell has the ability to undergo a productive infection by a particular retrovirus or not.
Proviral DNA[edit | edit source]
Proviral DNA is the copy of the retroviral RNA genome that is a double-stranded DNA copy.
Viral infectivity factor[edit | edit source]
The viral infectivity factor, also abbreviated as Vif, is a 21 kDa HIV-encoded protein with many domains in order to facilitate the interactions of protein-protein and protein-RNA.
Viral replication[edit | edit source]
Viral replication describes the entire viral life cycle, including but not limited to reverse transcription.
Discovery[edit | edit source]
A3G was first discovered by Jarmuz et al.[4] as a member of family of proteins APOBEC3A to 3G on chromosome 22 in 2002 and later also as a cellular factor able to restrict replication of HIV-1 lacking the viral accessory protein Vif. Soon after, it was shown that APOBEC3G belonged to a family of proteins grouped together due to their homology with the cytidine deaminase APOBEC1.
Structure[edit | edit source]
The structure of A3G is symmetrical, with two homologous catalytic domains (the N-terminal (CD1) and the C-terminal (CD2)). Each of these domains contains a Zn2+ coordination site, as well as typical His/Cys-X-Glu-X23–28-Pro-Cys-X2-Cys motif for cytidine deaminases. A3G also contains an alpha helix between two beta sheets in the catalytic domain that can potentially serve as a cofactor binding site[5].
The native form of A3G is composed of all sorts of monomers. Although it was originally thought that A3G functions as a dimer, it is possible that A3G actually functions as a mixture of monomers and oligomers[6].
There is an amino acid residue called D128 which lies embedded within the structure of A3G and is particularly important for A3G's interactions with Vif because if D128K becomes mutated, it would not be possible for Vif to completely deplete all traces of A3G[7].
Characteristics[edit | edit source]
A3G is a member of the family of cytidine deaminases named after apolipoprotein B editing catalytic subunit 1 (APOBEC1), which was the first enzyme discovered to have the capacity to have site-specific cytidine to uridine deamination of apoliproprotein B mRNA. The reason as to why a HIV protein known as the viral infectivity factor (Vif) was required for virus to infect non-permissive cells was that these cells expressed the A3G enzyme[8]. The viruses were able to penetrate the defense mechanisms of the host cell by using the Vif's to trigger the destruction of the A3G enzyme. Vif induced the degradation of A3G and neutralized the ability of A3G to hypermutate the single-stranded DNA (ssDNA) of HIV during reverse transcription after viral entry[9].
Without the presence of a functional Vif, A3G is able to catalyze dC to dU mutations, mainly in the minus strand reverse transcript, and this templates dG to dA transitions in the protein coding plus strand in viral replication. Some virions that have become mutated is then able to integrate themselves into the chromosome of the host cell, where they facilitate the expression of viral proteins by using missense substitutions or nonsense codons[10].
In a recent study, mice were humanized by injecting the human A3G enzyme into them and then infecting them with the HIV virus. The HIV genomes recovered from these mice showed a dG to dA mutation, consistent with A3G flanking sequence preference. In most cases, without the Vif, enough dC to dU mutations are produced to create a reduction in the number of proviral DNA, which is caused by the creation of abasic sites by uracil-DNA glycosylase, and then right after that DNA degradation. A3G-mediated mutations were initially thought to be only able to damage the virus because of their location in the HIV genome or their abundance.
It still remains unknown as to whether or not A3G is sufficient stop virus from replicating because from experiments, the mechanisms discovered were based on using either an over-expressed amount of A3G or a mutant of its form. So the system used in experiments are technically different from that of the human body. But it is known that with greater level of deaminase activity, it is more likely for A3G to inhibit viral replication.
[11].
Interactions with RNA[edit | edit source]
Although it is known that the A3G enzyme is not essential for cellular survival, they have important functions in their ability to facilitate the binding of RNA. More specifically, the A3G enzyme has the ability to regulate the functions of microRNA as well as suppress endogenous retroviral elements[12].
One of the most perplexing questions with regards to A3G is why a few particles of the enzyme is needed to be inserted into the viral particle in order to be able to successfully suppress it[13]. The reason is because the cellular A3G cannot enter the nucleoprotein core, where reverse transcription occurs, of the virus that it is attempting to attack without first having itself encapsidated or the ssDNA is protected from the attacking A3G. Furthermore, because A3G interacts with cellular RNA, cells might try to regulate it, thus limiting the ability of A3G to access the single-stranded proviral DNA[14].
It is quite a contradictory concept that the innate ability of A3G to be able to bind to RNA is essential for the deaminase-independent antiviral mechanism that A3G utilizes in order to bind to the viral particle, but simultaneously, inhibits A3G deaminase-dependent mutagenesis of ssDNA during reverse transcription[15]. The activity of A3G is regulated by cells by producing either the high molecular mass (HMM) of A3G, which has RNA binded to it in ribonucleoprotein complexes, or the low molecular mass (LMM) or A3G, which is relatively free of any sort of RNA. In the HMM form of A3G, the enzyme exhibits little to no antiviral activity, although Vif regulates the degradation of the enzyme in both its LMM and HMM forms.
