ACE2: the “Trojan horse” in SARS and COVID-19

The SARS-CoV-2 has disturbed many people's "original plans", which remind people of the SARS (Severe Acute Respiratory Syndrome) and the MERS (Middle East respiratory syndrome). The outbreak of the above two diseases are related to the coronavirus.

So what is coronavirus?

The diameter of the coronavirus is ranged from 60nm to 220nm. This coronavirus contains positive single stranded RNA with 27-32kb length. The single stranded RNA consist of two coding regions: replicase and structural protein. Replicase region consist of two genes: orf1a and orf1b, which are highly conserved in the coronavirus (Figure 1A). The structural region mainly encodes four proteins: Spike protein (S), for binding to receptor; Envelope protein (E), which helps virus enter to the host cell; Membrane glycoprotein (M), is responsible for the transmembrane transport of nutrients, the budding and releasing of progeny virus; Nucleocapsid protein (N) protects the stability of RNA^[1] (Figure 1B).

Figure 1. Genome and morphological structure of coronavirus

 Changed from Cui J, Li F, Shi ZL (2019). Origin and evolution of pathogenic coronaviruses. Nat Rev Microbio. 17(3):181-192.

How does coronavirus invade host cells?

Taking SARS-CoV as an example, spike protein on the surface of SARS-CoV consists of S1 and S2 subunit, S1 subunit binds to the receptor, and S2 transmembrane domain that mediates membrane fusion. The RBD (Receptor Binding Domain) domain , which is in the region of S1 subunit, binds to the receptor ACE2 (Angiotensin-Converting Enzyme 2) on the surface of host cells. Subsequently, SARS-CoV enters the host cell (Figure 2), then exposes viral RNA, translates viral RNA replicase, and forms RNA replicase transcriptase complex. In the process of transcription, 7-9 RNA subsets are produced by discontinuous transcription, including the structural proteins encoding RNA. Newly formed coronavirus RNA and structural proteins are assembled into new virus particles in the cytoplasm, which are released from the host cells to infect new cells, and then gradually spread to bronchi and lungs (Figure 3). In this process, ACE2  just like a ”Trojan horse“ to help SARS-CoV enter the human body.

Figure 2. Structure and function of SARS CoV spike protein

Changed from Zhu X, Liu Q, Du L, Lu L, Jiang S (2013).Receptor-binding domain as a target for developing SARS vaccines. J Thorac Dis. 5 Suppl 2:S142-8.

Figure 3. Life cycle of SARS-CoV in host cells

Changed from Zhu X, Liu Q, Du L, Lu L, Jiang S (2013).Receptor-binding domain as a target for developing SARS vaccines. J Thorac Dis. 5 Suppl 2:S142-8.

On February 3, 2020, the latest research results of SARS-CoV-2 from Zhengli Shi team of Wuhan Institute of Virology and Yongzhen Zhang  team of Fudan University, were published in Nature, respectively. The nucleotide similarity between SARS-CoV-2 and SARS-CoV is 89.1%^[2], and they used the same receptor ACE2 to enter the host cell^[3]. 

Now let‘s talk obout the ACE2.

ACE2 (about 120KD), is expressed in gastrointestinal tract, heart, kidney, lung, testis and brain, and works as a negative regulator of RAS systerm (renin-angiotension system, this system maintain the stability of human body environment, but overexpressed RAS will lead to the imbalance of blood pressure, water and electrolyte) to protect the cardiovascular system, ACE2 also function in amino acid absorption of kidney and intestine, besides, it plays a positive role in reducing stroke^[4,5]. After SARS-CoV ravaged China, scientists discovered that, ACE2 is the receptor of SARS-CoV ^[6], and then the research on the interaction between ACE2 and SARS-CoV never stopped.

Chengyu Jiang, professor of Chinese Academy of Medical Sciences, found out that, in the experiment of PAMAM nanoparticles induced lung injury in mice, gene inactivation of Ace2 can exacerbate lung injury, which indicated that ACE2 played an important role in lung function ^[7]. The Stanley Perlman team of Iowa University constructed a transgenic mouse expressing human ACE2 gene regulated by human K18 (cytokeratin 18) promoter. Four days after infected with SARS-Cov, the virus antigen were detected in K18-hACE2 mice, but not in wild-type mice ^[8,9]. Professor Ralph S. Baric from North Carolina university also proved the viewpoint: overexpression of human ACE2 in mice showed the susceptibility to SARS-CoV and WIV1-CoV (this artificial CoV play the similar effect with SARS-CoV, they used the same receptor ACE2, but the mortality rate of experimental mice is reduced, and more preclinical data can be obtained). Professor Ralph S. Baric used the lung ciliary epithelial cell promoter HFH4 to regulated the human ACE2 gene expression in mice, ACE2 did not show the expected lung specific expression, and it was also detected in the brain, liver, kidney and gastrointestinal tract. All HFH4-ACE2 mice lost >20% body weight, and maintained the strong replication ability in the lung and brain following infected with SARS-CoV. While the body weight loss> 10% of laboratory mice after 7 days of infection with WIV1-CoV, and detected robust replication in the brain, but the replication in the lung was significantly reduced. They also found a monoclonal antibody 227.14, which can protect HFH4-ACE2 mice from the SARS-CoV and WIV-CoV attacks. After antibody treatment, no virus was detected in the lungs of HFH4-ACE2 mice^[10](Figure 4).

