- Email: [email protected]
Hepatitis C NS3 helicase unwinds RNA in leaps and bounds
Comment
animals.3 Omnivorous species that eat fruits and insects in the wild could be expected to experience a similar discrepancy in available iron levels between natural and captive diets, and excessive iron deposits are often found in callitrichid primates and coatis,2 for example. Even in a piscivorous species, the northern fur seal, a difference in iron content between natural and captive diets has been suggested as a cause for observed cases of excessive iron deposition.6 These observations offer a set of testable hypotheses, linking a potential susceptibility to excessive iron storage to the biology of a species. Should such hypotheses be confirmed, the wealth of mammalian species offers many opportunities to learn about general absorption, transport, and storage of iron by comparing biochemical and genetic variables between species of different susceptibility. However, the use of necropsy diagnoses does not allow the quantitative testing of such hypotheses within a reasonable timeframe. The use of serum ferritin as an indicator of iron stores in live animals is limited because the reaction by which it is measured necessitates a species-specific immunological test. By contrast, serum transferrin saturation, as a functional test of serum iron and iron-binding capacity, is not species-specific. Serum transferrin saturation has been used in a limited variety of mammals (table),4,6–9 and reflects changes in the dietary content of available iron.9 This test could be readily applied to a multitude of captive mammalian species, preferably with a known dietary history. We want to encourage laboratories of human medicine to cooperate with zoological gardens in the establishment of a database of mammalian species susceptible and not susceptible to iron-storage disease. This cooperation would not only support conservation efforts for endangered species, but also identify species whose further investigation could yield valuable comparative insights into the genetics, mechanisms, and consequences of the uncontrolled absorption of iron, and the potential for dietary prevention.
Chris Wood, *Marcus Clauss College of Life Sciences, and Key Laboratory of Conservation Genetics and Reproductive Biology for Endangered Wild Animals of the Ministry of Education, Zhejiang University, Hangzhou, Zhejiang, China (CW); and Physiological Chemistry and Animal Nutrition, Institute of Animal Physiology, 5764 Oberschleissheim, Germany (MC)
Animal
Species
Dogs10 Black rhinoceros4 Sumatran rhinoceros4 White rhinoceros4 Domestic horse4 Tapirs7 Cattle8 Lemurs9,11,12
Canis lupus Diceros bicornis Diceros sumatrensis Ceratotherium simum Equus caballus Tapirus spp Bos taurus Different species
Northern fur seal6 Man13
Callorhinus ursinus Homo sapiens
Serum transferrin saturation (%TS) Normal absorption of iron
High absorption of iron
27% 28% (free-ranging) .. 37% (captive) 22–44% 33% (free-ranging) 32% (other breeds) 22% (weanlings), 30% (free-ranging), 47% (captive on special diet) 41% (males) <45%
.. 65–72% (captive) 91% (captive) .. .. 60–69% (captive) 92–100% (affected Salers-cross cattle) 57–83% (captive, conventional diet)
63% (females) >45%
Table: Serum transferrin saturation in mammals
[email protected] We thank S G Fang and Xi Yongmei for support and advice. We declare we have no conflict of interest. 1
