Biol. Chem. Hoppe-Seyler, Vol. 377, pp. 71 - 86, February 1996 · Copyright © by Walter de Gruyter & Co · Berlin · New York

Review Cystatins in Health and Disease

Yvonne M.C. Henskens,* Enno C.I. Veerman 1986). HIV-I also needs proteolytic processing by cysteine and Arie V. Nieuw Amerongen proteinases to regulate the expression of viral proteins Department of Oral Biochemistry, Academic Centre for (Guy ef a/., 1991). Protozoal cysteine proteinases are pro- Dentistry Amsterdam (ACTA), Van der Boechorstraat 7, duced by a number of protozoan parasites to facilitate NL-1081 BT Amsterdam, The Netherlands host invasion, metabolize host proteins, to degrade the host immune molecules or to use them for intracellular replication. Cysteine proteinases are produced by e.g. * Corresponding author Trypanosoma cruzi, the causative agent of American try- panosomiasis or by Leishmania species, causing one of the six major parasitic diseases. Furthermore, human ma- Proteolytic enzymes have many physiological func- tions in plants, bacteria, viruses, protozoa and mam- larial parasites produce a broad scala of proteinases in- mals. They play a role in processes such as food cluding cysteine proteinase which are involved in erythro- digestion, complement activation or blood coagula- cyte invasion and rupture (McKerrow, 1993). Mammalian tion. The action of proteolytic enzymes is biologically cysteine proteinases are present in the lysosomes of cells. controlled by proteinase inhibitors and increasing at- Lysosomes contain different cysteine proteinases, the tention is being paid to the physiological significance most important being the cathepsins B, H, L and S (Barrett of these natural inhibitors in pathological processes. and Kirschke, 1981, Barrett et a/., 1988). These cathep- The reason for this growing interest is that uncon- sins have common ancestors and are related to papain. Cathepsin B has been detected in every tissue or cell ex- trolled proteolysis can lead to irreversible damage e.g. amined ranging from macrophages to stratified squa- in chronic inflammation or tumor metastasis. This re- mous epithelial cells (Howie etal., 1985). Cathepsins B, H view focusses on the possible role of the cystatins, and L have a broad substrate specificity compared to natural and specific inhibitors of the cysteine protein- other lysosomal enzymes such as the aspartic proteinase ases, in pathological processes. cathepsin D. Lysosomal cysteine proteinases are well Key words: Cystatins / Cysteine proteinases / adapted to function under the acidic and reducing condi- Inflammation / Inhibitors / Pathological processes. tions of the lysosomal system and play an important role in the intracellular protein turnover (Kominami et a/., 1991). They are also involved in muscle protein turnover and Cysteine Proteinases cleavage of a number of precursor proteins, e.g. the liberation of thyroxins from iodinated thyroglobulin. In Cysteine Proteinases, synonymous with thiol proteinases, addition, they are, like collagenases, capable of degrad- are small proteins with molecular masses varying from 23 ing type I collagen. Although these enzymes show maxi- to 24 kDa and with two catalytic residues, Cys25 and mal activity at mildly acidic conditions, there are reports of His159, which are involved in the hydrolytic reaction. Cys- hydrolytic activity at neutral pH (Buttle et a/., 1988). teine proteinases catalyze the hydrolysis of various poly- Furthermore, it is well known that the local pH of metaboli- peptide substrates and are most active under reducing cally active macrophages during inflammatory reactions and mildly acidic conditions (pH 5 - 6.5). They have been or in the osteoclastic resorption lacuna is acidic, creating detected in and isolated from a large number of biological a suitable pH for the extracellular action of lysosomal cys- sources including plants, bacteria, animals and humans. teine proteinases. The action of human cysteine protei- Bacterial cysteine proteinases are produced by Clostri- nases can lead to irreversible damage if, under patholog- dium histolyticum, named clostripain, by hemolytic group ical conditions, lysosomal enzymes are secreted or re- A streptococci and by the pathogenic anaerobic Porphy- leased by autolysis. romonas gingivalis, a bacterium associated with perio- dontitis. Bacterial cysteine proteinases probably play a role in the penetration of normal tissues by the bacteria Cystatins, Natural Inhibitors of Cysteine and in the food digestion. Viral cysteine proteinases from Proteinases picornaviruses, e.g. the poliovirus and rhinovirus type I, are involved in proteolytic cleavage of precursor proteins Based on the amino acid sequences, the proteins with for virus replication and for the production of new virus cysteine proteinase inhibitory activities can be assigned particles. A virus-coded cysteine proteinase is involved in to three major families (Barrett etal., 1986a). The officially this process (Kay and Dunn, 1990; Körant et a/., 1985; assigned members of the cystatin superfamily and the 72 Y.M.C. Henskens et a/.

Table 1 The Officially Assigned Members of the Cystatin Superfamily (Rawlings and Barrett, 1990).

Family 1 Family 2 Family 3 Cystatin related proteins

Cystatin A (stefin A) human Cystatin C (-ã-trace) human L-kininogen C-Ha-ras-oncogene p21 Cystatin B (stefin B) human Cystatin D human human, rat, bovine Histidine-rich glycoprotein human Cystatin á rat Cystatin S (SAP-1) human H-kininogen aHS-glycoprotein human Cystatin β (cystatin B) rat Cystatin SA human human, rat bovine Oryzacystatin I Cystatin SN (SU, SA-I) human T-kininogen rat Oryzacystatin II Cystatin C rat Kininogen ox Cystatin S rat Cystatin C mouse Cystatin chicken egg-white Cystatin bovine colostrum Cystatin African puff adder Cystatin ox Cystatin Drosophila Sarcocystatin

Table 2 Characteristics of Human Cystatins.

Cystatin MW No of pi Reference Chromosomal Reference amino localization acids

Cystatin A 11,006 98 4.5-4.7 Machleidtefa/,,1983 STF1 3cen-q21 Fong etal., 1991; Hsiehefa/., 1991 Cystatin B 11,175 98 5.6-6.3 Ritonjaefa/,,1985 - - - Cystatin C 13,343 120 9.3 Abrahamson etal., 1987b CST3 20p11 .2 Abrahamson et a/.,, 1989; Saitoh et a/., 1989 Cystatin D 13,885 122 6.8-7.0 Freijeefa/,,1991 CST5 20p11.2 Freijeefa/., 1993b Cystatin S 14,189 121 4.4-4.6 lsemuraefa/.,1991 CST4 20p11.2 Saitoh etal.,1988; Saitoh etal., 1991 Cystatin SA 14,351 121 4.3 lsemuraefa/.,1991 CST2 20p1 1.2 Saitoh etal.,1988; Saitoh etal., 1991 Cystatin SN 14,316 121 6.6-7.0 lsemuraefa/.,1991 CST1 20p11.2 Saitoh etal.,1989; Saitoh etal., 1991 L-kininogen 68,000 409 4.5 Katoefa/,,1981 KNG 3q26-qter Fong et a/., 1991 H-kininogen 114,000 628 4.5 Katoefa/,,1981 KNG 3q26-qter Hsiehefa/., 1991

Inhibition constants of complexes with cathepsins Kj[nM]

CathepsinB CathepsinH CathepsinS CathepsinL

Cystatin A 8.2 0.31 1.3 0.05 Cystatin B 73 0.58 0.23 0.07

Cystatin C 0.25 0.28 < 0.005 0.008 Cystatin D >1000 8.5 25 0.24 Cystatin SN 19

H-kininogen 600 1.2 0.017 L-kininogen 400 1.1 0.109 -

References: Abrahamson, 1993; Abrahamson etal., 1994b; Barrett etal., 1986b. - is not determined.

Family 1 cystatin related proteins are summarized in Table 1, their characteristics in Table 2A and 2B and their concentra- Family 1 comprises cystatins consisting of 100 amino tions in various body fluids in Table 3. In the next part only acids residues, having a molecular mass of 11 kDa, no di- the human cystatins and rat cystatin S will be described in sulfide bonds or carbohydrate chains and found mainly in- more detail. tracellularly. The gene encoding for human cystatin A has Cystatins in Health and Disease 73

Table 3 Concentrations [/xg/ml] of Family 1 and 2 Cystatins in Human Body Fluids.

