Membrane fusion is involved in many biological events. As a result, two independent compartments, each enclosed in its own membrane, merge to form one compartment. Invasion of a host cell by enveloped viruses such as the human immunodeficiency virus type-1 (HIV-1), influenza virus, Ebola virus, and severe acute respiratory syndrome corona virus (SARS CoV) also relies on membrane fusion. During viral membrane fusion, the envelope proteins of viruses undergo substantial time-dependent conformational rearrangements. To elucidate the mechanisms of such dynamic viral membrane fusion processes, a method for real-time and quantitative monitoring of membrane fusion is essential.

A simple, easy-to-perform, cell-based membrane fusion assay system in which a positive reporter signal is produced only when membrane fusion takes place, has been developed by ProfessorMatsuda1 and colleagues from theChina-Japan Joint Laboratory of Structural Virology and Immunology, and is reported in a recent paper in the Journal of Biological Chemistry2. Their new assay system involves a pair of novel reporter proteins called dual split proteins (DSPs); split Renilla luciferase (spRL) and split green fluorescent protein (spGFP). spGFP not only facilitates stronger association of spRL but also provides a way to visually monitor the process of membrane fusion. Use of a membrane permeable substrate for RL allows researchers to monitor membrane fusion in a quantitative manner. Both GFP and RL modes of the DSP assay can be performed in living cells.

Prof Matsuda’s group applied this new assay system to investigate the dynamic nature of membrane fusion induced by the HIV-1 envelope glycoprotein (Env). They analyzed HIV-1 Env mutants whose membrane-spanning domains had been replaced with those derived from foreign proteins such as glycophorin A or vesicular stomatitis virus G-protein. Membrane fusion in these mutants showed a slower kinetics. The analysis of membrane fusion in the presence of the fusion inhibitors, soluble CD4 and C34, revealed that the mutants had a prolonged period of sensitivity to the inhibitors. Kondo et al. concluded that these mutations within the membrane-spanning domains exerted an allosteric effect on the HIV-1 Env, probably affecting receptor-induced conformational changes of the ectodomain of the protein.

1. Professor Matsuda’s group studies the structure-function relationship of viral proteins derived from HIV-1. HIV-1 is the causative retrovirus of acquired immunodeficiency syndrome (AIDS). One of the major targets of their current investigations is Env and its mechanism of membrane fusion. As reported here, they are employing protein-engineering techniques to develop versatile reporter proteins useful for analysing the processes of the viral life cycle.

2. Naoyuki Kondo, Kosuke Miyauchi, Fanxia Meng, Aikichi Iwamoto and Zene Matsuda (2010): Conformational Changes of the HIV-1 Envelope Protein during Membrane Fusion Are Inhibited by the Replacement of Its Membrane-spanning Domain. Journal of Biological Chemistry 285(19): 14681–14688.

Interview with Professor Zene Matsuda,  July, 2010

1. Professor Matsuda, tell us about the research focus of your group.  My group is part of the China-Japan Joint Laboratory of Structural Virology and Immunology which was established in 2006 as a joint program between the Institute of Medical Science at the University of Tokyo and the Institute of Biophysics. Our aim is to build strong partnerships between Japanese and Chinese scientists in the area of emerging and re-emerging infectious diseases. My group focuses on structure-function relationships in viral proteins and places strong emphasis on structural analysis. Currently we are particularly interested in viral proteins from HIV-1, especially the envelope protein (Env) and the core proteins.

I’ve been working on the HIV virus since 1987. HIV-AIDS is a global health issue. It is estimated that there are about 35 million people living with HIV/AIDS. It is difficult to obtain accurate statistics, but estimates suggest that there are about 10,000 people living with HIV/AIDS in Japan and about 0.7 million in China. Whereas in the early days the prognosis for individuals with AIDS was very poor, there are currently good anti-viral treatments available. In an ideal situation, such as if an individual’s infection is detected early enough and they adhere to a drug-treatment program without experiencing any deteriorating side effects, life expectancy can be extended several decades post-infection. There is currently no vaccine available, and development of a vaccine seems a difficult target since the rate of viral mutation is very high.

The HIV virus is a retrovirus. Once cells are infected, viral RNA is reverse transcribed into DNA. The DNA can then be integrated into the nuclear DNA of the host cell with a viral enzyme called integrase and thus become part of the genome which is replicated at every cell division. Once the DNA is integrated into the genome it is very difficult to eradicate. HIV-AIDS is a chronic disease with no specific initial symptoms. Detecting the infection early on, therefore, is important to ensure successful treatment.

One challenge in the design of treatment or prevention measures is that the mutation rate of the virus is high. This has resulted in the development of 9 major sub-types and their recombinants in different regions. The sequences of these sub-types can differ by as much as 20-30%. So called drug resistant viruses are also easily generated by this great diversity. It is thus important to know what kind of viruses an individual is infected with in order to choose an effective treatment strategy. To combat the effects of viral diversity we need to understand the virus better so that we can identify further drug targets. One recent target that researchers are interested in is integrase – this is the enzyme that is involved in splicing the viral genome into the host cell genome. Researchers believe that further analysis will reveal other potential drug targets.

2. What are the main breakthroughs/achievements your research group has made in the last three years?

1.　　  We have developed a new assay system for studying membrane fusion (see below).

2.　　  We have improved the function of split GFP proteins by varying the site at which the molecule is split to between the 7th and 8th b-sheets rather than between the 10th and 11th b-sheets.