The higher levels of deaminase activity in the LMM form of A3G suggests that the LMM form is already sufficient enough for cells to be able to be refractory to HIV. This, however, is not necessarily true. More recent studies has shown that even though all traces of LMM A3G were eliminated from the cell through either resting CD4+ T cells by RNA interference or Vif-mediated degradation of A3G, the cell itself was still able to repel an HIV infection[16]. This contradicts the theory that was previously stated, and proves that LMM A3G is not already present in cells in an abundance that is great enough to be able to effectively inhibit an HIV infection[17].
With this new hypothesis, a new question was raised: if LMM A3G is not able to defend its host cell from an impending HIV virus attack, then what, if any, is its mutagenic activity? Some studies have shown that, as opposed to fending off against a viral attack, the A3G enzyme actually has a role in helping the virus proliferate itself throughout the cell by promoting mutations that benefitted the HIV virus and inducing a phenotype of the virus that is prone to drugs. Moreover, this new data also suggests that the mutational activity of the LMM A3G enzyme might not be sufficient enough to stop the replication of the HIV virus in either its beginning or end of the life cycle.
A3G in HIV replication[edit | edit source]
a) Viral Replication During the early stage of HIV replication, the virus binds to the cell, and shred its virus coat. The HIV RNA are reservely transcribed into HIV double stranded DNA. During integration, the virus is inserted into the nucleus of the cell. Then there is transcription of the viral genomes by viral promoters. The HIV RNA is then exported to the cytoplasms. During the late stage, after more translation and processing of its RNA and protein, the virus are released from the cell.
b)b) This shows what happen when A3G is active and inactive, and the role of Vif in the degradation of A3G. In the early stage, the virus binds to the cell. The cellular TNAs and the A3G come together to form a complex. This complex is inactive and cannot interfere with replication of virus genome. After HIV replication, A3G’s LMM complex is active. Later on, there is the provirus DNA and HIV RNA inside the nucleus, with Vif being expressed. Then, Vif, other proteins, and A3G form a complex that leads to the degradation A3G.Viral proteins and HIV RNA are able to mature and exit the cell.
c) This is the current hypothesis of how A3G causes a mutation in viral genomes within the cell. A3G is incorporated into the virus and the virus binds to the cell. A3G’s antiviral activity will take over and A3G will make it so that the virus genomes are either not reversely transcribed, or the transcribed genome will be missense or nonsense.
d) This is the opinion of the author. The author thinks that A3G will lead to the mutations of viral genomes, which allows virus to be more drug resistant. He also thinks that A3G pre-exist in the cell, as opposed to the current hypothesis that they enter the cell along with the viral particles.
References[edit | edit source]
- ↑ Zhang, H. et al. (2003) The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424, 94–98
- ↑ Zhang, H. et al. (2003) The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424, 94–98
- ↑ Smith, H.C. (2009) The APOBEC1 Paradigm for Mammalian Cytidine Deaminases that Edit DNA and RNA, Landes bioScience
- ↑ Jarmuz A, Chester A, Bayliss J, Gisbourne J, Dunham I, Scott J, Navaratnam N (March 2002). An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics 79 (3): 285–96.
- ↑ Simon, V. et al. (2005) Natural variation in Vif: differential impact on APOBEC3G/3F and a potential role in HIV-1 diversification. PLoS Pathog. 1, e6
- ↑ Pace, C. et al. (2006) Population level analysis of human immunodeficiency virus type 1 hypermutation and its relationship with APOBEC3G and vif genetic variation. J. Virol. 80, 9259–9269
- ↑ Sato, K.I. et al. (2010) Remarkable lethal G-to-A mutations in vifproficient HIV-1 provirus by individual APOBEC3 proteins in humanized mice. J. Virol. 84, 9546–9556
- ↑ Chiu, Y.L. et al. (2005) Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 435, 108–114
- ↑ Chiu, Y.L. et al. (2010) Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 466, 276
- ↑ Pillai, S.K. et al. (2008) Turning up the volume on mutational pressure: is more of a good thing always better? (A case study of HIV-1 Vif and APOBEC3). Retrovirology 5, 1–8
- ↑ Albin, J.H. et al. (2010) Long-term restriction by APOBEC3F selects human immunodeficiency virus type 1 variants with restored Vif function. J. Virol. 84, 10209–10219
- ↑ Yu, X. et al. (2003) Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science 302, 1056– 1060
- ↑ Huthoff, H. and Malim, M.H. (2007) Identification of amino acid residues in APOBEC3G required for regulation by human immunodeficiency virus type 1 Vif and Virion encapsidation. J. Virol. 81, 3807–3815
- ↑ Zhang, L. et al. (2008) Function analysis of sequences in human APOBEC3G involved in Vif-mediated degradation. Virology 370, 113–121
- ↑ Santa-Marta, M. et al. (2005) HIV-1 Vif can directly inhibit apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G-mediated cytidine deamination by using a single amino acid interaction and without protein degradation. J. Biol. Chem. 280, 8765–8775
- ↑ Chen, G. et al. (2009) A patch of positively charged amino acids surrounding the human immunodeficiency virus type 1 Vif SLVx4Yx9Y motif influences its interaction with APOBEC3G. J. Virol. 83, 8674–8682
- ↑ Tian, C. et al. (2006) Differential requirement for conserved tryptophans in human immunodeficiency virus type 1 Vif for the selective suppression of APOBEC3G and APOBEC3F. J. Virol. 80, 3112–3115