Benirschke K, Miller C, Ippen R, Heldstab A. The pathology of prosimians, especially lemurs. Adv Vet Sci Comp Med 1985; 30: 167–208. 2 Lowenstine LJ, Munson L. Iron overload in the animal kingdom. In: Fowler ME, Miller ER, eds. Zoo and wild animal medicine: current therapy 4. Philadelphia: WB Saunders Co, 1999: 260–68. 3 Clauss M, Lechner-Doll M, Hänichen T, Hatt JM. Excessive iron storage in captive mammalian herbivore—a hypothesis for its evolutionary etiopathology. Proc Conf EAZWV 2002; 4: 123–31. 4 Paglia DE, Dennis P. Role of chronic iron overload in multiple disorders of captive black rhinoceros. Proc Conf AAZV 1999: 163–71. 5 Zhang H, Zhong N. Necrotic hepatitis in the giant panda. In: Zhang AJ, He GX, eds. Minutes of the International Symposium on the Protection of the Giant Panda Ailuropoda melanoleuca, Chengdu Foundation of Giant Panda Breeding, Chengdu, China, 1993: 351–53. 6 Mazzaro LM, Dunn JL, St. Aubin DJ, Andrews GA, Chavey PS. Serum indices of body stores of iron in northern fur seals (Callorhinus ursinus) and their relationship to hemochromatosis. Zoo Biol 2004; 23: 205–18. 7 Paglia DE, Miller CL, Foerster SH, Wynne JE, Tsu ICH, Kenny DE. Evidence for acquired iron overload in captive tapirs. Proc Conf AAZV 2000: 124–26. 8 O’Toole D, Kelly EJ, McAllister MM, et al. Hepatic failure and hemochromatosis of Salers and Salers-cross cattle. Vet Pathol 2001; 38: 372–89. 9 Wood C, Fang SG, Hunt A, Streich WJ, Clauss M. Increased iron absorption in lemurs: quantitative screening and assessment of dietary prevention. Am J Primatol 2003; 61: 101–10. 10 Weeks BR, Smith JE, Northrop JK. Relationship of serum ferritin and iron concentrations and serum total iron-binding capacity to nonheme iron stores in dogs. Am J Vet Res 1989; 50: 198–200. 11 Gonzales J, Benirschke K, Saltman P, Roberts J, Robinson PT. Hemosiderosis in lemurs. Zoo Biol 1984; 3: 255–65. 12 Dutton CJ, Junge RE, Louis EE. Biomedical evaluation of free-ranging ringtailed lemurs (Lemur catta) in Tsimanampetsotsa Strict Nature Reserve, Madagascar. J Zoo Wildl Med 2003; 34: 16–24. 13 Pietrangelo A. Hereditary hemochromatosis—a new look at an old disease. N Engl J Med 2004; 350: 2383–97.
Hepatitis C NS3 helicase unwinds RNA in leaps and bounds Recently, Victor Serebrov and Anna Marie Pyle used a novel approach to study how the hepatitis C NS3 helicase makes its way through double-stranded RNA.1 Surprisingly, the protein was found to have large unwinding translocation steps of equal size, which move in a discontinuous fashion. These characteristics suggest that www.thelancet.com Vol 364 October 16, 2004
nucleic acid motors can function in a manner analogous to kinesin and myosin. Hepatitis C virus (HCV) infections are a global health problem, affecting over 170 million individuals worldwide. 10–20% of those infected ultimately develop cirrhosis and 1–4% a year of the cirrhotic group develop liver cancer.2 1385
Comment
I
Tractor (T)
Plough (P)
T
P
II
ATP ADP
18 nt
18 bp T
P
VI "Rapid ripping"
III
ATP ADP
IV
T "Resting" "Rapid ripping"
P
V ATP ADP
Periodic motion of NS3 protein on duplex RNA Enzyme is depicted with two edges—leading edge which functions as tractor (T) and lagging edge which serves as plough (P). Enzyme is bound to RNA in stage I. Tractor translocates 18 bp and binds to duplex region, as indicated by hands (II). Plough unwinds 18 bp of duplex as it catches up to tractor (III and IV). Once it has caught up, protein “rests” briefly (V). Stages II–IV would correspond to “rapid ripping” phase of cycle, as suggested by Serebrov and Pyle.1 In VI, tractor translocates 18 bp again and enzyme repeats periodic cycle (III–V) until RNA is completely unwound. Energy for cycle comes from hydrolysis of ATP; it is unclear how many ATP molecules are hydrolysed per cycle. bp=basepairs, nt=nucleotides. Adapted from reference 10.