Body fluid Cystatin [/ig/ml]

B SA SN S+SA+SN D a a b _ _ _ _ _a Plasma 1.3 ;1.1 _ Urine -a -a 0.095b _ _ _a _a a Seminal plasma 0.6a 1.1a 40 + d +d -d 16a -a _ _a Milk -a -a 4a - _a a a a d d d a a Tears 1.1 - 3 + _+ + 57_ 0.7 Sweat + - - - Whole saliva 4.4a 1.3a; 1.1· 116e 11a 4a Submandibular/sublingual saliva - +d +d +d;90f 1309 - h h - - Submandibular saliva 1.5 177 _ Sublingual saliva - - 0.6h 37h - e e d d 9 - Parotid saliva C.4 1.0 ;+ _+ 1.1' _1.6 Palatinal saliva 1.5h 56h - _ _a a Amnioticfluid 2.2a 0.1a 1.3a - - _ _ _a Cerebrospinal fluid -a -a 14a - -a Synovial fluid -a -a 2.7a - _ __ _a __ a

- not detected; + qualitatively estimated no figure or symbol is not determined. a Derived from μΜ quantities using the molecular masses from Table 2A by Abrahamson et al., 1994b; b determined by L fberg and Grubb, 1979; c determined by Shomers etal., 1982a; 1982b; 1982c; d determined by Isemura etal., 1991; 6 determined by Henskens etal., 1996b; f determined by Aguirre etal, 1990; 9 determined by Aguirre etal., 1992; h determined by Veerman etal. 1996b.