(C and D) 24 week old HFH4-ACE2 mice were injected with monoclonal antibody 227.14 (solid line) or PBS (dotted line) for 1 day, then infected with SARS-CoV (black) or WIV-CoV (blue), and the (C) survival rate and (D) pulmonary virus replication ability were examined. Nd indicates no virus detected.

Changed from Menachery VD, et al. (2016). SARS-like WIV1-CoV poised for human emergence. Proc Natl Acad Sci USA. 113(11):3048-53.

How to control this ”trojan horse“ and what kind of ACE2 animal model is more suitable for preclinical research needs to be further explored. It is hoped that ACE2 will become a breakthrough in the treatment of SARS and 2019 ncov, and find an effective treatment as soon as possible.

[1] Jiang S. et al (2019). The spike protein of SARS-CoV—a target for vaccine and therapeutic development. Nat Rev Microbiol. 7:226-36.

[2] Wu, F. et al (2020). A new coronavirus associated with human respiratory disease in China. Nature https://doi. org/10.1038/s41586-020-2008-3.

[3] Zhou P. et al (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature.  https://doi.org/10.1038/s41586-020-2012-7.

[4] Alenina N, Bader M (2019). ACE2 in Brain Physiology and Pathophysiology: Evidence from Transgenic Animal Models. Neurochem Res. 44:1323-1329.

[5] Kuba K et al., (2010). Trilogy of ACE2: A peptidase in the renin–angiotensin system, a SARS receptor, and a partner for amino acid transporters. Pharmacol Ther. 128(1):119-28.  

[6] Li W et al., (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 426(6965):450-4.

[7] Sun Y, Guo F, Zou Z, Li C, Hong X, Zhao Y, Wang C, Wang H, Liu H, Yang P, Han Z, Liu K, Kuba K, Song B, Gao J, Mo Z, Li D, Li B, Li Q, Zhong N, Wang C, Penninger JM, Jiang C (2015). Cationic nanoparticles directly bind angiotensin-converting enzyme 2 and induce acute lung injury in mice. Part Fibre Toxicol. 7;12:4.

[8] Perlman S. et al (2007). Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. J Virol. 81(2):813-21.

[9]NetlandJ.etal(2008).Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2. J Virol. 82(15):7264-75.

[10] Menachery VD, et al. (2016). SARS-like WIV1-CoV poised for human emergence. Proc Natl Acad Sci USA. 113(11):3048-53. 

Previous studies have confirmed that, like SARS-CoV, the recognition receptor for new coronavirus (SARS-CoV-2) infection in humans is also angiotensin-converting enzyme 2, (ACE2). However, wild-type mice cannot be stably infected for a long time and develop symptoms due to different composition between human and mouse ACE2 amino acid, and low affinity of coronavirus to mouse ACE2. Therefore, wild-type mice are not suitable to study coronavirus directly. To obtain a mouse model capable of infecting SARS-CoV-2, we have to introduce the human ACE2, which can be recognized by the virus into wild-type mice.

GemPharmatech (GPT) utilizes the gene knock-in technology to develop ACE2 humanized mouse models. Testing results proved that the mice expressed integrated human fragments as expected. Now, these positive mice have been backcrossed to breed lines to obtain stable mouse strains.

Advantages of GPT’s Proprietary Humanized ACE2 Mice:

• Optimized genetic modification strategy to introduce the human ACE2 gene into the designated position;

• Expected to display similar symptoms to human after infection with SARS-CoV-2;

• Available on the C57BL/6JGpt, BALB/cJGpt, and NCG genetic background

• Customize single-, double-, triple-targeted humanized mouse models to accelerate the SARS-CoV-2 researches, such as humanized TMPRSS2 mice, and ACE2/TMPRSS2 mice, etc.

Development Strategy of GPT’s Proprietary Humanized ACE2 Mice:

In this model, chimeric CDS containing the extracellular domain of human ACE2 fused with mouse signal peptide, transmembrane, and the intracellular domain of mouse ACE2 is expressed under the transcriptional regulation of endogenous sequences. It mimics the physiological expression pattern of ACE2 state in tissues, including the kidney, lung, and intestine.

GPT’s Proprietary Humanized ACE2 Mouse Model is Ideal for SARS-CoV-2 Drug Screening, Vaccines and Test Kits R&D:

If the human ACE2 gene was randomly integrated into the mouse genome, once infected with SARS-CoV-2, the model usually shows severe symptoms and death, which were different from the progression after infection on humans often occurred. To solve these problems, GemPharmatech adopted a more optimized genetic modification strategy by knocking in human ACE2 fragments at the mouse-derived ACE2 locus. The fully proprietary humanized ACE2 model was expected to express human ACE2 at close to endogenous physiological levels and expression patterns to reproduce the disease progression, lung injury, and death after infection with coronavirus. GPT’s humanized ACE2 mouse model is ideal for drug screening (antibodies, viral protease inhibitors, etc.), vaccines and test kits' research and development.

ACE2 Related Mouse Strains: