April 23, 2015

April’s featured paper is "Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein,” published in the Journal of Virology. The paper is published by a team from the Dai and Nakai Labs.

Adeno-associated virus (AAV) is a small virus that doesn’t cause disease in humans and has a lot of natural variants, called serotypes. These attributes make it an ideal vector for gene therapy. Because it’s been used successfully in clinical trials, the biomedical research community sees great potential in uses for AAV yet there are still parts of the viral lifecycle that scientists don’t fully understand.

By learning more about AAV’s basic biology and how it interacts with human cells, scientists hope to gain knowledge that can be used to improve vector production and gene delivery methods for use in the clinic and generally advance the application of gene therapies in the treatment of various genetic and acquired diseases such as inborn errors of metabolism, hemophilia, muscular dystrophies, retinal diseases, neurodegenerative diseases, infectious diseases and cancers. 

A paper recently published by a team from the lab of Hiroyuki Nakai, M.D., Ph.D., associate professor of molecular and medical genetics in the OHSU School of Medicine, in collaboration with Mu-Shui Dai, M.D., Ph.D, assistant professor of molecular and medical genetics in the OHSU School of Medicine, sheds new light on some of that basic AAV biology, particularly how the viral protein shell, called a capsid, gets made inside infected cells.

“I chose this month’s featured paper because this work represents an important step in understanding the biology of the adeno-associated virus, with implications for both gene therapy and for the use of AAVs as research tools,” said Mary Heinricher, Ph.D., associate dean for basic research in the OHSU School of Medicine.

The role of AAP

For the AAV viral capsid to get made, there needs to be two viral components inside the cell, the proteins that make up that shell, called VP proteins, and the assembly-activating protein called AAP.

As it turns out, AAP is a newly discovered protein from this virus. When scientists first discovered AAP, they showed that if a cell expressed only the VP proteins, those VP proteins could be found everywhere except the nucleolus and there wouldn’t be any capsid produced. But if the cells also expressed AAP then the VP proteins could concentrate in the nucleolus and assemble into capsids.

Perhaps, one group theorized, AAP acts as a scaffolding protein that binds to the VP proteins and transports them into the nucleolus where capsid assembly takes place. That same group tried to identify the nuclear localization signal in AAP because there was a region near the end of the protein that perfectly matched a known nuclear localization signal, but when they removed it, AAP was still able to get into the nucleus.

Taking it a step further

“We already knew that the capsid was assembled inside the nucleolus, so we hypothesized that the AAP protein must contain a nuclear and nucleolar localization signal that would target it to these regions in the cell,” said Lauriel Earley, a fourth-year graduate student in the Nakai lab and the first author of this paper. “Localization signals are like address labels for proteins and a protein can’t properly perform its job if it can’t get to the right place inside the cell.”

“Our lab had previously found that there were at least two regions that could act as nuclear localization signals to target AAP to the nucleus, but we also wanted to know which regions were responsible for the nucleolar localization,” Dr. Nakai added. “We identified several regions near the end of AAP, which were rich in basic amino acids. It’s been known for a long time that basic amino acids are important for both nuclear and nucleolar localization.”

The team made 30 different AAP mutants by changing the basic rich regions from positively-charged amino acids to neutral amino acids and used immunofluorescence microscopy to find out if any of those changes prevented AAP from going to the nucleus or the nucleolus.

The result? The team learned that AAP actually has five signals, each of which by itself is sufficient for bringing AAP to the nucleus and nucleolus, and that these signals all overlap and form a very complex localization signal.

What’s next

“Unlike many other nuclear and nucleolar proteins that normally have only one or two signals, why does AAP carry five nuclear and nucleolar localization signals in a redundant manner?” said Dr. Nakai. “This question still needs to be fully solved, but our further experiments described in the paper provide a clue to this question through a novel finding that there is an alternative role of the signals in maintaining the AAP protein at high levels, which is critical for effective capsid assembly.”

“These findings are especially interesting because they touch on many aspects of basic biology, such as virus-cell interactions, and because this particular virus is so important for the current state of gene therapy,” he added.

Studies on AAP have mainly been done on the AAP derived from AAV serotype 2 and little is known about AAPs that are derived from other serotypes. So the team is expanding its work to other serotypes and also following up on hints that AAP might be doing more than just acting as a scaffolding protein to identify what components, both viral and host, are actually required for capsid assembly.

• Read the paper
• Read about the Nakai Lab 
• Read about the Dai Lab

CITATION

Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein
Earley LF, Kawano Y, Adachi K, Sun XX, Dai MS, Nakai H.J
Virol. 2015 Mar 15;89(6):3038-48. doi: 10.1128/JVI.03125-14.

MORE PUBLISHED PAPERS

• Previous OHSU School of Medicine Papers of the Month
• Recent OHSU published papers

Pictured above, from left to right: Dr. Dai, Lauriel Earley, Dr. Nakai, Dr. Kei Acachi