been assigned to chromosome 3cen-q21 (Hsieh et al., tered on a 1.2 Mb segment on chromosome 20 (Abraham- 1991); the human cystatin B gene has not been assigned son ef al., 1988; Freije et al., 1993a; Saitoh ef al., 1988; yet. Human cystatin A, also named stefin A by a group of 1989; 1991; Schnittger ef al., 1993). Human cystatin C the Stefan Institute in Ljubljana who first isolated and se- was first detected by Clausen (1961) as a trace protein quenced this inhibitor, is an acidic protein with a pi be- present in cerebrospinal fluid (CSF) but not in serum. This tween 4.5 and 4.7 and a molecular mass of 11 kDa (Mach- protein was originally named post-^-protein and later leidt et al., 1983). It was primarily detected in epithelial -/-trace (Hochwald andThorbecke, 1963; Hochwald ef al., cells and PMNs suggesting a primary defensive role 1967), post--/ (Laterre and Heremans, 1963) or post-ã- against cysteine proteinases produced by pathogens in- globulin (Colle ef a/., 1976). -ã-Trace was later also vading the body. Cystatin A was found in liver, spleen, detected in human biological fluids such as plasma, saliva placenta, oral mucosa and uterus (Davies and Barrett, and seminal plasma whereas urine contained only trace 1984; Brzin ef al., 1983) and in some body fluids. Human amounts (L fberg and Grubb, 1979; Grubb ef al., 1983). cystatin B is a more neutral protein with a pi between 5.7 The complete amino acid sequence of -ã-trace was first and 6.3 and a molecular mass of 11.2 kDa (Ritonja et al., presented in 1982 (Grubb and L fberg, 1982), together 1985). It is widely distributed in various cells and tissues: with the detection of -ã-trace in brain cortical neurons liver, spleen, placenta, epithelial cells, lymphocytes, mo- (L fberg ef al., 1981 a) and adenohypophysis (Grubb and nocytes and to a much lesser extent in PMNs (Brzin ef al., L fberg, 1982). The biological function of this protein re- 1982). This ubiquitous distribution of cystatin B indicates mained obscure until Barrett ef al. (1984) and Brzin etal. a general protective role for this cystatin against uncon- (1984) discovered that -ã-trace showed high amino acid trolled activities of host lysosomal cysteine proteinases. sequence homology with a cysteine proteinase inhibitor Cystatin B has been detected in only one body fluid, isolated from sera of patients suffering from autoimmune namely seminal plasma. disease as well as with cystatin from chicken egg-white. A new name, cystatin C, was proposed and inhibitory ac- tivities towards papain, cathepsins B, H and L were de- Family 2 termined. Recombinant cystatin C was expressed in Family 2 cystatins consist of 115 to 120 amino acids resi- Escherichia coli (Dalboge ef al., 1989) having the same dues and exhibit molecular masses of 13 to 14 kDa; they characteristics as native cystatin C (Abrahamson ef al., contain two disulfide loops near the carboxyl terminus. 1988). Because of the widespread extracellular distribu- This family of cystatins has been detected mainly extra- tion of cystatin C, it has been suggested to play a regula- cellularly. The genes encoding for these cystatins are clus- tory and defensive role against host or exogenous cys- 74 Y.M.C.Henskensefa/. teine proteinases present in body fluids. Human cystatins tin D expression was found only in parotid gland tissue S, SA, SN were first isolated from saliva by Juriaanse and and not in other tissues (seminal vesicle, liver, placenta) Booy (1979a, b) as acidic and neutral unglycosylated pro- (Freije ef al., 1991). Cystatin D was also detected in whole teins with affinity for hydroxyapatite. The three cysteine- saliva and tears whereas cystatin D concentrations in containing proteins isolated from human submandibular- seminal plasma, blood plasma, milk and cerebrospinal sublingual saliva by Shomers etal. (1982a, b, c) are proba- fluid were below sensitivity levels (Freije ef al., 1993a). bly identical to these cystatins as judged by their bio- These results indicate that the distribution of cystatin D is chemical properties. These proteins were also detected in more tissue restricted than that of cystatin C. Rat cystatin parotid saliva and tears but not in nasal secretions, labial S is hardly present in saliva or salivary glands of rats. How- salivary gland secretion or colostrum. However, their ever, after chronic sympathetic stimulation of the salivary function as cysteine proteinase inhibitors was not known glands by isoproterenol (IPR), a ß-adrenergic agonist, the at that time. The first report of the presence of cysteine submandibular gland produces large amounts of a cysta- proteinase inhibitors in human saliva was from Minakata tin that shares 40 - 50% amino acid sequence homology and Asano (1984). Isemura et al. (1984a) isolated and se- with human cystatin S (Shaw ef al., 1988; Shaw and quenced the salivary cystatin named SAP-1 and found Barka, 1989; Bedi, 1989a, b). Bedi (1990; 1991 a, b) 54% amino acid sequence homology with cystatin C. Its showed that the induction of cystatin S production in sub- inhibitory activity towards papain was analyzed and mandibular glands was 26 times the normal value, in sub- SAP-1 was subsequently renamed cystatin S (Isemura et lingual glands 28 times and in parotid glands 32 times. al., 1984b). Later the neutral non-phosphorylated cystatin Other tissues (liver, spleen) showed no cystatin S synthe- SN (Isemura et al., 1986) and the acidic non-phosphory- sis after IPR treatment whereas only trace amounts were lated cystatin SA (Isemura et al., 1987) were isolated and found in kidney and pancreas. sequenced. The three salivary cystatins share 90% amino acid sequence homology with each other. AI-Hashimi et Family 3 al. (1988) reported that the cysteine-containing phos- Kininogens are intravascular multifunctional proteins con- phoproteins were identical to the salivary cystatins. Cys- taining three cystatin-like domains. In mammals, three tatin S appears to consist of three isoforms with different types of kininogens have been found with different sizes phosphate content: cystatin S, containint no phosphate, and structures (Müller-Esterl ef al., 1986; Müller-Esterl, cystatin S1, which is monophosphorylated (Ser3) and cys- 1 3 1989; Kato ef al., 1981). The kininogen gene has been tatin S2, which is diphosphorylated (Ser , Ser ) (Isemura et mapped to the long arm of chromosome 3 and further lo- al., 1991; Ramasubbu et al., 1991). Isolation of the three calized to 3q26-qter (Fong ef al., 1991). High (H) and low full sized isoforms of cystatin S is difficult because of their (L) molecular weight kininogens were first known as the susceptibility to degradation. Screening of other secret- biosynthetic precursors of vasoactive kinins which also ory fluids for presence of cystatins demonstrated that play a role in the intrinsic blood coagulation cascade. La- tears contained cystatin SN, SA and S1, whereas seminal ter it was discovered that the previously isolated «1 - and plasma and parotid saliva contained minor amounts of SA a2-cysteine proteinase inhibitors present in human and S1 (Isemura et al., 1991; Barka ef al., 1991). Cystatins plasma (Ohkubo ef al., 1984) are identical to these kini- S, SA and SN in whole saliva are predominantly derived nogens (Müller-Esterl ef al., 1985); Sueyoshi ef al., 1985). from the submandibular-sublingual gland and to a lesser The heavy chains of L- and H-kininogen harbor three do- extent from the parotid gland (Aguirre ef al., 1990; Rath- mains (D1, 02, 03) of mutual sequence homology (Sal- man etal., 1990a). Immunochemical analysis of subman- vesen ef al., 1989). Two of these are functionally active in- dibular, sublingual and palatal saliva, using individual hibitors of cysteine proteinases (D2, D3), whereas the third segregators, shows that submandibular saliva contains one (D1) displays no inhibitory activity. The kininogens are more cystatin S than sublingual or palatal saliva (Veerman synthesized in the liver and secreted into blood plasma. ef al., 1996). The differences in secretion of cystatins by Their concentration is highest in blood plasma and syno- the submandibular and parotid glands have been con- vial fluid, whereas in other body fluids such as colostrum, firmed by immunolocalization (Isemura ef al., 1984b; cerebrospinal fluid, seminal plasma and tears only trace Rathman ef al., 1990b) and by in situ hybridization (Saba- amounts were detected (Abrahamson ef al., 1986). The tini ef al., 1989; Bobek ef al., 1991). Escherichia co//ex- kininogens, together with a2-macroglobulin, are the major pression systems were used for the expression of cDNA inhibitors of cysteine proteinases in blood plasma. clones encoding human cystatins SN and S (Bobek ef al., 1993,1994). The biological function of the cystatins in sa- Inhibitory Mechanism and Stability liva, tears or seminal plasma is not yet established. It is hy- pothesized that they play a role in the protection of the oral The amino acid sequences of cystatins are highly con- cavity and the eyes against the proteolytic activity of cys- served in three domains which are important for the inhib- teine proteinases of bacteria, viruses or host inflammatory itory activity. The first domain is the N-terminal Gly9 which cells. Human cystatin D, a recently discovered neutral seems to be important for the optimal orientation of the cystatin species, has been cloned from a genomic library N-terminal region towards the cysteine proteinase (Hall ef using a cystatin C cDNA probe (Freije ef al., 1991). Cysta- a/.,1993). The second and third cystatin domain involved Cystatins in Health and Disease 75 in the inhibition are the first and second hairpin loops. The A, B and chicken egg-white cystatin (Lenarcic et a/., first hairpin loop consists of the highly conserved amino 1988b). Cystatin C in cerebrospinal fluid and urine is ef- acid residues Gln53 to Gly57 (QXVXG-region) while the fectively degraded by host or bacterial proteolytic en- second hairpin loop contains the conserved Pro103 and zymes (Abrahamson et a/., 1986). Human PMN elastase Trp104. Several studies using synthetic peptides or re- rapidly cleaves the Val8-Gly9 bond of human cystatin C at combinant cystatins having single mutations showed the neutral pH, resulting in a decreased affinity for papain as importance of the three cystatin domains in the inhibition well as for cathepsins B, H and L (Abrahamson et al.t of cysteine proteinases. Truncated forms of cystatin C 1991 a). Other PMN serine proteinases, cathepsin G and lacking the first 11 amino acids display at least 1000-fold proteinase 4, do not hydrolyse peptide bonds in cystatin C weaker inhibition of papain (Abrahamson et a/., 1987a, b) (Abrahamson etal., 1991b). This suggests that truncated underlining the important role of the N-terminusforthe in- cystatin C in vivo plays practically no role in the inhibition hibitory interaction. Studies using chemically or geneti- of cathepsin B and that the PMN elastase might play a role cally modified chicken egg-white cystatin or cystatin C in the regulation of cystatin activity. Cystatin C is much have confirmed these findings (Machleidt et a/., 1989; more stable in blood plasma than in CSF and urine, proba- 1991; Grubb et a/., 1990; Abrahamson et a/., 1991 a, b; bly due to the presence of proteinase inhibitors in blood- Genenger et a/., 1991; Lindahl et a/., 1992; Hall et a/., plasma. Enzymes in the culture supernatant of Porphyro- 1992; Lalmanach et a/., 1993). Cystatin A is also reported monas gingivalis degraded cystatin S 14 kDa to a 12 kDa to be inactivated after removal of the first 15 N-terminal re- band, without affecting the biological activity (Rathman ef sidues (Samejima etal., 1986). Amino acid substitutions in a/., 1990c). In contrast, human cystatin A is cleaved and the QXVXG region of recombinant chicken egg-white cys- inactivated by the isolated aspartic proteinase of Candida tatin reduced the efficiency of inhibition of papain and ca- albicans (Tsushima et a/., 1994). The inhibitory activities of thepsin B by 10- to 1000-fold (Auerswald et a/., 1992). On kininogens are stable at neutral and alkaline pH but not at the other hand, inhibition of cathepsin L was unaffected pH values below 4 or elevated temperatures (50-90 °C) by amino acid substitutions in the QXVXG region, sug- (Sasaki ef a/., 1981). gesting differences in proteinase-inhibitor interactions between closely related cysteine proteinases. Modifica- tion of the Trp104 in chicken egg-white cystatin reduces the affinity of the inhibitor for papain (Lindahl ef a/., 1988). Cystatins in Pathological Processes Reduction of the disulfide bond between Cys71 and Cys81 has no effect on the inhibitory activity of chicken Hereditary Cystatin C Amyloid Angiopathy (HCCAA) egg-white cystatin. In contrast, the less accessible Icelandic hereditary cystatin C amyloid angiopathy Cys95-Cys115 disulfide bond is of importance for main- (HCCAA) is an autosomal dominant disorder character- taining the native conformation (Björk and Ylinenjärvi, ized by amyloid deposition of a cystatin C variant in almost 1992). Cystatin C is the most universal and potent inhibi- all tissues including the cerebral arteries. This deposition tor, binding tightly to all cysteine proteinases investigated, results in fatal cerebral hemorrhages in young non-hyper- including the lysosomal cathepsins. It is feasable that the tensive adults and is the main cause of death before 40 cystatins A, B and SN, which bind weakly to cathepsin B, years of age in these individuals. The cystatin C variant play no role in the inhibition of this cysteine proteinase in has a single amino acid substitution caused by a T->A vivo. The L- and H-kininogens are strong inhibitors of pa- point mutation in the codon for leucine at position 68, pain and cathepsin L and weaker inhibitors of cathepsin H which is replaced by glutamine in the mutated cystatin C and particularly of cathepsin B. Domain 2 of L-kininogen (Ghiso etal., 1986; Palsdottir etal., 1988; Levy etal., 1989; displays inhibitory activity against the calcium-activated Abrahamsson etal., 1992). HCCAA can be diagnosed by cysteine proteinase, calpain, which is not shared by Fam- the direct demonstration of the loss of an Alu I restriction ily 1 or Family 2 cystatins. Regarding stability, the cysta- site in exon 2 of the cystatin C gene (Palsdottir etal., 1988; tins are very stable to conditions of extreme pH and high