The Peking University team discovers a new mechanism of virus-like particles, wh

2024-07-02

The results of this study are of significant importance for vaccine development. Compared to the commonly used live attenuated vaccines or inactivated vaccines, vaccines based on virus-like particles (VLPs) not only have the advantages of higher safety and easier manufacturing, but also can accommodate more antigens, thus offering the potential for stronger therapeutic efficacy, said Chen Kuangshi, a researcher at the Institute of Future Technology at Peking University.

Recently, he and his team found that the assembly density of Gag can affect the recruitment of cellular membrane proteins on the viral membrane. The higher the assembly density of Gag, the lower the enrichment of membrane proteins on the viral membrane.

Compared to the Gag assembly of viral particles, the assembly of VLPs is more loose, thus allowing for the accommodation of more antigens, which is expected to bring about stronger therapeutic efficacy.

Furthermore, compared to the commonly used live attenuated vaccines and inactivated vaccines, VLPs, due to the absence of the viral genome, have higher safety and are easier to manufacture.

The study of the different assembly mechanisms of viral particles and VLPs, and the discovery of their regulation of subsequent membrane protein recruitment, will help in the development of a vaccine platform based on VLPs.The Gag protein is a structural protein of the HIV-1 virus, as well as other retroviruses. The Gag protein is also a multifunctional protein with multiple structural domains.

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These structural domains are as follows: the Matrix domain that interacts with the cell membrane, the Capsid domain that interacts with itself, the nucleocapsid domain that interacts with RNA, and the p6 domain responsible for virus release.

The assembly of the HIV-1 virus particle is the result of extensive interactions on the cell membrane between thousands of viral structural proteins Gag and dimerized viral genomic RNA (gRNA).

Only a small portion (dozens) of Gag can specifically interact with a small special sequence on gRNA (i.e., the packaging signal Ψ), thereby mediating the precise packaging of the virus for gRNA.

The vast majority of Gag, on the other hand, interacts non-specifically (electrostatically) with the rest of gRNA, using gRNA as a long chain scaffold, which not only promotes Gag polymerization but also packages gRNA.Therefore, in addition to being able to interact with gRNA, Gag has been proven to bind with various types of RNA, such as mRNA, microRNA, and even artificially synthesized oligonucleotides.

Interestingly, in cells infected with HIV, that is, when gRNA is present, although the quantity of gRNA is much less than the cell's own mRNA, approximately 90% of the released particles carry gRNA.

In the absence of gRNA, that is, when Gag is expressed alone, Gag can randomly interact with various different cellular mRNAs and undergo polyadenylation, ultimately forming non-infectious but morphologically normal particles—virus-like particles (VLPs).

It is reported that why Gag can efficiently select viral RNA in the complex cellular environment and use it for efficient polyadenylation has always been a key biological issue in the field.

The interaction between Gag and the viral packaging signal Ψ is considered an important reason why Gag can efficiently package viral RNA even in the presence of numerous cellular mRNAs.However, is the packaging of gRNA by Gag and cellular mRNA achieved through a similar polyadenylation pathway? In this regard, the academic community remains unclear.

Understanding these two assembly processes is of great significance for basic research in RNA virology, the development of RNA drugs, and even the research and development of inactivated virus and pseudovirus vaccines.

Currently, the study of Gag assembly on the cell membrane is mainly achieved through biochemical means. However, these methods rely on cell lysis, and thus cannot dynamically explore the assembly process in situ and in real-time.

Fluorescence microscopic imaging technology can make up for this deficiency. However, current research mainly uses wide-field microscopy technology that can image target molecules in living cells.

This technology is limited by the diffraction limit of 250nm, and therefore still cannot deeply explore the specific assembly process of the virus particles with a diameter of only about 150nm.These technical issues have led to the current consensus in research that Gag can precisely package viral RNA because of the interaction between Gag and Ψ, which forms a nucleation site capable of driving assembly.

Most of the subsequent gRNA assembly processes in which Gag is involved are indistinguishable from the assembly process of Gag interacting with cellular mRNA.

Chen Kuangshi's research direction is RNA nanobiotechnology and single-molecule imaging and its application in RNA virology.

Over the past decade, he has focused on developing new imaging techniques to study the role of RNA in the assembly of the HIV-1 virus.

During this period, the main technology he has used is single-molecule localization microscopy (SMLM), which can accurately locate specific molecules at a spatial scale of 20nm.For instance, he has utilized single-molecule localization microscopy (SMLM) technology to elucidate the complete assembly process of Gag with guide RNA (gRNA), discovering that microRNAs can compete with viral RNA and cellular messenger RNA (mRNA) to bind with Gag.

This work has paved the way for intervening in the Gag assembly process and has also led to the development of RNA self-assembling nanomaterials capable of disrupting viral assembly.

These efforts have laid the groundwork for using SMLM technology to analyze the assembly patterns of viral particles and virus-like particles.

Accordingly, he proposed the idea of using SMLM to differentiate between the assembly of Gag using viral RNA and cellular mRNA.

To verify the feasibility, he and his team constructed plasmids that can express fluorescently labeled Gag and viral RNA simultaneously in cells, as well as plasmids that express only Gag.By expressing these two plasmids separately in cells and verifying that they can be properly assembled and released within the cells, the research group further utilized Single-Molecule Localization Microscopy (SMLM) technology to study the assembly behavior of Gag under the two experimental conditions mentioned above.

Specifically, they observed Gag polymers of different sizes on the cell membrane and analyzed the density of each polymer to obtain the relationship between assembly density and the size of the assembly platform.

The experimental results showed that when packaging viral RNA, the assembly of Gag is more compact and greatly depends on the interactions between Gag molecules and between Gag and RNA.

