Jennifer Doudna (UC Berkeley Professor) – The fate of viral genomes (Jun 2013)

Chapters

Abstract

Unraveling the Mysteries of Nucleic Acid Sensing and HIV Replication: Pioneering Insights from Doudna’s Lab

In the field of molecular biology, Jennifer Doudna’s lab stands as a beacon of innovation, particularly in understanding how cells detect foreign nucleic acids and the intricacies of HIV replication. Central to this exploration is the discovery of host cell mechanisms for sensing viral DNA, epitomized by the study of the C6-ORF150 gene, now identified as the human ortholog of mouse cGAS, and the groundbreaking research into the HIV Rev-RRE complex. This updated article delves into the latest findings from Doudna’s lab, emphasizing the structural comparison of C-gas and OAS enzymes, the unique cyclic dinucleotide chemistry of C-gas, and the application of shape chemistry in analyzing RNA structures, specifically in the context of HIV’s replication cycle. The article also incorporates additional information on the molecular mechanisms underlying viral DNA and RNA detection in human cells, the unexpected discovery of a DNA sensor, the crystallization of cGAS, and insights into HIV RNA structure and the REV-RRE complex.

Host Cell Detection of Foreign DNA:

Jennifer Doudna’s lab has made significant strides in understanding how human cells detect viral DNA. Interferon-inducible transmembrane proteins and effector molecules play crucial roles in recognizing foreign DNA and RNA molecules. The detection of DNA in the cytoplasm has been less clear, with many potential DNA sensor molecules identified but not thoroughly validated.

A notable finding is the identification of the C6-ORF150 protein as the human ortholog of mouse cGAS, challenging previous assumptions about its function. This protein plays a pivotal role in the DNA sensing pathway, synthesizing a unique cyclic dinucleotide in response to double-stranded DNA. The crystal structure of human cGAS reveals a distinctive zinc ribbon structure, critical for DNA recognition and signaling in the interferon response pathway.

Unexpected Discovery of a DNA Sensor:

Philip Kranzich, a postdoc in Doudna’s lab, initially studied genes stimulated by interferon in response to viral infection. He focused on a gene named C6-ORF150, predicted to encode an enzyme involved in RNA maturation. Upon purification, the protein bound to DNA instead of RNA, prompting further investigation.

A paper from the James Chen Laboratory identified an enzyme, cyclic GMP AMP synthase (cGAS), involved in the DNA-sensing pathway. cGAS connects DNA recognition to the activation of STING, an ER-associated protein that stimulates the interferon response. Doudna’s lab realized that the human ortholog of cGAS was the protein they had been studying, C6-ORF150.

Confirmation of cGAS Activity:

Philip Kranzich conducted experiments to determine if his enzyme possessed cGAS activity. Using thin-layer chromatography, he demonstrated that the purified human cGAS enzyme synthesized a cyclic dinucleotide in response to double-stranded DNA. The enzyme exhibited selectivity for double-stranded DNA, showing no activity with single-stranded DNA or RNA.

Crystal Structure of cGAS:

Kranzich successfully crystallized and solved the crystal structure of cGAS at 2.5 angstrom resolution. The structure revealed an overall architecture similar to other nucleotidal transferases. A prominent zinc ion was located in a non-canonical zinc finger or zinc ribbon structure, positioned at one end of a cleft.

Structural Comparison of C-gas and OAS Enzymes:

C-Gas and OAS enzymes share a similar architecture, but their ligand-binding properties and functions differ markedly. The unique zinc ribbon motif in C-gas, absent in OAS, is instrumental in distinguishing the types of nucleic acids they recognize, highlighting the specificity of cellular response mechanisms.

Cyclic Dinucleotide Chemistry:

The study of cyclic dinucleotide chemistry in C-gas has revealed its role in synthesizing a distinctive cyclic dinucleotide, crucial in the interferon response pathway. This finding has profound implications for our understanding of cellular defense mechanisms against pathogens.

Zinc Ribbon and Nucleic Acid Recognition:

The zinc ribbon structure in C-gas is vital for its ability to recognize double-stranded DNA, a function critical for triggering interferon signaling. Mutations in this region have been shown to impair DNA binding and downstream signaling, underscoring its importance in innate immunity.

Viral RNA Folding and Trafficking:

The lab is also exploring the folding and trafficking of viral RNA molecules within cells. This research is crucial for understanding viral replication and pathogenesis, with Doudna’s team investigating the interaction of viral RNA with cellular machinery. Such insights are not only academically fascinating but also have practical implications in the development of antiviral therapies.

Shape Chemistry for RNA Structure Analysis:

Utilizing shape chemistry to modify RNA molecules has allowed Doudna’s lab to study the structure and dynamics of RNA, particularly in the context of HIV. This technique has proven instrumental in understanding the interaction between the HIV-RRE rev response element and the REV protein, shedding light on viral RNA trafficking and assembly processes.

Challenges and Future Directions:

While Doudna’s lab has made substantial contributions to our understanding of nucleic acid sensing and HIV replication, challenges remain. Isolating specific strands of HIV RNA and understanding the in vivo dynamics of the REV-RRE interaction are areas that require further exploration. Collaborative efforts with experts like Alan Frankel, David Booth, and Howard Chang, among others, are crucial for advancing this research frontier.



Jennifer Doudna’s lab’s groundbreaking research into nucleic acid sensing mechanisms and HIV replication provides invaluable insights into viral infection and immunity. These findings not only enhance our understanding of molecular biology but also pave the way for the development of novel antiviral strategies, marking a significant step forward in the fight against viral diseases.

Incorporations of Supplemental Information:

HIV and the Rev-RRE Complex:

Doudna’s research also extends to HIV, particularly the challenge the virus faces in evading the host’s immune response. The study of the Rev-RRE complex in HIV-infected cells offers insights into the virus’s replication cycle and potential therapeutic targets.

Shape Chemistry and High-Throughput Sequencing to Study RNA Structure and Protein Interactions:

Jennifer Doudna’s lab has employed shape chemistry to modify RNA molecules, revealing exposed and dynamic regions of their structure. This approach, coupled with high-throughput sequencing, enables the study of RNA structure and protein interactions in a comprehensive manner. This methodology has been applied to investigate the interaction between the HIV-RRE rev response element and the REV protein, providing insights into the viral RNA assembly process.

Deeper Understanding of REV-RRE Interaction Dynamics:

Further research is needed to understand the dynamics of the REV-RRE interaction in vivo. Doudna’s team aims to analyze the assembly of the REV-RRE complex in cells and compare it with in vitro observations to uncover potential differences and changes between the two environments.

Investigating RRE Interacting Partners and Structural Changes:

The research team also plans to identify other RRE interacting partners within cells and examine structural changes in the REV-RRE complex before and after nuclear export. This will provide a more comprehensive understanding of the assembly and dynamics of the viral RNA complex.

Collaborations and Contributors:

Doudna acknowledges the contributions of James Berger, Philip, and Amy to the sea gas project and Yoon and Akshay for their work on the HIV project. She also recognizes collaborations with the Vance and Hammond labs and support from the Hark Center, Center for RNA Systems Biology, and Howard Hughes Medical Institute.

Notes by: Rogue_Atom