Abrahamson etal., 1992; Jonsdottir and Palsdottir, 1993). temperature. For example, human cystatin A is stable to At present, 191 persons in Iceland have been screened for boiling for 20 minutes, treatment with 5% trichloroacetic the HCCAA mutation showing that 36 of these individuals acid, 4M NH4OH (pH 11.9) and 20mM Na3PO4 (pH 12.1) have the disease. Furthermore, cystatin C levels of cere- (Järvinen, 1978). Human cystatin B is stable during stor- brospinal fluid (CSF) from HCCAA patients are signif- age at-20 °C or 4 °C and, like cystatin A, heat-stable even icantly lower compared to CSF from healthy individuals for 10 min at 80 °C (Green et a/., 1984). Cystatin C is com- (2^g/ml versus 7^g/ml) (Grubb, 1984). The pa- pletely stable at 80 °C for 10 min, pH 6.5 (Brzin etal., 1984) thogenic mechanism behind the deposition of mutant and at pH 2.0 at 25 °C for 10 min (Barrett etal., 1984). The cystatin C might be that the substitution of one amino acid salivary cystatins are also stable to extremes of pH (2-10) changes the physical characteristics of cystatin C in such and heat (60 min 85 °C or 15 min 100 °C) Minakata and a way that it cannot be processed regularly. This could in- Asano, 1984). Proteolytic degradation of cystatins could volve the intracellular processing of cystatin C or its ex- be of physiological importance. The aspartic proteinase tracellular breakdown (Löfberg etal., 1987; Thorsteinsson cathepsin D for example cleaves and inactivates cystatins etal., 1992). Abrahamson etal. (1994a) published a paper 76 Y.M.C. Henskens etal. in which they produced the mutant cystatin C in E. coil. lowest cystatin activities (Lah efa/., 1992a, b). Scaddan et This mutant cystatin C lost its biological activity rapidly al. (1993) investigated cystatin activity in culture super- and formed aggregates of dimers when the temperature natant of two breast cancer cell lines, MCF-7 (low metas- was increased from 37 °C to 40 °C. This may have clinical tatic), and MCF 7/AdrR (highly metastatic), and showed significance for the treatment of HCCAA because preven- that cystatin activity was lowest in supernatant of the tion of fever episodes might reduce the formation of ag- highly metastatic cell line. Since epithelial cells in various gregates. Benedikz et al. (1989) showed that 5 out of 36 organs are rich in cystatin A a number of studies have patients suffering from the more common cerebral amy- been performed on the comparison of epithelial carcino- loid angiopathy, present in Alzheimer's disease, had cys- mas and normal tissues. In general these studies suggest tatin C, next to the omnipresent ß-protein, in their senile that levels of cystatin A are diminished in malignant situ- plaques indicating that the native cystatin C also has a ations. For example, ovarian carcinoma, a tissue of epi- tendency to deposit as amyloid (Li et al., 1993; Maruyama thelial origin, contained negligible amounts of cystatin A efa/., 1992). compared to various normal tissues, whereas cystatin B was present in normal quantities (Lah efa/., 1990; Kastelic ef al., 1994). Similarly, benign prostate epithelium con- Tumors and Metastasis tained less cystatin A than normal prostate tissue and Tumor malignancy has been linked to increased activities adenocarcinoma cells contained no cystatin A at all of the lysosomal cathepsins B, H, and L Cysteine protei- (Söderström efa/., 1995). In this respect the authors pro- nases secreted by cancer cells may facilitate tumor inva- pose that cystatin A could function as a marker for histolo- sion and metastasis by assisting tumor cell penetration gical differential diagnosis of prostatic adenocarcinoma through stromal tissue and by degrading biological bar- and benign lesions. Järvinen efa/. (1987) investigated cys- riers such as basement membranes. For example, plas- tatin A in normal and malignant epithelial tissues by immu- ma-membrane fractions of several human tumors contain nohistochemical staining. They also found lower amounts cathepsin B-like (Sloane efa/., 1987) and cathepsin L-like of cystatin A in malignant squamous epithelia. Hawley- (Rozhin et al., 1989) activities and cathepsin L mRNA was Nelson efa/. (1988) found highest expression of cystatin in expressed in higher levels in carcinoma of breast, ovary, mice skin papillomas followed by mice primary epithelial colon, adrenal gland or bladder, compared to normal tis- cells. In agreement with the human studies they also sues (Chauhan et al., 1991). Buck et al. (1992) demon- observed lowest expression of cystatin A in mice skin strated that increased malignancy of several types of hu- carcinomas. Based on these results they suggested that man and animal tumors is associated with increased ca- cystatin A may be important for the regulation of cell dif- thepsin B activity and gene expression. Moreover, Lah et ferentiation. Furthermore, Lah et al. (1992a, b) demon- al. (1989 a) and Guinec efa/. (1993) showed that cathepsin strated that decreased immunostaining of cystatin A in B and L degrade basement membrane in vitro by liber- human breast carcinoma was correlated with an in- ating collagen IV, laminin and fibrinonectin fragments. It creased degree of malignancy and differentiation of the has been stated that cathepsins should be regulated ex- tumor. In addition to decreased amounts of cystatin A in tracellularly by cystatin C and intracellularly by cystatins A malignant tissues, Lah efa/. (1989b, 1990) reported an al- and B. Therefore, various investigations havefocussed on tered cystatin A activity after detecting that cystatin ex- the question whether impaired regulation by cystatins tract from various sarcomas was less effective towards may play a role in tumor metastasis. On the other hand papain than healthy liver extract, whereas there was no Collella ef al. (1993) suggested an opposite effect of change in cystatin concentration. Characterization of cystatins in the process of malignancy, considering that both cystatin A and B isolated from sarcomas indicates an excess production of i.e. cystatin C could inhibit the that inhibitory activity of cystatin A towards cathepsin B is proteolytic attack of cathepsins on the cancer cell by decreased compared to normal cystatin A. In contrast, suppressing the host inflammatory response (Nishida ef isolated tumor cystatin B exhibited the same affinity to- al., 1984; Järvinen efa/., 1987; Leung-Tack efa/., 1990a, wards papain, cathepsin B and Las the normal cystatin B. b) and in this way enhancing the oncogenicity of the The molecular basis underlying this altered cystatin A ac- cell. Studies on the determination of cysteine proteinase tivity has not been elucidated yet. Besides cystatin A, B inhibitory activity have been focussed on a possible im- and kininogen, cystatin C has been detected in ascites balance between the proteinases and their inhibitors in fluid from patients with ovarian cancer. The inhibitory ac- malignant tissues. Human lung tumors displayed an im- tivity of cystatin C isolated from ascites towards cathepsin balance between cathepsin B levels and cystatin activity B was unaltered (Lah efa/., 1990; 1991). A number of car- compared to normal tissue (Knoch efa/., 1994) which was cinoma cell lines have been reported to secrete cystatin C, also shown in human amelanotic melanoma: Rapidly including a human colon carcinoma cell line, f ibrosarcoma growing tumors contained an excess of cathepsin B and cell lines (Keppler efa/., 1994, Corticchiato efa/., 1992) too little cystatin activity for complete inhibition (Sloane ef and a human medullary thyroid carcinoma cell line (Barka al., 1990). Cystatin activity present in breast carcinoma efa/., 1992). Cystatin C has been investigated as a possi- was also decreased compared to normal tissue. Besides, ble marker for tumor cells although Jacobsson efa/. tissues with the highest cathepsin activities displayed the (1995) concluded that cystatin C determined in normal or Cystatins in Health and Disease 77 tumor proximal tubular cells could not function as a activity of whole saliva was highest in patients suffering marker of renal carcinomas. In line with this Lignelid etal. from severe periodontitis when compared to healthy sub- (1992) found that cystatin C was expressed not only in hu- jects (Henskens ef al., 1993a). This enhanced activity ap- man pancreatic and gut carcinoid tumors but also in all pears to be at least partially caused by an increased cys- other normal tissues investigated, indicating that it is also tatin C concentration (Henskens ef al., 1993b; 1994). not a reliable marker for endocrine gastro-entero pan- When the origin of cystatin C in whole saliva was studied, creatic tumors. In brief, malignant tissues appear to have increased values of cystatin C, correlating with enhanced less cystatin activity compared to healthy tissues resulting cystatin activity, were detected in parotid secretions of in an imbalance between the cysteine proteinases and periodontitis subjects (Henskens etal., 1996b) and whole their natural inhibitors. Lower levels of cystatin A or an im- salivary cystatin C levels were reduced after periodontal paired functioning cystatin A might be involved in this im- treatment (Henskens etal., 1996a). Furthermore, in a case balance whereas the amounts and biochemical properties study on experimental gingivitis an induction of cystatin C of cystatins B and C appear to be unaltered. secretion by the submandibular and parotid glands was observed when the degree of gingival inflammation was maximal (Henskens et al., 1994). This suggests that the Inflammatory Diseases salivary glands respond to inflammatory conditions of the Due to its epidermal origin cystatin A was extensively oral cavity by enhancing the protective potential and se- studied in inflammatory skin diseases. For example, Jär- creting more cystatin C. Inducible cystatins have also vinen etal. (1987) investigated normal and diseased tis- been described in the rat salivary gland which secretes sues of psoriasis patients who suffered from abnormal hardly any cystatin S under normal conditions but secre- keratinization and inflammatory reactions of the epidermis tion is induced after treatment with a ß-adrenergic agonist and demonstrated increased amounts of cystatin A in (Bedi, 1989a, b). Induction of glandular cystatin S expres- both the inflammatory skin samples and psoriatic epi- sion was also found after treating rats with inflammatory dermis. On the other hand, a cysteine proteinase inhibitor, stimuli (Cohen etal., 1989; 1990) or by irritant actions such probably cystatin A, isolated from psoriatic skin was less as incisor amputation (Yagil etal., 1986). Oral administra- stable and less active towards papain compared to the in- tion of papain, the cysteine proteinase from Papaya latex, hibitorfrom normal cells (Ohtani etal., 1982). In contrast to to rats caused a dramatic increase in the salivary level of this, and in line with Järvinen etal. (1987). Hopsu-Havu et cystatin S (Naito ef al., 1992) suggesting a biological re- al. (1983) reported that the total cystatin activity of sponse which might prevent tissue damage caused by an psoriatic skin was higher than in normal epidermis. In ad- exogenous cysteine proteinase. Comparable results were dition, the more neutral inhibitor, probably cystatin B, was reported by Alves ef al. (1994) who found an induction of only present in psoriatic skin and not in normal skin ex- cystatin S in saliva of rats experimentally infected by Try- tracts. panosoma cruzi. The authors propose that the detection Periodontal inflammatory diseases which affect the tis- of the inducible cystatin might serve as a sensitive marker sues that support the teeth have been associated with of tissue damage in the salivary glands as described for high levels of cathepsins B, H and L in gingival tissues and Chagas disease. Regarding the salivary gland derived gingival crevicular fluid (Cox and Eley, 1987; 1989; Eisen- cystatins in humans, Aguirre ef al. (1992) found no differ- houwer etal., 1983; Eley and Cox, 1992a, b). The origin of ences in the total concentration of cystatins S, SN and SA these cathepsins during inflammatory reactions are most between a healthy and a periodontitis group. The only pa- probably the monocytes or macrophages. Since cysteine per on cystatins in gingival crevicular fluid, another possi- proteinases are suspected to play a role in the pa- ble source of cystatins in whole saliva, demonstrated that thogenesis of inflammatory periodontal disease, cystatins inhibitory activity against papain was present in gingival in gingival tissue, saliva and crevicular fluid have been crevicular fluid (GCF) of one gingivitis and three periodon- studied as possible protective proteins in the process of titis patients (Ichimaru ef al., 1992). tissue and bone destruction. Inflamed gingival tissue ho- A cysteine proteinase, similar but not identical to lyso- mogenates contain cystatin A, cystatin C and kininogen somal cathepsin B, was isolated from human purulent (Babnik et al., 1988) and cystatin C concentration was sputum from patients with inflammatory lung diseases negatively correlated to pocket depth (Skaleric et al., (Buttle ef al., 1988). Another study indicated a high corre- 1989). Despite the low concentration of cystatin C present lation between sputum cathepsin B and markers of in- in gingival tissues, compared to cystatin A, it should be flammation such as elastase and myoeloperoxidase (But- able to inhibit cathepsin B under pathophysiological con- tle ef al., 1990). Cystatins A, C and S were also present in ditions (Skaleric et al., 1989). Cystatin C in gingival tissues these samples. Cystatin C in sputum might originate from originates most probable from inflammatory cells such as the bronchial epithelial cells (Burnett ef al., 1995) or alveo- monocytes and macrophages which have been demon- lar macrophages (Warfel ef al., 1991). The cystatin C levels strated to secrete cystatin C in wfro(Chapman etal., 1990; were negatively correlated to the level of myeloperoxidase Warfel ef al., 1987; 1991). Salivary cystatins could play a indicating that highest cystatin C levels were found in protective role towards cysteine proteinases from en- non-inflamed sputum samples. It has been suggested, dogenous or exogenous sources. Accordingly, cystatin therefore, that cystatin C in purulent sputum samples is 78 Y.M.C. Henskens et a/.

