Arch Virol. 2015 Sep;160(9):2293-300.

Inability of rat DPP4 to allow MERS-CoV infection revealed by using a VSV pseudotype bearing truncated MERS-CoV spike protein.

 

Fukuma A, Tani H, Taniguchi S, Shimojima M, Saijo M, Fukushi S.

Department of Virology 1, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo, 208-0011, Japan.

 

Abstract

Middle East respiratory syndrome (MERS) coronavirus (Co-V) contains a single spike (S) protein, which binds to a receptor molecule, dipeptidyl peptidase 4 (DPP4; also known as CD26), and serves as a neutralizing antigen. Pseudotyped viruses are useful for measuring neutralization titers against highly infectious viruses as well as for studying their mechanism of entry. In this study, we constructed a series of cytoplasmic deletion mutants of MERS-CoV S and compared the efficiency with which they formed pseudotypes with vesicular stomatitis virus. A pseudotype bearing an S protein with the C-terminal 16 amino acids deleted (MERSpv-St16) reached a maximum titer that was approximately tenfold higher than that of a pseudotype bearing a non-truncated full-length S protein. Using MERSpv-St16, we demonstrated the inability of rat DPP4 to serve as a functional receptor for MERS-CoV, suggesting that rats are not susceptible to MERS-CoV infection. This study provides novel information that enhances our understanding of the host range of MERS-CoV.

PMID: 26138557

 

Supplementary

A small animal model of human viral infection may facilitate not only understanding of the viral pathogenesis but also the evaluation of anti-viral drugs as well as vaccine development. Severe acute respiratory syndrome (SARS)-coronavirus (CoV), the causative agent of SARS, first emerged in China and spread worldwide between 2002 and 2003. It has been shown that an efficient approach to developing small animal models of SARS is an adaptation of the virus to rodents. Indeed, a serial passage of the virus by experimental infection of mice or rats with SARS-CoV results in enhanced permissiveness and disease (1-4). Since the infectivity of SARS-CoV is highly dependent on its cellular receptor molecule angiotensin-converting enzyme II (ACE2), an alternative approach for developing small animal models of SARS is the establishment of transgenic mice expressing human ACE2. Such human ACE2-transgenic mice develop severe illnesses with a high mortality rate (5, 6).

Since the first identification of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in 2012, many researchers have attempted to develop a small animal model of MERS. It has been reported that MERS-CoV utilizes human dipeptidyl peptidase 4 (DPP4) as a cellular receptor. DPP4 from camel, goat, cow, and sheep can also be recognized by MERS-CoV and can support MERS-CoV infection (7). However, DPP4 of mice and hamsters is not a functional receptor for MERS-CoV infection (7,8). Analysis of the function of DPP4 from other small animal species on MERS-CoV infection might provide novel information regarding a host range of MERS-CoV. In the paper described above, we examined whether the expression of DPP4 from rats, another traditional small animal model, supports the infection of MERS-CoV receptor. The results clearly indicate that rat DPP4 does not function as a receptor for MERS-CoV infection. Furthermore, from mutational analyses of rat DPP4, we presented data indicating that three or more amino acid substitutions in rat DPP4 are required to gain the function as a receptor. This finding is in contrast to those of SARS-CoV susceptibility to rats; that is, first, although inefficient in comparison to human ACE2, rat ACE2 supports some degree of SARS-CoV infection, and second, only one glycosylated amino acid in rat ACE2 might be responsible for the limited receptor function (9). Previous studies have shown the enhanced infectivity of rat-adapted SARS-CoV on rat ACE2-expressing cells, and have identified amino acid substitutions of the S protein responsible for an enhanced ability to utilize rat ACE2 (2, 10). Until now, there has been no report showing a successful adaptation of MERS-CoV to rodent DPP4.

Although mice are not susceptible to MERS-CoV infection, the transient infection of an adenovirus vector expressing human DPP4 in the respiratory tract mimics some of the aspects of human disease (11). Human DPP4-transgenic mice exhibit severe disease upon inoculation of MERS-CoV (12). These small animal models might be useful for the evaluation of anti-viral drugs and vaccines. The continuous development of appropriate small animal models of MERS will provide a new insight into the molecular mechanisms underlying MERS-CoV pathogenesis.

The importance of DPP4 in MERS-CoV tropism has been shown (7,8). The findings that mouse, hamster, and rat DPP4 do not function as a receptor for MERS-CoV suggests that rodents are unlikely the host of MERS-CoV. Interestingly, glycosylation of DPP4 might be an important limitation in the ability of MERS-CoV to infect mouse DPP4-expressing cells (13). In the case of rat DPP4, there exists a glycosylation motif that differs from human DPP4. Further studies are needed to determine whether the glycosylation of rat-DPP4 has an interference effect against MERS-CoV infection.

Our study has also shown that a vesicular stomatitis virus (VSV)-based pseudotype bearing the 16 amino acid-truncated S protein of MERS-CoV (MERSpv-St16) is useful to analyze the entry process of the virus without using a highly infectious live MERS-CoV (see Figure). The MERSpv-St16 might also be useful for determining the neutralizing antibody against MERS-CoV. Our recent study has shown the application of a VSV pseudotype to high-throughput screening of neutralizing antibodies (14). Since viral neutralization assays are the gold standard for detecting antibodies against MERS-CoV in serum samples with high sensitivity and specificity, MERSpvSt16 will be an important research tool not only for the sero-diagnosis of MERS but also for the investigation of animal hosts of MERS-CoV.

 

SF FIG1

Figure: VSV pseudotype. The VSV pseudotype system is useful for mimicing the infection of highly pathogenic viruses since it has been proved to be a safe viral entry model because of its inability to produce infectious progeny viruses. The VSV has MERS-CoV S protein on its viral surface and a GFP gene instead of a glycoprotein G gene.

 

Acknowledgements:

This study was supported in part by a Grant-in-Aid from the Ministry of Health, Labor, and Welfare of Japan (H25 Shinko-Ippan-008), and JSPS KAKENHI Grant Number 26450418

 

Contact:

Aiko Fukuma, Ph.D. <fukuma@nih.go.jp>

Shuetsu Fukushi, Ph.D.<fukushi@nih.go.jp>

Department of Virology 1

National Institute of Infectious Diseases

4-7-1 Gakuen, Musashimurayama

Tokyo 208-0011, Japan 

 

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