Conversely, when packaging cellular RNA, more assembly platforms are formed. However, the assembly of Gag on each platform is relatively loose, and its density does not increase with the growth of the assembly platform. This indicates that the multiple random mRNAs being packaged cannot effectively coordinate to promote the assembly of Gag.

By using time-correlated photoactivated localization microscopy (tc-PALM), HMM-Bayes, and other analytical methods, they analyzed the motion trajectory, frequency, and other parameters of single-molecule Gag, and further found that Gag is more unstable when packaging cellular mRNA.Based on these results, the research group proposed an assembly model: when Gag packages gRNA (approximately 18kb in length) existing in a dimeric form, gRNA can act as a scaffold for long chains to recruit all Gag proteins, driving the polymerization of Gag proteins in a coordinated manner to form a compact cluster.

When Gag packages multiple, relatively shorter cellular mRNAs, Gag will form smaller and relatively scattered clusters in a polymerized manner on different mRNAs. Although the assembly platform can still be formed, the tightness of this platform is lower than that of the Gag assembly platform packaging viral RNA.

Subsequently, they also demonstrated that these two assembly differences have a certain impact on the membrane components of HIV.

There is already a large amount of evidence showing that Gag polymerization can actively reshape the structure of the plasma membrane, forming a unique HIV membrane with an ordered liquid structure, which is rich in cholesterol and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], etc.

In addition, studies from other research groups have also shown that lipid reorganization mediated by Gag polymerization may also serve as a driving force, thereby helping to selectively recruit specific plasma membrane proteins that have affinity for an ordered lipid environment.However, whether the packaging of different RNAs affects the selective lipid and protein sorting process on the HIV membrane has not been explored before.

 

Based on the high density of Gag during the packaging of viral RNA, the team speculated that such assembly environment might have steric hindrance effects, thus inhibiting the virus's recruitment of other proteins to some extent.

 

Through microscopic imaging, the research group confirmed that the two assembly modes have similar effects on the remodeling of the cell membrane. However, in the Gag assembly platform packaging cellular mRNA, the enrichment level of transmembrane proteins such as MLV-Env is higher.

 

Based on this, they proposed the concept that the packaged RNA can regulate the proteome of the HIV membrane by mediating the tightness of Gag assembly.

 

Ultimately, the related paper was published in Science Advances with the title "Roles of RNA scaffolding in nanoscale Gag multimerization and selective protein sorting at HIV membranes" [1].Ying Yichen is the first author, Yang Yantao is the second author, and Chen Kuangshi is the corresponding author.

Chen Kuangshi said: "The successful publication of this paper is inseparable from the efforts of the laboratory members. Ying Yichen, the doctoral student who served as the first author, has demonstrated excellent scientific research ability and perseverance, and has basically completed all the experiments and data analysis involved in this study independently. Dr. Yang Yantao, who graduated in 2021, once participated in the establishment of the laboratory's super-resolution microscopic imaging platform, which laid an important foundation for the completion of the subject."

For this paper, the reviewer said that the result of analyzing the different assembly modes of Gag packaging gRNA and cellular mRNA at the nano level through precise microscopic imaging technology is very attractive.

HIV Gag assembles more efficiently on gRNA than on cellular mRNA, forming denser aggregates, and the formation of these aggregates has a synergistic effect.

These findings answer an unresolved question in the field: why the viral genome needs to be packaged in a dimeric form to be efficiently packaged.In addition, this discovery strongly supports the hypothesis proposed by previous studies based on biochemical and aqueous Gag assembly experiments, that is, gRNA can drive Gag assembly more effectively than cellular mRNA.

Finally, since people often do not specifically distinguish between viral particles and viroid particles when studying particles, the reviewers believe that the differences in Gag and envelope protein components of these two types of particles discovered in this study are a disruptive finding.

As mentioned earlier, gRNA is packaged into viral particles in a dimeric form, and the gRNA dimer is a condition for driving viral assembly. However, how gRNA dimers are formed in cells is still a controversial issue.

To answer this question, the team plans to develop imaging techniques that can efficiently label HIV gRNA dimers and, by combining them with super-resolution imaging techniques, to further study the mechanism of gRNA dimer formation and its role in viral assembly.

It is also reported that Chen Kuangshi found that Gag can not only interact with natural mRNA but also with artificially synthesized oligonucleotides.Based on these findings, the research team plans to develop an RNA delivery system based on pseudovirus particles (virus-like particles).

Compared to delivery systems that rely on chemical synthesis, protein-based virus-like particles have the advantages of higher safety, easier synthesis, and better controllable targeting.

In contrast to viral particles, the team found that the Gag assembly platform of virus-like particles is more loose. This allows more transmembrane proteins to be recruited to the virus-like particle membrane due to lower steric hindrance.

This phenomenon suggests that virus-like particles can accommodate more antigens. Based on this discovery, they plan to develop a series of virus-like particle systems with different antigens.

It is also reported that Chen Kuangshi was born in Taipei, Taiwan, China, with the English name: ANTONY KUANG-SHIH CHEN, where "KUANG-SHIH" is the Taiwan phonetic spelling of "Kuangshi".At the age of 15, Chen Kuangshi went to the United States to study abroad, and successively completed high school, undergraduate, and graduate education in the United States.

 

He obtained a bachelor's and master's degree from the University of California, San Diego, and a doctoral degree from the University of Pennsylvania.

 

Subsequently, he completed postdoctoral research at the National Institute of Standards and Technology and the National Institutes of Health in the United States.

 

In 2013, Chen Kuangshi returned to China and joined Peking University as a researcher, currently mainly researching RNA nanotechnology, single-molecule imaging, and HIV-1 assembly biology.

 

 

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