Table 4 An Overview on the Studies of Mammalian Family 1 and 2 Cystatins in Pathological Processes.

Cystatin Disease / process Human / animal / tissue / fluid Result (compared to Reference normal tissues or fluids) Cystatin Tumors Human breast carcinoma Decreased activity and imbalance Lahefa/.,1992a;1992b activity Human breast cancer cell lines between cystatin activity and Scaddanefa/.,1993 Human amelanotic melanoma cathepsin B Sloane era/., 1990 Human lung tumors Knochefa/,,1994 Skin inflammation Human psoriatic skin Higher activity Hopsu-Havu et a/., 1983 Inflammatory Human whole and glandular Higher activity Henskens et a/., 1993a; periodontal disease saliva 1993b; 1994; 1995 Gingival crevicular fluid Cystatin activity was detected Ichimaru era/., 1992 Cystatin A Tumors Human epithelial carcinomas Decreased immunostaining Lahefa/.,1990;1992a;1992b i.e. ovarian, prostate, Kastelicefa/.,1994 squamous epithelia, Söderströmefa/.,1995 Human breast tumors Järvinenefa/.,1987 Mice skin carcinoma Lower expression Hawley Nelson et a/., 1988 Various human sarcomas Decreased activity of isolated Lahefa/.,1989a,1989b cystatin Human ascites/ Cystatin A was detected Lahefa/.,1990;1991 ovarian carcinoma Inflammatory Inflamed human gingival Present in gingival tissue Babnikefa/.,1988 periodontal disease tissue Skin inflammation Human psoriatic skin Increased immunostaining Jarvineneta/.,1987 Decreased activity/stability of Ohtaniefa/.,1982 isolated cystatin Inflammatory lung Human purulent sputum Higher concentrations Buttle et a/., 1990 disease Bacterial growth Staphylococcus aureus Inhibition of growth by rat Takahashiefa/,,1994 cystatin A Cystatin B Tumors Human epithelial carcinoma, Normal immunostaining Lahefa/.,1990; ovarian Kastelicefa/,,1994 Human ascites/ Cystatin B was detected Lahefa/.,1990;1991 ovarian carcinoma Skin inflammation Human psoriatic skin Increased immunostaining Jarvinenefa/,,1987; Hopsu-Havu efa/.,1983 degraded by PMN elastase which is present in high con- migration. Cystatin C is also involved in complement acti- centrations under inflammatory conditions (Buttle et a/., vation because of its interaction with complement com- 1991). Another explanation for the lower levels of cystatin ponent C4 (Ghiso et a/., 1990) - and modulation of inflam- C in purulent sputum samples might be a down-regulation matory reactions via its small bioactive tetrapeptide of cystatin C synthesis by the alveolar macrophages. The Lys-Pro-Pro-Arg at the N-terminus (Leung-Tack et al., effect of inflammatory stimuli on cystatin C synthesis and 1990a). expression by human alveolar macrophages in vitro was Cysteine proteinases might also play a role in the de- studied by several investigators (Chapman et a/., 1990; struction of articular cartilage and collagen since synovial Warfel etal., 1987; 1991). They found that cultured human fluid of patients with inflammatory joint diseases contains alveolar macrophages release cystatin C and that macro- high cathepsin B levels. Patients suffering from rheuma- phages derived from smokers or stimulated by zymosan, toid arthritis had also highest levels of cystatin C in their an inflammatory mediator, released less cystatin C com- synovial fluid (Lenarcic et a/., 1988a). A possible role for pared to non-smokers. In vitro experiments, in which mo- cystatin C in bone resorption in vitro was investigated by nocytes and macrophages were stimulated with lipopoly- Lerner and Grubb (1992) by analyzing the parathyroid hor- saccharide or - -interferon, also showed that the cystatin mone stimulated release of 45Ca and 3H from prelabeled C release of these cells was down-regulated by inflamma- mouse calvarial bones. Using recombinant cystatin C they tory stimuli. Remarkably, human cystatin C modulates showed a significant reduction in the release of 45Ca and neutrophilic (PMN) chemotactic acivity in vitro (Leung- 3H. Tack etal., 1990b) and therefore might play a role in PMN Cystatins in Health and Disease 79

Table 4 Continued

Cystatin Disease / process Human / animal / tissue / fluid Result (compared to Reference normal tissues or fluids)

Cystatin C Tumors Human renal cell carcinomas Normal expression Jacobsson etal., 1995 and gastro-entero pancreatic Lignelid era/., 1992 carcinomas Human ascites/ Cystatin C was detected in ascites Lah er a/., 1990; 1991 ovarian carcinoma and supernatant Barka etal., 1992 Human carcinoma cell lines Keppler etal., 1994 Inflammatory Human whole and glandular Higher concentrations Henskens era/., 1994,1996b periodontal disease saliva Inflamed human gingival Cystatin C was detected and Babnik etal., 1988 tissue negatively correlated with Skaleric et a/., 1989 pocketdepth Inflammatory Human purulent sputum Lower concentrations/ Buttle et a/., 1990; 1991 lung disease degraded by elastase Inflammatory Human alveolar Lower secretion Warfelefa/.,1987;1991 stimulus macrophages Chapman etal., 1990 Human monocytes/ macrophages Inflammatory Human synovial fluid Higher concentration Lenarcic ef a/., 1988a joint disease Multiple sclerosis Human cerebrospinal fluid Lower concentration Bollengier ef a/., 1987; MacPherson.1965 Normal concentration Lofbergefa/,,1980 Hereditary cystatin C Human cerebrospinal fluid Lower concentration Grubber a/., 1984 amyloid angiopathy amyloid deposits Detection of mutant cystatin C Ghiso era/., 1986 Failure of renal Human serum Higher concentration Löfberg and Grubb, 1979 function Brzinefa/,,1984 Kabandaefa/,,1994 Bone resorption Mouse calverial bones Cystatin C inhibits bone resorption Lerner and Grubb, 1992 Virus replication Poliovirus Inhibition of replication Korantefa/,,1986 Corona viruses Collins and Grubb, 1991 Herpes simplex virus Bjorckefa/.,1990 Bacterial growth Group A Streptococci Inhibition of growth Bjorckefa/,,1989