3. You’ve recently published a paper in the Journal of Biological Chemistry[J1] . Where does this fit in the work your lab is doing? The initial event in the HIV infection process is fusion of the viral membrane with the host cell membrane. Inhibitors targeting this process are available, but they are peptide-based drugs and their recognition by the immune system may be a problem. If we can understand the membrane fusion process more fully, we should be able to design other effective strategies for targeting this process. However, studying the membrane fusion process in detail has been difficult because of the lack of good assay systems. We need a system that allows continuous monitoring of the process that is simple, rapid, and safe. This paper describes how we developed such a system.

4. What are the major findings?

1.　　  Development of a simple, rapid, and safe assay system that can be used to continuously monitor membrane fusion processes. Our assay uses novel reporter proteins called dual split proteins (DSPs); a chimera between split Renilla luciferase (spRL) and split green fluorescent protein (spGFP). The principle of the assay relies on the fact that these split proteins are inactive on their own but become functional once they reassoicate. Thus, if membrane fusion between two compartments containing different DSPs occurs, the DSPs can reassociate, thus generating a signal. spGFP not only facilitates association of spRL but also provides a way for visual monitoring of the process of membrane fusion. Using a membrane-permeable substrate for RL makes quantitative monitoring of membrane fusion achievable in living cells.

2.　　  Use of our new assay system to investigate the dynamic nature of membrane fusion induced by the HIV-1 envelope glycoprotein (Env). We analyzed HIV-1 Env mutants whose membrane-spanning domains had been replaced with those derived from foreign proteins such as glycophorin A or vesicular stomatitis virus G-protein. Membrane fusion in these mutants showed a slower kinetics. The analysis of membrane fusion in the presence of the fusion inhibitors, soluble CD4 and C34, revealed that the mutants had a prolonged period of sensitivity to the inhibitors. These mutations within the membrane-spanning domains exerted an allosteric effect on the HIV-1 Env, probably affecting receptor-induced conformational changes of the envelope protein.

5. Can you summarise the significance of your paper in layman’s terms?

We have developed a simple, safe assay system for monitoring membrane fusion using a pair of novel reporter proteins called DSPs. This method has wide applications in addition to its use here for studying membrane fusion in HIV-1. It is applicable to viral envelope proteins derived from viruses such as influenza virus, SARS corona virus, and hepatitis C virus. Of course, this method can be applied to non-viral membrane fusion as well.

6. What are the next steps in your work and what strategy do you plan to use?

1.　　  Our assay system can be used to screen for inhibitors of the membrane fusion process. We will search for drugs that inhibit the fusion of HIV-1 with its host cell.

2.　　  During the process of infection, the virus surface glycoprotein, gp120, initially attaches to CD4 cell surface receptors and subsequently to cell surface co-receptors, such as CCR5 (R5) and CXCR4 (X4). The latter determines the so-called tropisms of the HIV-1 virus; R5 or X4 tropisms. Currently drug inhibitors in clinical use are only available against R5-tropic viruses, so it is important to know the tropism of viruses in infected individuals in order to offer appropriate treatment. Furthermore viral gp120 proteins may evolve during the progression of the disease, and the switch from CCR5 to CXCR4 co-receptor usage may be linked with accelerated disease progression. Our assay system can be used to determine the tropism of Env proteins derived from HIV-1 infected individuals. We have applied for a patent for this assay and are looking for industrial partners to develop it.

3.　　  Now that we have a good assay system, we will use it to analyse the mechanism of envelope fusion in greater detail.

7. Where do you see your research leading in the future? We plan to analyse more Env mutants to investigate structure-function relationships. We hope our assay system can be used by researchers in other fields as well.

8. What are the implications of your research for society? Our work on the mechanism of HIV-1 virus infection will help to identify drug targets and will have implications for the design of drugs for the treatment of AIDS. We hope that the assay system we have developed can be used to screen for new drugs, and also to determine the virus profiles of individuals with AIDS, thus allowing the selection of more appropriate drug treatment regimes. Our assay is rapid, so the determination of the tropism of viruses in an individual can be achieved faster than with currently available methods. This should facilitate the earlier initiation of treatment, increasing the likelihood that treatment will be successful. Again our assay is applicable to other viruses.

9. What else is your lab working on now? We’re looking at the early events of HIV-1 infection such as membrane fusion and disassembly processes. We are interested in structural analysis of the core proteins and studying viral disassembly following membrane fusion. Much effort has been invested within this field to determine the native structure (trimer) of the envelope proteins, but it has not yet been elucidated. We are trying to capture intermediate structures during membrane fusion.

10.  How did you get into this area of research? I owe a great debt to my former mentors. Prof. Isao Miyoshi, one of the discoverers of HTLV-1, introduced me to the filed of human retroviruses. He referred me to Prof. Max Essex’s lab at Harvard where I started to study HIV-1.

11. Which of your professional achievements brings you the most satisfaction? Laboratory work, such as the development of this fusion assay system, has brought me great satisfaction. The development and training of young scientists, though, gives me an equal sense of achievement. I have really enjoyed working cross-culturally and building relationships between Japanese and Chinese scientists. I want to thank all my lab members and colleagues for giving me such a precious experience.

12. What are the big issues/outstanding problems that have to be addressed in your field? In the field of HIV-AIDS research, one of the biggest problems is still the lack of understanding of this disease in the community. This sometimes leads to unfair treatment of people living with HIV/AIDS. I do not want to see a world where awareness of such chronic sexually-transmitted diseases is missing and where ignorance of the disease puts many infected people in a hopeless position.