Unlike hepatitis B virus, no vaccine is available. The current standard treatment for chronic HCV infection, interferon alfa and ribavirin, succeeds in only 50% of patients and can cause serious side-effects.3,4 The development of new antiviral agents with minimum side-effects, designed to inhibit the activities of proteins essential to HCV proliferation, is thus essential to curbing this viral pandemic. Helicases are essential nanoscale motor-proteins that translocate or motor their way in one direction along DNA or RNA tracks, with energy liberated from ATP hydrolysis to drive their motion.5 Often, helicases are covalently coupled to, or associated with, proteins whose activities are unrelated to duplex-strand separation.6 The non-structural protein 3 (NS3) of HCV is one such helicase. NS3 is a multifunctional protein with a serine protease domain located in its N-terminal third, coupled to a helicase domain in its C-terminal two-thirds.7 Both the serine protease and helicase activities are essential for HCV replication.8 Thus NS3 has become a target for anti-HCV treatment, and understanding the biochemical mechanism of its helicase activity is of great importance.9 Helicases can function as bulldozers, driving their way into duplex molecules where they translocate along the unwound strands after they unwind the duplex. Alternatively, 1386
helicases can function as tractor-plough combinations: the tractor motors along the duplex and the plough separates the strands from behind. Regardless of the mechanism, the helicase must have a leading edge and a lagging edge and, in the two mechanisms mentioned, the roles of these edges are reversed. In the tractor-plough analogy, the leading edge binds the duplex while the lagging edge unwinds the nucleic acid strands. Each of these two edges can move in different increments. For example, the leading edge of the Escherichia coli RecBC enzyme translocates first, in 23 bp steps,10 while the lagging edge unwinds the duplex in 4 bp steps11 as it catches up to the leading edge. Cycles of large translocation steps followed by smaller unwinding steps repeat until the DNA is unwound or the enzyme dissociates. Serebrov and Pyle1 used a novel combinatorial timeresolved approach to gain insight into the helicase mechanism of NS3. The approach uses a large family of duplex RNA substrates containing, on average, a single nick at each position along only one strand of the RNA duplex. They monitored the microscopic behaviour of NS3 helicase at each nucleotide in duplex RNA by analysing the products of unwinding in gels. They found that the translocation and unwinding domains of the NS3 helicase move in equal 18 bp steps. These large equal-sized translocation unwinding steps are a new discovery for nucleic acid motors. More detailed quench-flow kinetic analyses showed that these domains progressed through periods of fast ripping followed by local pausing, and that these periods of movement and rest occurred with regular spacing along the duplex RNA molecule (figure). This periodic motion is analogous to the stepping behaviour observed for the cytoskeletal motor proteins kinesin and myosin.12 For helicases and cytoskeletal motor proteins, the conformational changes associated with the binding of ATP, its hydrolysis, and product release are coupled to large conformational changes that provide the driving force for the mechanical work used to separate the strands of the nucleic acid duplex, transport intracellular cargo, or contract muscle.12,13 Because ATPase activity is coupled to strand separation, researchers are actively pursuing potential specific inhibitors that target ATPase and helicase activities of NS3 protein.9 Some inhibitors target the helicase activity while others target the ATPase activity.14 These inhibitors show promise because they are specific potent inhibitors of viral replication and they also act on other viruses in addition to HCV. The success of these inhibitor studies requires detailed knowledge of the biochemical mechanism of the NS3 helicase.
Piero R Bianco Center for Single Molecule Biophysics, Department of Microbiology and Immunology and Department of Biochemistry, University at Buffalo, Buffalo, NY 14214, USA [email protected]
www.thelancet.com Vol 364 October 16, 2004
Comment
I thank Cindy Dlugos for comments. I am supported by NIH grant GM66831–01. I declare I have no conflict of interest. 1 2 3
4 5
6
Serebrov V, Pyle AM. Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 2004; 430: 476–80. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001; 345: 41–52. McHutchison JG, Gordon SC, Schiff ER, for the Hepatitis Interventional Therapy Group. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 1998; 339: 1485–92. Dev A, Patel K, McHutchison JG. New therapies for chronic hepatitis C virus infection. Curr Gastroenterol Rep 2004; 6: 77–86. Delagoutte E, von Hippel PH. Helicase mechanisms and the coupling of helicases within macromolecular machines, part I: structures and properties of isolated helicases. Q Rev Biophys 2002; 35: 431–78. Delagoutte E, von Hippel PH. Helicase mechanisms and the coupling of helicases within macromolecular machines, part II: integration of helicases into cellular processes. Q Rev Biophys 2003; 36: 1–69.