Cystatin Periodontal disease Human whole and Lower cystatin S concentration Henskens eta/., 1996 b S.SA.SN glandular saliva Normal cystatin S + SA + SN Aguirreefa/., 1992 concentration Infection with T. cruzi Rat salivary glands and saliva Induction of cystatin S Alves era/., 1994 papain feeding Naito era/., 1992 Inflammatory Human purulent sputum Lower concentration Buttle ef. a/., 1990; 1991 lung disease Virus replication Herpes simplex Inhibition by recombinant Weaver et a/., 1995 cystatin SN Bacterial growth Porphyromonas gingival is Inhibition by rat cystatin S Naito era/., 1995

Infections Moreover, exposure to chicken cystatin or cystatin C prior Protein cleavage of precursor proteins in the cytoplasm of to infection resulted in absence of viral protein synthesis virus-infected cells is essential for the replication of some (Korant ef a/., 1985). In contrast, large inhibitors, i.e. viruses (e.g. poliovirus, rhinovirus, herpes simplex virus) a2-macroglobulin, have no effect on virus replication, and production of new virus particles. Cysteine protei- probably because they cannot penetrate the cells (Korant nases are also involved in these cleavages. Several inves- etal., 1986; Björck ef a/., 1990; Collins and Grubb, 1991). tigators determined the effects of cysteine proteinase in- Both cystatin C and chicken egg-white cystatin bind to, hibitors in vitro when added to the culture medium of and inhibit the isolated cysteine proteinase from the polio- virus-infected cells. Chicken egg-white cystatin caused a virus (Korant etal., 1988). Also the oryzacystatins both in- reduction in virus production of poliovirus-infected cells. hibited poliovirus replication in infected Vero cells in vitro 80 YM.C. Henskensefa/. and the truncated oryzacystatin I, lacking the first 25 the earlier studies did not take into account the age de- amino acids, was even more effective (Kondo et al., 1992). pendency of the cystatin level in cerebrospinal fluid. In Similarly, Aoki et al. (1995) demonstrated antiviral effects contrast to Löfberg ei al. (1980) but in agreement with of oryzacystatin against herpes simplex type I virus. The McPherson (1965), Bollengier et al. (1987) demonstrated recombinant salivary cystatin SN and its mutants were al- not only significantly lower cystatin C in CSF in a numeri- ready tested on herpes simplex type 1 showing that the cally larger group of MS patients but also the absence of a full length recombinant cystatin SN inhibits viral replica- correlation between age and cystatin C. tion better than the mutants (Weaver et al., 1995). It might be possible that the salivary cystatins are among the HIV Serum Cystatin C as a Marker of Glomerular inhibitory factors present in whole saliva (Fultz et al., 1986) Filtration Rate which are derived mainly from the submandibular gland Low molecular weight proteins are eliminated from the cir- and to a lesser extent from the parotid gland (Fox et al., culation by glomerular filtration followed by reabsorption 1988; Malamud and Friedman 1993). It has already been and catabolism in proximal tubule cells. In healthy individ- shown that a cysteine proteinase of HIV-1 is involved in uals the blood cystatin C level is constant and therefore it the processing of certain viral proteins (Guy et al., 1991). could function as a measure of glomerular filtration rate Clones that do not produce this cysteine proteinase (GFR). Because of the low molecular weight and the posit- showed a decrease in infectivity towards susceptible ive charge at physiological pH, cystatin C easily passes CD4* cell lines. These results suggest an important role of the glomerular filter. The proximal tubular cells reabsorb cystatins as anti-viral agents. Cysteine proteinases also and catabolize practically all of the filtered cystatin C so play a role in the penetration of normal tissues by bacteria that the normal concentration of cystatin C in urine is only such as group A Streptococci. Björck et al. (1989) syn- 0.03-0.3 mg/l (Löfberg and Grubb, 1979). As serum levels thesized peptides that mimickthe proteinase binding cen- of cystatin C are much more constant than creatinine ter of cystatin C and found that they specifically inhibited levels, they would be more reliable indicators of GFR growth of all group A Streptococci in vitro. These cystatin (Simonsen etal., 1985; Grubb et al., 1985; Grubb, 1992). like-peptides were also effective in vivo: Mice injected The plasma level of cystatin C only rises as renal function with a lethal doses of group A Streptococci were cured by fails. For example: Patients suffering from glomerulone- one injection of a cystatin-like peptide. The growth of phritis have serum levels of cystatin C that are ten times Porphyromonas gingivalis, a periopathogen secreting a higher than normal (Brzin, 1984), blood plasma of patients large number of proteolytic enzymes including cysteine with renal tubular disorders contains up to 14.5 g/ml cys- proteinases, is inhibited by rat cystatin S (Naito et al., tatin C, normal is 1.1 g/ml (Löfberg and Grubb, 1979), 1995). In line with this, the phosphorylated rat cystatin A and patients on maintenance hemodialysis have serum was used successfully to inhibit growth of Staphylococ- cystatin C levels which are 10 to 12 times higher than nor- cus aureus and its cysteine proteinase activity (Takahashi mal (Kabanda ef al., 1994). Newman et al. (1990) deve- et al., 1994). This could be interesting since cystatin A is loped and validated an assay for serum cystatin C using an epidermal cystatin and S. aureus is well known to be latex particle-enhanced immunoturbidimetry (PETIA) increased in the skin surface of lesions in atopic dermati- showing more sensitivity as a screening test for early renal tis. damage than creatinine. Neurological Disorders Since cystatin C was first detected as a cerebrospinal fluid Resümee protein (CSF) there have been numerous reports dealing with the role of cystatin C in the etiology of neurological Cystatins have received much attention during the last disorders. In this respect multiple sclerosis (MS) has re- decade due to their potential regulating function for an im- ceived the most attention since this disease involves de- portant group of proteolytic enzymes, the cysteine pro- myelination which might be caused by proteolytic en- teinases. These investigations are of major importance zymes. Bollengier et al. (1987) suggest that the predomin- since there is growing evidence that cysteine proteinases ance of macrophages in the lesions of inflammatory play a prominent role in patho-physiological processes demyelating diseases such as MS could imply a role for such as invasion of normal tissues by tumors or micro- cysteine proteinases. Of all inflammatory cells, macro- organisms, viral infections and inflammation. Most stud- phages produce the highest amounts of cathepsins B, H, ies described in this review focussed on the quantification L and S which could be involved in the degradation of of cystatins or cystatin activity in tissues or body fluids to myelin. Two studies on the cystatin C level in CSF from pa- demonstrate an imbalance between the inhibitor and the tients with multiple sclerosis or other demyelinating disor- cysteine proteinases or to investigate the cystatins as ders (McPherson 1965, Pepe and Hochwald, 1967) de- possible markers for disease. Future research activity monstrated lower values of cystatin C in CSF. In contrast should focus on the regulation of the cystatin production, to these studies Löfberg et al. (1980) found no significant the biological significance of the synthesis of different decrease in the CSF cystatin C concentration of MS pa- cystatin species as well as on their actual effectiveness as tients. Löfberg attributes this discrepancy to the fact that inhibitors of viral replication and bacterial growth in vivo. Cystatins in Health and Disease 81

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Review Repeat-Induced Gene Silencing: Common Mechanisms in Plants and Fungi

