杭州市科学技术协会

The cumulative number of confirmed cases of COVID-19 throughout the world now exceeds 330 million with more than 5.5 million deaths according to the latest statistics from Johns Hopkins University [1]. The epidemic has continued for more than two years with a huge impact all around the world as coronavirus has been burdening the health care system, affecting economic growth, and decreasing overall quality of life.

Figure | Global Transmission Map of COVID-19. (Source: Johns Hopkins University)

With the long-lasting COVID-19 epidemic and the challenge in developing efficient drugs, vaccination is thought to be the most promising approach to contain virus spread through herd immunity achieved by mass vaccination, thus ultimately eliminating the virus. At present, the number of vaccinations administered worldwide is about to exceed 10 billion doses.

The newly emerged omicron and IHU mutant strains harbor multiple mutations in the spike protein on the surface of the virus, which greatly weakens the immune protection provided by currently existing vaccines. These new variants carry multiple mutations, especially in the receptor binding domain of the spike protein, requiring constant updates for vaccine design. However, the speed at which some of these variants spread throughout the world adds an extra challenge for vaccines. As far as the current situation is concerned, it has been rather challenging to contain the COVID-19 epidemic, and the fight between humans and the SARS-CoV-2 will most likely be a long-lasting one. Therefore, developing effective and quickly scalable approaches against new virus variants has become an important problem that needs to be urgently addressed.

Heat treatment destabilizes the RNA polymerase of wild type and omicron variant of SARS-CoV-2

On January 12, 2022, the joint team of Professors Hua Naranmandura and Chih-Hung Hsu from Zhejiang University School of Medicine published a paper entitled "Heat Treatment Promotes Ubiquitin-Mediated Proteolysis of SARS-CoV-2 RNA Polymerase and Decreses Viral Load" on journal Research, proposing for the first time that heat treatment specifically degrades the RNA polymerase of SARS-CoV-2 and inhibits virus replication [2].

Figure | Related publication (Source: Research)

RNA-dependent RNA polymerase (RdRp) complex is the main effector involved in the replication and transcription of SARS-CoV-2 genome. Its core component is the catalytic subunit NSP12 protein, which exerts polymerase activity by forming a replication-transcription complex (RdRp complex) with two cofactors including NSP7 and NSP8. Therefore, NSP12 plays an important role in the replication and transcription of SARS-CoV-2 viral RNA, and it is also the main target for the development of antiviral drugs including nucleoside analogs such as Remdesivir.

In this study, authors found that a relatively mild heat shock (40°C) could induce significant degradation of the RNA-dependent RNA polymerase (NSP12) of SARS-CoV-2. More importantly, P323L mutant, which is concurrently identified on NSP12 in several SARS-CoV-2 variants including omicron and IHU, displays similar heat sensitivity as wild type NSP12, suggesting that heat treatment could be a potential intervention against SARS-CoV-2 variants regardless of their RNA polymerase mutation status. Further, the team also found that treating cells at 40°C for half an hour every day could maintain low levels of both wild type and P323L mutant of NSP12, indicating strong clinical potential. The authors further identified the mechanism how the RNA polymerase is degraded. They showed that heat treatment promotes E3 ubiquitin ligase ZNF598-dependent NSP12 ubiquitination leading to proteasomal degradation and significantly decreases SARS-CoV-2 RNA copy number and viral titer.

Figure | Mechanism by which heat treatment inhibits SARS-CoV-2 virulence（Source：Research）

Bases on the above results, the team cooperated with the biosafety level 3 (P3) laboratory of the Zhejiang Provincial Center for Disease Control and Prevention to validate the effects of heat treatment on Vero E6 cells infected with SARS-CoV-2. They found that a mild heat treatment (40°C) also effectively reduces the RNA level of SARS-CoV-2 virus (~95% reduction) and virus titer (more than 99.5% reduction) without compromising host cell viability. These results suggest that heat treatment induced degradation of NSP12 leads to reduction of viral RNA load and downregulation of viral titer (see figure above).

The research behind the discovery that heat treatment affects SARS-CoV-2 RNA polymerase was inspired by bat body temperature

It is well known that bats are natural hosts of viruses such as SARS-CoV-2, SARS-CoV, MERS-CoV, and Ebola, but these deadly viruses do not cause diseases in bats. Instead, the virus might enter the human body through other intermediate hosts, triggering massive outbreaks of infectious diseases.