7
8 9 10 11
12 13 14
Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol 2000; 242: 55–84. Bartenschlager R, Lohmann V. Replication of hepatitis C virus. J Gen Virol 2000; 81: 1631–48. Tan SL, Pause A, Shi Y, Sonenberg N. Hepatitis C therapeutics: current status and emerging strategies. Nat Rev Drug Discov 2002; 1: 867–81. Bianco PR, Kowalczykowski SC. Translocation step size and mechanism of the RecBC DNA helicase. Nature 2000; 405: 368–72. Lucius AL, Vindigni A, Gregorian R, et al. DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies. J Mol Biol 2002; 324: 409–28. Vale RD, Milligan RA. The way things move: looking under the hood of molecular motor proteins. Science 2000; 288: 88–95. Hall MC, Matson SW. Helicase motifs: the engine that powers DNA unwinding. Mol Microbiol 1999; 34: 867–77. Borowski P, Deinert J, Schalinski S, et al. Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses. Eur J Biochem 2003; 270: 1645–53.
Chlamydia, gonorrhoea, and injectable progesterone
www.thelancet.com Vol 364 October 16, 2004
while a change led to reassignment to their new chosen arm for subsequent data collection. The women who initially chose DMPA were different at the outset: they were substantially more likely to attend the inner city clinic, and they were more likely to be older, black, and parous (all p<0·01).1 Most tellingly, at the start of therapy, the DMPA choosers were already more likely to be infected with chlamydia: 8·9% compared with 3·1% in oral contraceptive users and 4·6% in controls (p<0·05). Although the investigators attempted to statistically correct for later behaviours that could confound the apparent risk of acquiring infections, including numbers of sexual partners and condom use, the women who chose DMPA were clearly linked to different sexual networks than
Rights were not granted to include this image in electronic media. Please refer to the printed journal.
Science Photo Library
Biological hypotheses to explain how progesterone could increase risk of cervical infections are unconvincing. Thus the recent finding by Charles Morrison and colleagues1 that the injection of depot medroxyprogesterone acetate (DMPA) was independently associated with a four-fold increase in chlamydial infection (hazard ratio 4·3, 95% CI 1·7–11·1) demands careful scrutiny. Chlamydia rates are rising in many countries2 and, 50 years after it was first developed, use of DMPA is growing in popularity. DMPA is a high-efficacy contraceptive that is administered as a 3-monthly injection. For many years, DMPA’s use was restricted because of concerns about possible long-term carcinogenic effects that have since been dispelled.3 Nevertheless, our impression is that DMPA maintains a residual aura of being a second-line contraceptive choice in the minds of many clinicians in some countries. Morrison and colleagues’ prospective study, of 819 women attending two reproductive health clinics in Baltimore, USA, and with no desire to become pregnant in the next year, was well conducted. Particular strengths were the ongoing monitoring of sexual behaviour and contraceptive methods, the use of sensitive tests for chlamydia, and the careful quantification of cervical ectopy. There was no significant association between combined oral contraceptives and cervical infections, nor was DMPA associated with an increased risk of gonorrhoea. Although cervical ectopy appeared to have a limited, if any, role in the increased risk of chlamydia with DMPA use, it was a marginally significant risk factor for cervical infections in its own right (hazard ratio 2·3, 95% CI 0·9–6·0). However, Morrison and colleagues did an observational cohort study, not a randomised trial. For ethical reasons the women were allowed to choose their method of contraception: combined oral, DMPA, or no hormonal contraception. There is no mention of the level of persuasion by the clinicians that might have affected this choice. About two-thirds of the women used the same method throughout the study,
Coloured transmision electron-micrograph of Chlamydia trachomatis in cells of Fallopian tube Bacteria (red) occur in inclusions (yellow) inside cells.