Peter Meyer transgene had undergone an epigenetic modification in- University of Leeds, Centre for Plant Biochemistry & itiated by the homologous silencer locus. Trans-inactiva- Biotechnology and Department of Biology, tion did not depend on the transformation process, be- Leeds LS2 9JT, U.K. cause it could also be achieved when the silencer locus was introduced in a genetic cross (Matzke et al., 1993). However, not every homologous secondary transgene integrated into the genome of the primary transformant One of the most surprising observations made in plant was capable of frans-inactivating the primary trans- science in recent years is the inactivation of trans- gene, which clearly hinted at the importance of the genes triggered by interactions between DMA repeats. chromosomal location in the frans-inactivation process. In plants, we can differentiate between transcriptional Moreover, not every primary transgene could be frans-in- silencing, most likely reflecting a regulation at the activated (Neuhuber et al., 1994). This implies that the sus- DMA level, and post-transcriptional silencing that af- ceptibility of transgenes for homology-induced silencing fects steady state RNA levels. In the filamentous fungi is influenced by transgene-specific features, such as the Ascobolus immersus and Neurospora crassa, we find arrangement or modification of transgene sequences, two premeiotic silencing processes that are also their secondary structure or their genomic location. based on the interaction of repeated sequences. A Apart from the exciting challenge to understand the reg- common feature of transcriptional silencing in plants ulation and biological significance of this totally unex- and premeiotic gene inactivation in filamentous fungi pected silencing mechanism, these results caused con- is that the repeated sequences undergo cytosine siderable concern about the control of gene expression in methylation. DMA methylation, which is either the transgenic plants (Finnegan and McElroy, 1994). Most re- cause or the consequence of gene silencing, can combinant constructs introduced into crop plants to im- be associated with changes in chromatin structure. prove disease resistance, growth performance or food These structural changes are reminiscent of homol- quality derive from identical or related transformation vec- ogy-based silencing mechanisms in Drosophila, an or- tors, thus sharing a significant degree of homology within ganism that lacks DMA methylation. Repeat-induced enhancer, promoter or terminator sequences and within silencing may therefore reflect the activity of an the selectable marker genes. Marketing of a transgenic endogenous mechanism, present in some species, plant already involves tedious and expensive field trials to which screens for homology and has significant imp- select lines that stably express a transgene under various lications for the organization and evolution of the environmental conditions. However, it can be anticipated genome. that with a growing number of transgenic lines entering Key words: Cosuppression / DNA methylation / MIP / the market, breeders will wish to combine lines that Paramutation / Repeat-induced gene silencing / RIP / display different, novel features which are transgene- Transinactivation. mediated. The possibility of an interaction between newly combined and partly homologous transgenes abolishing transgene activity, generated some anxiety about the use Introduction of transgenic plants in breeding programmes. An inverse correlation between transgene copy number In 1989, Matzke et al. published a report on the reversible and gene activity had already been observed previously methylation and inactivation of a marker construct in (Jones et al., 1987), but the significance of this result had Nicotiana tabacum after introduction of a second recom- not been noticed by the scientific community. Not until the binant gene that shared common homologies with the in- description of frans-inactivation by Matzke ef a/., 1989 activated marker gene (Matzke et al., 1989). Trans-inacti- and the publication of other homology-dependent gene vation of the marker was clearly dependent on the pres- silencing phenomena in Nicotiana tabacum (Hobbs et al., ence of its homologous counterpart which was integrated 1990), Petunia hybrida (Linn et a/., 1990) and Arabidopsis at a different position in the tobacco chromosome. When thaliana (Mittelsten Scheid ef al., 1991; Assad ef al., 1993) both transgenes were segregated away from each other, was the general importance of this mechanism noticed. the frans-inactivated copy could become reactivated during subsequent generations. Apparently, the silenced 88 R Meyer

Homology-Dependent Inactivation Can Occur Silencing in Concatameric DMA Repeats at the Transcriptional and at the Posttranscriptional Level We can differentiate between silencing of homologous copies arranged as tandem repeats at allelic positions and An even broader interest in homology-dependent gene at ectopic locations. Studies of gene silencing in tandem silencing was raised when two laboratories reported an repeats yielded convincing evidence that inactivation is di- effect that was later termed cosuppression (Jorgensen, rectly dependent on the presence of multiple homologous 1990), displaying a mutual inactivation of a transgene and copies. In Arabidopsis, lines have been identified that con- its endogenous homologue (Napoli et al., 1990; Van der tained inactivated marker transgene integrated as multi- Krol et al., 1990). The first gene for which cosuppression ple tandem repeats. In derivatives of these lines that had became noticeable was chalcone synthase (CHS), which lost a portion of tandem repeats, the frequency of trans- encodes a key enzyme in the anthocyanin pathway re- gene inactivation was significantly decreased (Assaad sponsible for the pigmentation of the flowers. When, as a and Signer, 1992; Assad et al., 1993), and reactivated control for an antisense expression experiment, a sense states of the transgene were stably inherited to the next construct of the CHS gene was introduced into petunia generation (Mittelsten Scheid et al., 1994). plants that displayed fully pigmented, purple flowers, up Single copy transgenes are not exempt from transcrip- to half the transformants that contained a CHS sense tional inactivation (Pröls and Meyer, 1992), but the integra- copy produced white flowers or floral sectors due to the tion of multiple copies at the same locus clearly enhances loss of CHS activity. Run-on analysis showed normal CHS the probability of a transgene to be inactivated. It is un- transcription rates, but a reduction in steady state levels of clear whether the presence of direct repeats is sufficient CHS mRNA due to post-transcriptional effects (Flavell, for the induction of silencing or whether silencing is espe- 1994; Van Blokland et al., 1994). Individual branches of cially triggered by inverted repeats forming stem-loops or transgenic plants displayed different cosuppression pat- even more complex secondary structures. An interesting terns that remained very stable among the flowers of each feature of certain multiple repeats is the progressive de- branch. This suggested that cosupression patterns were velopment or enhancement of silencing over subsequent generated during the formation of the meristem, and that generations (Kilby et al., 1992; Assad et al., 1993). As they were subsequently stably inherited in somatic cells Agrobacterium-meo\ateo DMA transfer into plant fre- (Flavell, 1994). Today, cosupression phenomena have quently leads to the integration of multiple copies at single been documented for several endogenous genes in vari- loci, it is very likely that many transgene inactivation ous plants species, including petunia (Van der Krol et al., phenomena reported in early literature (Amasino et al., 1990; Angenent et al., 1994), tomato (Smith et al., 1990; 1984; Van Slogteren etal., 1984) represent repeat-induced Fray and Grierson, 1993; Seymour et al., 1993), Arabidop- silencing events of concatameric transgenes. s/s(Brusslan etal., 1993; Elkind etal., 1990) and tobacco (De Carvalho et al., 1992; Hart et al., 1992; Dorlhac de Borne et al., 1994; Boerjan et al., 1994; Brandle et al., 1995). Paramutation Based on transcription and RNA turnover analysis, we can distinguish cosuppression, which includes post- Silencing of homologous sequences at allelic positions transcriptional degradation but stable transcription rates, is probably related to the effect of paramutation, a from transcriptional silencing, which is frequently as- phenomenon that has been known for more than 60 years sociated with changes in the methylation pattern. The (Winkler, 1930). The term paramutation (Brink, 1956) de- general value of this classification is still questionable, be- scribes the induction of a heritable epigenetic effect in a cause we cannot exclude that transcriptional and post- gene due to its interaction with a homologous allele. When transcriptional silencing represent individual steps within a paramutagenic allele is combined with a paramutable common silencing pathways. In fact, there is evidence allele in a genetic cross, an epigenetic modification is in- that posttranscriptional silencing can include hyperme- duced in the paramutable allele, which converts it into a thylation of the silenced gene (Ingelbrecht et al., 1994), paramutant allele. The paramutant allele is metastable, and that it can also involve a reduction in transcription because it can retain its paramutant state if it is separated rates (Dehio and Schell, 1994). As a working hypothesis, from the paramutagenic allele. Stability and reversion however, it seems justified to focus on those silencing frequencies vary for individual loci and are dependent on phenomena that include transcriptional inactivation and environmental and developmental factors. epigenetic modifications of the silenced DMA in order to The link between repeat-dependent silencing and understand the effects of repeated sequences at the DMA paramutation becomes obvious if one cross-references level. the terminology. The paramutagenic allele represents the silencer locus, the paramutable allele is the frans-inacti- vated target gene and its conversion into the metastable paramutant state represents the metastable silenced state of the target gene. Repeat-Induced Gene Silencing 89

