In the context of the ongoing pandemic of coronavirus disease 2019 (COVID-19), much research has focused on understanding how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses work to overcome the host’s innate immune response.
An interesting new study describes the activities of a family of viral enzymes called two-histidine-phosphoesterases (2H-PEs), found in coronaviruses (CoVs) and rotaviruses, as well as a mammalian A-kinase anchoring protein enzyme, AKAP7, that shares the same function. Together called 2’,5’-PEs, these enzymes all break down 2’,5’-oligoadenylate compounds that activate the antiviral enzyme ribonuclease L (RNase L).
In this, they use a different mechanism of action from other 2H-PE enzymes and other phosphodiesterases.
A preprint version of the study is available on the bioRxiv* server, while the article undergoes peer review.
Unique biochemical activity
The team of scientists at the Lerner Research Institute, Cleveland, and the Perlman School of Medicine at the University of Pennsylvania, Philadelphia, found that these enzymes do not show dependence for their catalytic activity on the presence of a metal ion such as magnesium and cleave only 3’,5’-phosphodiester bonds. Thus, the breakdown products always terminate in cyclic 2’,3’-phosphates.
In mammals, the cell often detects the presence of a virus because of the production of certain peculiar molecules during viral replication. Such molecules present pathogen-associated molecular patterns (PAMPs), one common example being viral double-stranded ribonucleic acid (dsRNA).
In response, the cell secretes type I and type III interferons (IFNs), which cause several hundred genes to be expressed at higher levels. These IFN stimulated genes (ISGs) include antiviral effector proteins, such as, in humans, 2’,5’-oligoadenylate (2-5A) synthetases 1-3 (OAS1-3). The number of OAS units in each of these differs from one to three, respectively.
The OAS molecules are enzymes that act to transfer 2’-nucleotides, using ATP (adenosine triphosphate) as a substrate, and also resulting in the production of many different molecules linked by 2’,5’ bonds.
When these bind to viral dsRNA, they begin to synthesize 2-5A, which causes RNase L to dimerize and become active. Activated RNase L breaks down RNA indiscriminately, thus halting protein synthesis and inducing cell death and inflammasome activation.
RNase L also causes cells to enter apoptosis when exposed to dsRNA synthesized from repetitive DNA sequences in the host cell nucleus, as occurs with ADAR 1 (adenosine deaminase acting on RNA-1) or when cells are treated with the DNA demethylating drug 5-aza-cytidine.
Having the right levels of 2-5A is therefore essential for the health of the host cell and to counter viral infections and limit the damage they cause. Interestingly, SARS-CoV-2 lacks a 2’,5’-PE, causing it to activate RNase L-mediated pathways that inhibit its replication.
What were the findings?
The researchers found 2’,5’-PE activity in the mouse hepatitis virus and Middle East respiratory syndrome coronavirus, among the coronaviruses; rotavirus group A, and mouse AKAP7. All were remarkable for cleaving only 2’,5’ linked oligoribonucleotides in linear but not cyclic molecules.
However, among these, the first two specifically cleaved 2’,5’-linked phosphodiester bonds, and especially split 2’,5’-linked oligoadenylates. In contrast, the others also cleaved other 2’,5’-oligonucleotides.
None of these cleaved 3’,5’-oligoribonucleotides and seem to have only one function, that of eliminating 2-5A by cleaving the 2-5A trimer, yielding mono- and deadenylates ending in phosphoryl groups. This enables the coronaviruses and rotaviruses to counter the host cell’s RNase L-mediated antiviral activity.
The viral and mammalian 2’,5’-PEs family members differ from the human phosphodiesterase 12 (PDE12) in that the latter breaks down trimer 2-5A into ATP (adenosine triphosphate) and 2 (5’-AMP)s (adenosine monophosphate) in the presence of magnesium ions.
Conversely, the former cleaves anti-viral 2-5A into 5’ products ending in 2’,3’ cyclic phosphates. These are not able to activate RNase L since they lack the required three or more adenylyl residues.
Their action is also independent of metal ions.
What are the implications?
More research is required to identify the acceptors for the 2’,5’-AMP residues and alternative 2-5A-like molecules that may act as substrates for these enzymes. Such enzymes are present in invertebrates as well, though their role is unknown.
In short, the 2’,5’-PEs are part of the 2H-PE superfamily of enzymes but show a different and unique cleavage pattern. While the viral 2’,5’-PEs antagonize innate immunity and thus promote viral replication, the mammalian AKAP7 does not have this function. Besides the antiviral function, these enzymes also regulate the levels of dsRNA from viral or host origin to limit the adverse cytotoxic impact on the host.
Hopefully, this explanation of the mechanism of action of 2’,5’-PEs will eventually suggest a new approach to developing antivirals against CoVs and rotaviruses, among others.
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