1387
animals.3 Omnivorous species that eat fruits and insects in the wild could be expected to experience a similar discrepancy in available iron levels between natural and captive diets, and excessive iron deposits are often found in callitrichid primates and coatis,2 for example. Even in a piscivorous species, the northern fur seal, a difference in iron content between natural and captive diets has been suggested as a cause for observed cases of excessive iron deposition.6 These observations offer a set of testable hypotheses, linking a potential susceptibility to excessive iron storage to the biology of a species. Should such hypotheses be confirmed, the wealth of mammalian species offers many opportunities to learn about general absorption, transport, and storage of iron by comparing biochemical and genetic variables between species of different susceptibility. However, the use of necropsy diagnoses does not allow the quantitative testing of such hypotheses within a reasonable timeframe. The use of serum ferritin as an indicator of iron stores in live animals is limited because the reaction by which it is measured necessitates a species-specific immunological test. By contrast, serum transferrin saturation, as a functional test of serum iron and iron-binding capacity, is not species-specific. Serum transferrin saturation has been used in a limited variety of mammals (table),4,6–9 and reflects changes in the dietary content of available iron.9 This test could be readily applied to a multitude of captive mammalian species, preferably with a known dietary history. We want to encourage laboratories of human medicine to cooperate with zoological gardens in the establishment of a database of mammalian species susceptible and not susceptible to iron-storage disease. This cooperation would not only support conservation efforts for endangered species, but also identify species whose further investigation could yield valuable comparative insights into the genetics, mechanisms, and consequences of the uncontrolled absorption of iron, and the potential for dietary prevention.
Chris Wood, *Marcus Clauss College of Life Sciences, and Key Laboratory of Conservation Genetics and Reproductive Biology for Endangered Wild Animals of the Ministry of Education, Zhejiang University, Hangzhou, Zhejiang, China (CW); and Physiological Chemistry and Animal Nutrition, Institute of Animal Physiology, 5764 Oberschleissheim, Germany (MC)
Animal
Species
Dogs10 Black rhinoceros4 Sumatran rhinoceros4 White rhinoceros4 Domestic horse4 Tapirs7 Cattle8 Lemurs9,11,12
Canis lupus Diceros bicornis Diceros sumatrensis Ceratotherium simum Equus caballus Tapirus spp Bos taurus Different species
Northern fur seal6 Man13
Callorhinus ursinus Homo sapiens
Serum transferrin saturation (%TS) Normal absorption of iron
High absorption of iron
27% 28% (free-ranging) .. 37% (captive) 22–44% 33% (free-ranging) 32% (other breeds) 22% (weanlings), 30% (free-ranging), 47% (captive on special diet) 41% (males) <45%
.. 65–72% (captive) 91% (captive) .. .. 60–69% (captive) 92–100% (affected Salers-cross cattle) 57–83% (captive, conventional diet)
63% (females) >45%
Table: Serum transferrin saturation in mammals
[email protected] We thank S G Fang and Xi Yongmei for support and advice. We declare we have no conflict of interest. 1