Figure | Bat（Source：National Geographic）

How do bats defend against these deadly viruses? It has been shown that the body temperature of some bat species reaches as high as 40 degrees during long hours of night flight, while the normal body temperature of humans is 36.5 degrees. In addition, a previous statistical association study showed that COVID-19 patients with higher body temperature at the initial presentation show lower mortality rate. Epidemiological studies have further demonstrated a reverse correlation between COVID-19 transmission rate and environmental temperatures. These findings suggest that an elevated temperature might affect SARS-CoV-2 virulence, which was the inspiration for this study.

Heat treatment (Hyperthermia, HT) can cause changes in the stability of some exogenous or abnormal proteins. In the beginning of this study, the team screened a variety of key SARS-CoV-2 effector proteins, such as nucleocapsid protein (N protein), non-structural protein 7 (NSP7), non-structural protein 8 (NSP8), and non-structural protein 12 (NSP12) among others. After extensive screening and validation, it was found that heat treatment specifically degrades NSP12.

The key to validate this finding was to directly determine the effect of heat treatment on viral RNA replication by conducting experiments on SARS-CoV-2 infected cells. Professor Naranmandura said that their laboratory did not meet the strict requirements for conducting experiments with live coronaviruses as stringent laboratory biosafety protection conditions are required to carry out such virus experiments. Subsequently, authors established cooperation with P3 laboratory in Zhejiang Provincial Center for Disease Control and Prevention to conduct viral infection experiments. The experiments in P3 laboratory showed that heat treatment indeed inhibits SARS-CoV-2 RNA replication and decreases viral load.

Yasen Maimaitiyiming (Postdoctoral Fellow), Tao Yang (PhD candidate) and Qian Qian Wang (Postdoctoral Fellow) are co-first authors of this study. This team has also received support and help from other parties, including Professor Fudi Wang and Professor Mikael Bjorklund of Zhejiang University. Professor H. Eric Xu from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and Professor Pei-Hui Wang from Institute of Advanced Medicine, Shandong University, generously provided SARS-CoV-2 viral RNA plasmids.

The team is committed to hyperthermia research, aiming to promote clinical translation of their findings

Hyperthermia or heat treatment, as a non-invasive, economical, simple, and easy-to-operate physical therapy, has been applied in the treatment of various diseases in combination with chemotherapy and radiotherapy. The emergence of novel technologies such as high-frequency hyperthermia and infrared-based hyperthermia now also provide new ways for clinical application of hyperthermia. However, lack of understanding of the molecular mechanisms how hyperthermia benefits patients is largely unclear, which currently restricts its more widespread application.

In summary, in the current study the authors demonstrated the mechanism by which heat treatment inhibits the virulence of SARS-COV-2, opening up the possibility of fighting COVID-19 with heat-based approches. Such mechanistic studies will facilitate application of heat treatment in the clinic.

Figure | The research team of professor Naranmandura（Source: Prof. Naranmandura）

Professor Naranmandura's team has been focusing on research in the field of heat treatment for more than ten years. In addition to the current study, the previous publication of the group also revealed the favorable effects of hyperthermia combined with arsenic trioxide in the treatment of refractory relapsed acute promyelocytic leukemia by degrading a key oncogenic protein necessary for this cancer [3]. Currently, research on other refractory diseases is also underway. The team is committed to the basic research oriented by clinical problems, with the goal of promoting basic medicine from bench to bedside, facilitating the transformation of scientific research, and contributing to the greater cause of human health. Young talents who are interested in this direction are welcome to join the team, please contact narenman@zju.edu.cn.

References:

[1] https://coronavirus.jhu.edu/map.html

[2] Heat Treatment Promotes Ubiquitin-Mediated Proteolysis of SARS-CoV-2 RNA Polymerase and Decreases Viral Load, Article in Press, https://spj.sciencemag.org/journals/research/aip/9802969/

[3] Hyperthermia Selectively Destabilizes Oncogenic Fusion Protein, Blood Cancer Discov July 1 2021 (2) (4) 388-401. DOI: 10.1158/2643-3230.BCD-20-0188

Tag: Health Science