Respiratory infections, pink eye, or an upset stomach may seem like unrelated, mild inconveniences, but all of these ailments share one root cause: adenovirus infection. Many of us will encounter some type of adenovirus in our life and make a speedy recovery thanks to our immune systems. However, adenovirus infections can be life threatening for people with weakened immune systems like the elderly or the very young. Because they can pose such a high risk to these vulnerable populations, understanding the molecular details of how they are able to enter and hijack human cells is key to developing new drugs to fight adenovirus infection.
Adenoviruses enter human cells by touching specific receptors on cell membranes. When this contact is made, the adenovirus is cloaked in a piece of membrane and brought inside the cell. Once in the cell, the virus escapes the membrane disguise and rushes to invade the cell’s nucleus. Inside the nucleus, the virus unpacks its viral DNA and proteins. The foreign DNA and proteins hijack human cell components to create new viral molecules. When enough viral molecules are synthesized, the virus particles pack themselves up and escape from the first cell to infect other cells where the cycle continues.
Adenovirus DNA encodes genes that guarantee viral replication and survival. Adenovirus proteins escort viral DNA to the host nucleus and protect it from degradation by the host cell. One of these proteins, protein VII, is crucial to virus entry into the host cells. Without it, virus becomes trapped in their membrane cloaks and cannot establish infection. Protein VII regulates viral gene expression by changing the structure of viral and host DNA to change which genes are expressed. Tight regulation of the interactions between protein VII and viral DNA is crucial for efficient viral replication. Protein VII association with DNA is regulated by the addition of post-translational modifications (PTMs) like acetyl groups or phosphate groups at different sites on protein VII. The precise effects of these modifications are unclear. Dr. Edward Arnold, a graduate student in the Avgousti lab, led critical studies to define the purpose of these modifications in adenoviral replication.
To start, the group created adenoviruses with protein VII lacking all PTMs by mutating residues that are modified or by mutating only the first acetylation site. Next, they infected cells in a dish with normal and mutant virus to track where protein VII was localized in the cell over time. They found that both mutant versions of protein VII localize to the nucleus more quickly than proteins with correct PTMs, indicating that protein VII PTMs are crucial for timing the adenovirus entry into the nucleus. They found that the increased entry of protein VII into the nucleus was matched by an increase in transcription factor E1A levels. This indicates that protein VII entry and induction of viral gene expression depends on the addition of PTMs.
After confirming that mutant protein VII localized to the nucleus more efficiently, the group wanted to confirm that the mutations led to faster nuclear localization of the viral DNA, a crucial step to early viral infection. To do this, they stained normal and mutant viral DNA, infected cells, and tracked viral DNA location in the hosts over time. They found that viral DNA in the protein VII mutants localized to the nucleus at the same time as protein VII, indicating that nuclear entry of adenovirus genes is controlled by protein VII PTMs. Mechanistically, they found that both mutant viruses induced the expression of early viral genes more quickly than the normal virus. This is likely due to the increase in viral genes present in the host nucleus at these early timepoints, again highlighting the importance of protein VII modifications in early infection.
Despite the important role for protein VII PTMs in early infection, the group found no differences in the ability of the virus to replicate, create proteins, or produce new infectious virus. Previous literature has demonstrated that protein VII is not acetylated in virus particles. This indicates that protein VII acetyl marks likely come from host interactions with the protein. Because the PTMs are likely produced by the host, these marks could represent an underexplored antiviral defense mechanism. In the future, identifying the host machinery responsible for the viral PTMs could be leveraged to disentangle immune responses to adenoviruses and improve therapeutics targeting these common bugs.
This work was supported by grants from the NIH, the University of Washington Helen Riaboff Whiteley Fellowship, and the Magnuson Scholarship.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium Members Drs. Jason Smith and Daphne Avgousti contributed to this work.
Arnold EA, Smith JR, Leung K, Nguyen DH, Kelnhofer-Millevolte LE, Guo MS, Smith JG, Avgousti DC. 2024. Post-translational modifications on protein VII are important during the early stages of adenovirus infection. J Virol. 99:e01462-24
Kelsey Woodruff is a PhD candidate in the Termini Lab at Fred Hutch Cancer Center. She studies how acute myeloid leukemia cells remodel the sugars on their membranes to reprogram cancer cell signaling. Originally from Indiana, she holds a bachelor's degree in Biochemistry from Ball State University. Outside of lab, you can find her crocheting and enjoying the Seattle summers.
For the Media