Benirschke K, Miller C, Ippen R, Heldstab A. The pathology of prosimians, especially lemurs. Adv Vet Sci Comp Med 1985; 30: 167–208. 2 Lowenstine LJ, Munson L. Iron overload in the animal kingdom. In: Fowler ME, Miller ER, eds. Zoo and wild animal medicine: current therapy 4. Philadelphia: WB Saunders Co, 1999: 260–68. 3 Clauss M, Lechner-Doll M, Hänichen T, Hatt JM. Excessive iron storage in captive mammalian herbivore—a hypothesis for its evolutionary etiopathology. Proc Conf EAZWV 2002; 4: 123–31. 4 Paglia DE, Dennis P. Role of chronic iron overload in multiple disorders of captive black rhinoceros. Proc Conf AAZV 1999: 163–71. 5 Zhang H, Zhong N. Necrotic hepatitis in the giant panda. In: Zhang AJ, He GX, eds. Minutes of the International Symposium on the Protection of the Giant Panda Ailuropoda melanoleuca, Chengdu Foundation of Giant Panda Breeding, Chengdu, China, 1993: 351–53. 6 Mazzaro LM, Dunn JL, St. Aubin DJ, Andrews GA, Chavey PS. Serum indices of body stores of iron in northern fur seals (Callorhinus ursinus) and their relationship to hemochromatosis. Zoo Biol 2004; 23: 205–18. 7 Paglia DE, Miller CL, Foerster SH, Wynne JE, Tsu ICH, Kenny DE. Evidence for acquired iron overload in captive tapirs. Proc Conf AAZV 2000: 124–26. 8 O’Toole D, Kelly EJ, McAllister MM, et al. Hepatic failure and hemochromatosis of Salers and Salers-cross cattle. Vet Pathol 2001; 38: 372–89. 9 Wood C, Fang SG, Hunt A, Streich WJ, Clauss M. Increased iron absorption in lemurs: quantitative screening and assessment of dietary prevention. Am J Primatol 2003; 61: 101–10. 10 Weeks BR, Smith JE, Northrop JK. Relationship of serum ferritin and iron concentrations and serum total iron-binding capacity to nonheme iron stores in dogs. Am J Vet Res 1989; 50: 198–200. 11 Gonzales J, Benirschke K, Saltman P, Roberts J, Robinson PT. Hemosiderosis in lemurs. Zoo Biol 1984; 3: 255–65. 12 Dutton CJ, Junge RE, Louis EE. Biomedical evaluation of free-ranging ringtailed lemurs (Lemur catta) in Tsimanampetsotsa Strict Nature Reserve, Madagascar. J Zoo Wildl Med 2003; 34: 16–24. 13 Pietrangelo A. Hereditary hemochromatosis—a new look at an old disease. N Engl J Med 2004; 350: 2383–97.
Hepatitis C NS3 helicase unwinds RNA in leaps and bounds Recently, Victor Serebrov and Anna Marie Pyle used a novel approach to study how the hepatitis C NS3 helicase makes its way through double-stranded RNA.1 Surprisingly, the protein was found to have large unwinding translocation steps of equal size, which move in a discontinuous fashion. These characteristics suggest that www.thelancet.com Vol 364 October 16, 2004
nucleic acid motors can function in a manner analogous to kinesin and myosin. Hepatitis C virus (HCV) infections are a global health problem, affecting over 170 million individuals worldwide. 10–20% of those infected ultimately develop cirrhosis and 1–4% a year of the cirrhotic group develop liver cancer.2 1385
Comment
I
Tractor (T)
Plough (P)
T
P
II
ATP ADP
18 nt
18 bp T
P
VI "Rapid ripping"
III
ATP ADP
IV
T "Resting" "Rapid ripping"
P
V ATP ADP
Periodic motion of NS3 protein on duplex RNA Enzyme is depicted with two edges—leading edge which functions as tractor (T) and lagging edge which serves as plough (P). Enzyme is bound to RNA in stage I. Tractor translocates 18 bp and binds to duplex region, as indicated by hands (II). Plough unwinds 18 bp of duplex as it catches up to tractor (III and IV). Once it has caught up, protein “rests” briefly (V). Stages II–IV would correspond to “rapid ripping” phase of cycle, as suggested by Serebrov and Pyle.1 In VI, tractor translocates 18 bp again and enzyme repeats periodic cycle (III–V) until RNA is completely unwound. Energy for cycle comes from hydrolysis of ATP; it is unclear how many ATP molecules are hydrolysed per cycle. bp=basepairs, nt=nucleotides. Adapted from reference 10.
