Picture of Linda Chelico

Linda Chelico Professor Biochemistry, Microbiology & Immunology

Address
6B63, Health Sciences

Research Area(s)

  • HIV restriction factors, DNA deaminases, Mutagenesis, Enzyme mechanisms

About

Areas of Expertise

HIV restriction factors, DNA deaminases, mutagenesis, enzyme mechanisms

In the News

 

Research Interests

Our lab has two main projects that examine the host intrinsic immunity to HIV-1 infection and the origins of mutations in cancer cells. These seemingly disparate topics are unified by the enzyme family involved in both processes, the APOBEC3 family of deoxycytidine deaminases.

Retrotransposons and endogenous retroviruses have been genomic parasites in organisms throughout evolution and have contributed to both species evolution and disease. The APOBEC (Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide) family of enzymes present in their earliest form in bony fish acted as a defense to retroelements. Due to expansion of retroelements through evolution there was a corresponding expansion in the APOBEC family. The most recent expansion in placental mammals formed the APOBEC-like 3 (APOBEC3) family in response to ancient pathogenic retroviruses. Humans contain seven APOBEC3 (A3) enzymes (A3A, A3B, A3C, A3D, A3F, A3G, and A3H).

The A3 enzymes act as host restriction factors to inhibit retroelement replication through either RNA binding ability or activity as single-stranded (ss) DNA cytosine deaminases that catalyze the formation of promutagenic uracils. Our lab studies from a biochemical perspective how A3 enzymes restrict the replication of the retrovirus HIV-1.

Restriction of the replication of HIV-1 by A3 enzymes occurs through the deoxycytidine deamination activity of A3 enzymes which results in hypermutated and inactivated viral genomes. HIV can overcome A3 restriction factors by encoding the accessory protein Vif that hijacks the host ubiquitination system to induce polyubiquitination and proteasomal degradation of A3 enzymes.

Inhibition of HIV-1 by A3G that escapes Vif mediated degradation. In an HIV-1 producer cell, the HIV-1 virus infectivity factor (Vif) interacts with the cotranscription factor CBF-β and a cellular ubiquitin ligase complex to become the substrate recognition subunit of an E3 ubiquitin ligase. (1) The Vif-E3 complex recruits an E2 enzyme that transfers ubiquitin molecules to A3G, thereby signaling it for degradation through the proteasome pathway. (2) A3G that escapes this fate, either fortuitously or in the presence of a Vif-defective HIV-1 strain, can enter an assembling virus particle through interactions with RNA (host 7SL RNA or HIV-1 genomic RNA) and the nucleocapsid portion of Gag. Then, A3G travels with the HIV-1 particle to a target cell where it waits for reverse transcription of the HIV-1 genomic RNA to (-)DNA to ensue. A3G, a single-stranded DNA deaminase is able to deaminate cytosine (C) to uracil (U) in (-)DNA, which causes the reverse transcriptase to introduce guanine (G) to adenine (A) mutations upon using uracil-containing (-)DNA as a template to synthesize (+)DNA. This creates a hypermutated and likely inactivated virus. Other A3 enzymes, such as A3D, A3F and A3H, can restrict HIV replication in the same manner, although not all A3 enzymes are equally sensitive to Vif-mediated degradation.
Figure credit: Robin P. Love.

Our lab studies:

(1) The biochemistry of A3 enzymes and how A3 enzymes scan nascently produced single-stranded DNA during HIV reverse transcription for cytosines to deaminate.

(2) The biochemical interface of Vif and A3 enzymes and the biochemical determinants of Vif-mediated degradation of A3 enzymes.

Despite these benefits of A3 enzymes for suppression of retroelements, HIV-1 restriction, and even lesser characterized roles in restricting replication of other viruses, there is evidence that there is a cost to this defense system in the form of off-target A3-catalyzed deaminations that occurs in our genomes during our lifetime. This occurs when the expression of A3 enzymes occurs in the wrong cell and at the wrong time. Usually, redundant DNA repair mechanisms can remove uracils and negate most of these promutagenic lesions. However, with the development of Next Generation Sequencing technology to obtain greater sequencing depth, it is clear that many cancer genomes have a bias of A3-induced mutations at cytosines and most cancer cells or tumors show overexpression of A3B or A3H mRNA, suggesting that some uracils persist and develop into mutations.

Our lab studies:

(1)  The mechanisms by which A3 enzymes gain access to transiently single-stranded DNA in the genome.

(2)  The ability of A3 enzymes to cause cellular transformation versus providing a mutator phenotype to cancer cells.

Figure Legend:
At the replication fork, single stranded DNA in both the leading and lagging strand is protected by bound RPA. There is more RPA bound ssDNA in the lagging strand than the leading strand due to discontinuous synthesis of the lagging strand. In order for A3 enzymes to access the ssDNA formed in the lagging strand, they must be able to compete with RPA.

Recent Publications