The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/Helicase Belongs to a Distinct Class of 5′ to 3′ Viral Helicases

Tanner, Julian A.; Watt, RM; Chai, Yubo; Lu, Lin-Yu; Lin, Marie C.M.; Peiris, J. S. Malik; Poon, Leo L. M.; Kung, Hsiang‐Fu; Huang, Jian‐Dong

doi:10.1074/jbc.c300328200

Cited by 200 publications

(262 citation statements)

References 31 publications

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“…To fuel strand separation, SARS-CoV helicase hydrolyzes ATP or any of the eight canonical NTPs [53]. SARS-CoV helicase also has the ability to cleave the terminal phosphate from a triphosphate moiety linked to the 5′ end of a RNA molecule [52,54].…”

Section: Sars Coronavirus Helicasementioning

confidence: 99%

Understanding Helicases as a Means of Virus Control

Frick¹,

Lam²

2006

CPD

101

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Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a required cofactor.

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Section: Sars Coronavirus Helicasementioning

confidence: 99%

Understanding Helicases as a Means of Virus Control

Frick¹,

Lam²

2006

CPD

101

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“…This is consistent with previous report describing the ATPase activity stimulation effect was most enhanced when poly(U) was used. 23 However, it is not clear why poly(U) is better ATPase activity stimulator than other homopolynucleotide RNAs for SARS-CoV helicase.…”

Section: Resultsmentioning

confidence: 99%

“…Previous dsDNA unwinding experiment with fully dsDNA (blunt ended) did not show dsDNA unwinding activity, which supports our current results. 23 In general, the binding of ssNA stimulates the NTPase activity of helicases, which is believed that ssNA induces protein conformational change and allow the NTP hydrolysis to proceed. 27 Comparison of the kinetic parameters in the presence of poly(U) and M13 ssDNA suggests that SARS-CoV helicase does not show strict selectivity for ssNA, although k cat value of poly(C) is lower than other ssNA.…”

Section: Resultsmentioning

confidence: 99%

ATP Hydrolysis Analysis of Severe Acute Respiratory Syndrome (SARS) Coronavirus Helicase

2009

Bulletin of the Korean Chemical Society

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Severe acute respiratory syndrome coronavirus (SARS-CoV) helicase separates the double-stranded nucleic acids using the energy from ATP hydrolysis. We have measured ATPase activity of SARS-CoV helicase in the presence of various types of nucleic acids. Steady state ATPase analysis showed that poly(U) has two-times higher turnover number than poly(C) with lower Michaelis constant. When M13 single-stranded DNA is used as substrate, the Michaelis constant was about twenty-times lower than poly(U), whereas turnover numbers were similar. However, stimulation of ATPase activity was not observed in the presence of double-stranded DNA. pH dependent profiles of ATP hydrolysis with the helicase showed that the optimal ATPase activities were in a range of pH 6.2 ~ 6.6. In addition, ATP hydrolysis activity assays performed in the presence of various divalent cations exhibited that Mg 2+ stimulated the ATPase activity with the highest rate and Mn 2+ with about 40% rate as compared to the Mg 2+ .

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“…As discussed above, the NTP-binding site of E. coli DnaB does not appear sufficiently flexible to accommodate NTPs other than ATP or dATP (34). Interestingly, helicases from many parasites can use multiple NTPs in the unwinding reaction (17,35,36). It is assumed that T7 helicase might use dTTP in vivo for its helicase activity because dTTP is the most effective NTP in most helicase assays examined in vitro.…”

Section: Role Of Residues In Ntp-binding Site Of T7mentioning

confidence: 99%

“…Some helicases can use dATP, GTP, or dCTP (13,16,17) almost as efficiently as ATP. However, preferential use of dTTP over ATP has not been observed by any helicase other than the T7 helicase.…”

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confidence: 99%

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

Lee

Richardson

2010

Journal of Biological Chemistry

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The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.Helicases are ubiquitous enzymes that play pivotal roles in diverse cellular activities (1). Reactions mediated by helicases require their ability to bind and move along a DNA or RNA strand. Such a fundamental feature is coupled to the binding of nucleoside triphosphates (NTPs) 2 and their hydrolysis. An underlying mechanism in helicase action is a change in contact of the protein with the nucleic acid strand depending on the state of the bound NTP (1). Binding and subsequent hydrolysis of NTP induce conformational shifts in the nucleic acid-interacting part of the helicase that enable the enzyme to move unidirectionally. The unidirectional movement enables the helicase to translocate on the strand to which it is bound, and upon encountering duplex DNA, its continued movement results in unwinding of the dsDNA. Consequently, despite the divergence in their structure and mechanism of action, all helicases require the binding and hydrolysis of NTP to fulfill these multiple roles (2). Accordingly, signature structural motifs of helicase for NTP binding, for example the Walker motifs A and B, are conserved throughout the helicase family (3). These motifs interact with the phosphate moiety of NTP and influence DNA binding upon hydrolysis of NTP. In the case of hexameric helicases such as the T7 DNA helicase, complexity of helicase action is increased because the oligomerization of the subunits is a prerequisite for all the steps described above. In helicases of this family, binding and hydrolysis of NTP oc...

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The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/Helicase Belongs to a Distinct Class of 5′ to 3′ Viral Helicases

Cited by 200 publications

References 31 publications

Understanding Helicases as a Means of Virus Control

Understanding Helicases as a Means of Virus Control

ATP Hydrolysis Analysis of Severe Acute Respiratory Syndrome (SARS) Coronavirus Helicase

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

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