Unlike hepatitis B virus, no vaccine is available. The current standard treatment for chronic HCV infection, interferon alfa and ribavirin, succeeds in only 50% of patients and can cause serious side-effects.3,4 The development of new antiviral agents with minimum side-effects, designed to inhibit the activities of proteins essential to HCV proliferation, is thus essential to curbing this viral pandemic. Helicases are essential nanoscale motor-proteins that translocate or motor their way in one direction along DNA or RNA tracks, with energy liberated from ATP hydrolysis to drive their motion.5 Often, helicases are covalently coupled to, or associated with, proteins whose activities are unrelated to duplex-strand separation.6 The non-structural protein 3 (NS3) of HCV is one such helicase. NS3 is a multifunctional protein with a serine protease domain located in its N-terminal third, coupled to a helicase domain in its C-terminal two-thirds.7 Both the serine protease and helicase activities are essential for HCV replication.8 Thus NS3 has become a target for anti-HCV treatment, and understanding the biochemical mechanism of its helicase activity is of great importance.9 Helicases can function as bulldozers, driving their way into duplex molecules where they translocate along the unwound strands after they unwind the duplex. Alternatively, 1386
helicases can function as tractor-plough combinations: the tractor motors along the duplex and the plough separates the strands from behind. Regardless of the mechanism, the helicase must have a leading edge and a lagging edge and, in the two mechanisms mentioned, the roles of these edges are reversed. In the tractor-plough analogy, the leading edge binds the duplex while the lagging edge unwinds the nucleic acid strands. Each of these two edges can move in different increments. For example, the leading edge of the Escherichia coli RecBC enzyme translocates first, in 23 bp steps,10 while the lagging edge unwinds the duplex in 4 bp steps11 as it catches up to the leading edge. Cycles of large translocation steps followed by smaller unwinding steps repeat until the DNA is unwound or the enzyme dissociates. Serebrov and Pyle1 used a novel combinatorial timeresolved approach to gain insight into the helicase mechanism of NS3. The approach uses a large family of duplex RNA substrates containing, on average, a single nick at each position along only one strand of the RNA duplex. They monitored the microscopic behaviour of NS3 helicase at each nucleotide in duplex RNA by analysing the products of unwinding in gels. They found that the translocation and unwinding domains of the NS3 helicase move in equal 18 bp steps. These large equal-sized translocation unwinding steps are a new discovery for nucleic acid motors. More detailed quench-flow kinetic analyses showed that these domains progressed through periods of fast ripping followed by local pausing, and that these periods of movement and rest occurred with regular spacing along the duplex RNA molecule (figure). This periodic motion is analogous to the stepping behaviour observed for the cytoskeletal motor proteins kinesin and myosin.12 For helicases and cytoskeletal motor proteins, the conformational changes associated with the binding of ATP, its hydrolysis, and product release are coupled to large conformational changes that provide the driving force for the mechanical work used to separate the strands of the nucleic acid duplex, transport intracellular cargo, or contract muscle.12,13 Because ATPase activity is coupled to strand separation, researchers are actively pursuing potential specific inhibitors that target ATPase and helicase activities of NS3 protein.9 Some inhibitors target the helicase activity while others target the ATPase activity.14 These inhibitors show promise because they are specific potent inhibitors of viral replication and they also act on other viruses in addition to HCV. The success of these inhibitor studies requires detailed knowledge of the biochemical mechanism of the NS3 helicase.
Piero R Bianco Center for Single Molecule Biophysics, Department of Microbiology and Immunology and Department of Biochemistry, University at Buffalo, Buffalo, NY 14214, USA [email protected]
www.thelancet.com Vol 364 October 16, 2004
Comment
I thank Cindy Dlugos for comments. I am supported by NIH grant GM66831–01. I declare I have no conflict of interest. 1 2 3
4 5
6
Serebrov V, Pyle AM. Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 2004; 430: 476–80. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001; 345: 41–52. McHutchison JG, Gordon SC, Schiff ER, for the Hepatitis Interventional Therapy Group. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 1998; 339: 1485–92. Dev A, Patel K, McHutchison JG. New therapies for chronic hepatitis C virus infection. Curr Gastroenterol Rep 2004; 6: 77–86. Delagoutte E, von Hippel PH. Helicase mechanisms and the coupling of helicases within macromolecular machines, part I: structures and properties of isolated helicases. Q Rev Biophys 2002; 35: 431–78. Delagoutte E, von Hippel PH. Helicase mechanisms and the coupling of helicases within macromolecular machines, part II: integration of helicases into cellular processes. Q Rev Biophys 2003; 36: 1–69.
7
8 9 10 11
12 13 14
Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol 2000; 242: 55–84. Bartenschlager R, Lohmann V. Replication of hepatitis C virus. J Gen Virol 2000; 81: 1631–48. Tan SL, Pause A, Shi Y, Sonenberg N. Hepatitis C therapeutics: current status and emerging strategies. Nat Rev Drug Discov 2002; 1: 867–81. Bianco PR, Kowalczykowski SC. Translocation step size and mechanism of the RecBC DNA helicase. Nature 2000; 405: 368–72. Lucius AL, Vindigni A, Gregorian R, et al. DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies. J Mol Biol 2002; 324: 409–28. Vale RD, Milligan RA. The way things move: looking under the hood of molecular motor proteins. Science 2000; 288: 88–95. Hall MC, Matson SW. Helicase motifs: the engine that powers DNA unwinding. Mol Microbiol 1999; 34: 867–77. Borowski P, Deinert J, Schalinski S, et al. Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses. Eur J Biochem 2003; 270: 1645–53.
Chlamydia, gonorrhoea, and injectable progesterone
www.thelancet.com Vol 364 October 16, 2004
while a change led to reassignment to their new chosen arm for subsequent data collection. The women who initially chose DMPA were different at the outset: they were substantially more likely to attend the inner city clinic, and they were more likely to be older, black, and parous (all p<0·01).1 Most tellingly, at the start of therapy, the DMPA choosers were already more likely to be infected with chlamydia: 8·9% compared with 3·1% in oral contraceptive users and 4·6% in controls (p<0·05). Although the investigators attempted to statistically correct for later behaviours that could confound the apparent risk of acquiring infections, including numbers of sexual partners and condom use, the women who chose DMPA were clearly linked to different sexual networks than
Rights were not granted to include this image in electronic media. Please refer to the printed journal.
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Biological hypotheses to explain how progesterone could increase risk of cervical infections are unconvincing. Thus the recent finding by Charles Morrison and colleagues1 that the injection of depot medroxyprogesterone acetate (DMPA) was independently associated with a four-fold increase in chlamydial infection (hazard ratio 4·3, 95% CI 1·7–11·1) demands careful scrutiny. Chlamydia rates are rising in many countries2 and, 50 years after it was first developed, use of DMPA is growing in popularity. DMPA is a high-efficacy contraceptive that is administered as a 3-monthly injection. For many years, DMPA’s use was restricted because of concerns about possible long-term carcinogenic effects that have since been dispelled.3 Nevertheless, our impression is that DMPA maintains a residual aura of being a second-line contraceptive choice in the minds of many clinicians in some countries. Morrison and colleagues’ prospective study, of 819 women attending two reproductive health clinics in Baltimore, USA, and with no desire to become pregnant in the next year, was well conducted. Particular strengths were the ongoing monitoring of sexual behaviour and contraceptive methods, the use of sensitive tests for chlamydia, and the careful quantification of cervical ectopy. There was no significant association between combined oral contraceptives and cervical infections, nor was DMPA associated with an increased risk of gonorrhoea. Although cervical ectopy appeared to have a limited, if any, role in the increased risk of chlamydia with DMPA use, it was a marginally significant risk factor for cervical infections in its own right (hazard ratio 2·3, 95% CI 0·9–6·0). However, Morrison and colleagues did an observational cohort study, not a randomised trial. For ethical reasons the women were allowed to choose their method of contraception: combined oral, DMPA, or no hormonal contraception. There is no mention of the level of persuasion by the clinicians that might have affected this choice. About two-thirds of the women used the same method throughout the study,
Coloured transmision electron-micrograph of Chlamydia trachomatis in cells of Fallopian tube Bacteria (red) occur in inclusions (yellow) inside cells.
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