Paramutation has been described for several loci in vari- rying at least the 90 bp homology was finally inactivated ous species (Hagemann, 1958); evidence for its regulation by the silencer locus, the onset of inactivation differed for at the molecular level mainly come from studies in snap- individual ectopic target transgenes (pers. com. H. Vau- dragon (Krebbers et a/., 1987; Coen and Carpenter, 1988; cheret), suggesting differences in susceptibility for trans- Bollmann et a/., 1991), maize (Patterson ef a/., 1993) and inactivation. petunia (Meyer ef a/., 1993). Like most silencer loci, several paramutagenic loci contain complex repeat structures. In maize, multiple homologous r genes are clustered at the The Role of DNA-DNA Pairing in Transcriptional paramutagenic R locus, in Antirrhinum majus, a similar Silencing complexity has been found for two semi-dominant alleles of the nivea alleles, which show structural rearrangements A plausible explanation for homology-mediated silencing such as inverted duplications or concatamerisation of is the presence of a DNA pairing mechanism (Matzke ef truncated copies of the nivea gene (Bollmann et a/., 1991). a/., 1994). A comparison of the different transcriptional A correlation between the expression of the paramutable silencing systems implies that homology-based silencing gene and its methylation state was observed for the R is mediated by three major factors: the chromosomal loca- locus in maize (Dooner ef a/., 1991) and an A1 transgene in tion of the interacting homologues, the length and com- petunia (Meyer ef a/., 1993). An interesting exception is plexity of homologous sequences and the nature of the transcriptional silencing at the B locus of maize which common homologous sequence. These factors define the does not contain any repeat elements. Despite an exten- efficiency of the silencing process, as they regulate the sive analysis over a distance of 12 kb (Patterson and Chan- two crucial steps of the pairing mechanism: the capacity dler, 1995), no differences in cytosine methylation could of homologous sequences to undergo transient somatic be detected between paramutagenic and paramutable al- pairing and the establishment of a molecular modification leles at the B locus. This does not necessarily exclude any at the silencer locus that is responsible for transcriptional role of DMA modification in paramutation, as certain types inactivation and that is transferred to the homologous se- of nucleotide modifications, such as hydroxy-methyl- quence during the pairing process. cytosine, A-methylation or methylcytosines located in This molecular modification is most likely to be non-symmetrical positions would have gone undetected. mediated by cellular components that associate with the Nevertheless, it strongly suggests either that methylation silencer locus. The nature of these cellular factors is still is not the cause but a secondary effect of paramutation, or dubious, but potential candidates are transcription fac- that different classes and mechanisms of paramutation tors that bind to enhancer or promoter (Muiznieks and exist. The latter aspect is supported by the fact that Doerfler, 1994) or proteins involved in chromatin formation paramutated b alleles are much more stable compared to (Lorch ef a/., 1987; Locke ef a/., 1988; Travers, 1994; Or- other loci. lando and Paro, 1995). The identification of the regulatory factors involved is a crucial requirement to improve our un- derstanding of homology-based silencing. The search for Interactions between Repeats at Ectopic Sites modifier genes of epigenetic effect has proved successful in mammals (Allen ef a/., 1990; Engler ef a/., 1991), but the Two well-characterized examples for silencing between isolation of such genes will be difficult due to the complex- homologous repeats at nonallelic locations derive from ity of the genome of higher eukaryotes. The study of studies of transgenic tobacco plants. The first system homology-specific silencing phenomena in filamentous comprises the already mentioned silencer transgene that fungi may therefore be a valuable approach to identify was capable of inactivating another transgene inserted at common molecular factors that regulate DNA-mediated an ectopic position (Matzke ef a/., 1989). The two trans- silencing in both systems. gene loci contained different gene constructs, but shared a common homology of a 300 bp promoter region. Trans- inactivation was associated with an increase in DNA Related Repeat-Induced Silencing Phenomena methylation within the promoter region of the silenced in Filamentous Fungi locus. The presence of multiple transgene copies at the si- lencer locus was responsible for its ability to frans-inacti- In filamentous fungi, two probably related silencing pro- vate the ectopic locus, because the reduction in the cesses have been identified that are based on the pres- number of repeats also reduced silencing efficiencies ence of repeated elements. In Neurospora crassa, a (Matzke ef a/., 1994). In another system, as little as 90 bp mechanism termed Repeat-Induced Point mutation (RIP) of homology within a promoter was sufficient for a silencer was discovered when plasmid sequences were integrated locus to inactivate ectopic homologues (Vaucheret, 1993). into the genome that were homologous to an endogenous Again, the silencer locus was hypermethylated and con- sequence. RIP-specific modifications occurred in all sisted of a complex multimeric structure, and methylation homologous integration events where both homologous was imposed on homologous promoter regions located at copies were arranged as tandem duplications. When the ectopic positions. Although every ectopic transgene car- two homologous sequences were located at ectopic pos- 90 P. Meyer itions, both homologues were modified in 50% of the strand, if the template strand carries a 5mC residue at a transformants (Selker et a/., 1987). The alterations in- symmetrical position (Holliday and Pugh, 1975). cluded methylation of cytosine residues and G-C to A-T Homology-dependent silencing, however, can include mutations. methylation of additional C residues. Both RIP (Selker et In Ascobolus immersus a phenomenon called Methyla- a/., 1993) and MIP (Goyon et a/., 1994) induced methyla- tion Induced Premeiotically (MIP) was discovered in a 5.7 tion affects C residues located outside of symmetrical se- kb duplication of the met2 gene that had been generated quences. Non-symmetrical methylation patterns were by homologous recombination of a plasmid carrying the also found in a hypermethylated transgene in petunia that met2 allele into a met2~ allele of a recipient strain (Goyon could frans-inactivate a homologous allele (Meyer et a/., and Faugeron, 1989). After sexual reproduction, the Met"1" 1993; Meyer et a/., 1994). Within the promoter region of the phenotype was lost in almost all progeny cells. The dupli- transgene, methylated C residues in a non-symmetrical cated sequences were still present, but had undergone sequence context were clustered in certain regions. It was extensive methylation of the cytosine residues. Similar to not always the same C residue, but one or several C re- RIP, silencing efficiencies reached about 100% in linked sidues within a particular region that were affected. duplications, while unlinked duplications showed a 50% These results imply that the induction and conservation of silencing efficiency (Rhounim etal., 1992). However, unlike non-symmetrical methylation patterns is probably not en- RIP, MIP was not accompanied by G-C to A-T mutations, coded in the sequence, but more likely in the secondary and silencing was reversible after vegetative growth of si- structure of a sequence, a feature that can also be found lenced cells under selective conditions. for the methylation of RNA. A tRNA cytosine-5 methyl- Both RIP and MIP occur premeiotically, and are transferase recognizes the secondary structure of tran- triggered by the presence of more than one copy of a DMA scripts (Sakamoto and Okata, 1985) generating clustered segment, irrespective of whether the segment contained methylated residues. Clustering of m5A residues was also foreign or endogenous sequences (Faugeron et a/., 1990; observed at three positions of Rous sarcoma virus RNA Selker, 1990b). An interesting difference between RIP and (Kane and Beemon, 1985). It is therefore possible that MIP is the capacity of silenced copies to induce silencing methylation of non-symmetrical sites results from the ac- in additional homologous copies. Sequences altered by tivity of DNA methyltransferases or even RNA methyl- RIP show a decreased sensitivity to RIP in subsequent transferases that detect a particular secondary structure generations, while sequences that have undergone MIP or chromatin conformation. can still efficiently pair with and inactivate homologous se- Non-symmetrical methylation patterns have also been quences. This suggests that M IP-associated methylation found in mammals when two chromosomal replication does not interfere with the pairing mechanism, while RIP- origins became densely methylated after replication associated mutations decrease the potential for pairing (Tasheva and Roufa, 1994). The biological function of (Faugeron et a/., 1990). these methylation patterns remains to be determined, but as methylation follows replication, it is possible that the conformational changes associated with the replication Common Characteristics of process provide the signal for de novo methylation. Homology-Dependent Silencing in Plants For Neurospora it has been suggested (Selker, 1990a) and Fungi that the presence or absence of methylation reflects alter- nate structural states of chromatin. The 'collapsed The similarities between premeiotically induced silencing chromatin' model proposes that a loss of binding of non- processes in filamentous fungi and transcriptional, homol- histone proteins results in the collapse of a chromosomal ogy-dependent silencing phenomena in plants suggest region into a condensed form, which is further stabilized that these phenomena share common molecular features by DNA-methylation. DNA methylation would then be the which may even be relevant for other eukaryotes. second step following an initial modification at the The most obvious common feature of silencing effects chromosomal level. in plants and fungi is the association of hypermethylation In Drosophila, an organism lacking DNA methylation, patterns with silenced states. Moreover, we find a new we find homology-dependent silencing mechanisms that type of non-symmetrical methylation pattern in silenced are mediated by changes at the chromosomal level. Posi- genes in plants and fungi. Until recently, methylation in tion effect variegation (PEV) is an example for repeat-de- eukaryotes was thought to be limited to CG dinucleotides pendent gene inactivation in cis. Genes are inactivated if and CNG trinucleotides (Gruenbaum et a/., 1981). The they are located next to sequence repeats that induce a symmetry of the target sequences CG and CNG provides condensed chromatin state spreading into adjacent se- the information for the faithful propagation of methylation quences (Dorer and Henikoff, 1994). An example for pair- patterns after replication. As hemimethylated DMA is the ing-dependent frans-inactivation is dominant PEV at the preferred substrate for DMA methyltransferase, it is as- brown locus. Heterochromatin that was imposed on a sumed that the enzyme recognizes 5mC residues in the rearranged brown allele was transmitted to the unrear- * template strand of semiconservatively replicated DMA ranged homologous copy, which also became inactivated I and that it methylates C residues in the synthesized (Dreesen et a/., 1991). Other pairing-dependent modula- Repeat-Induced Gene Silencing 91 tions of gene expression have been described under the methylation compared with native sequences. Further- term transvection (Lewis, 1954). Like paramutation, trans- more, in mutated sequence that had been subject to RIP, vection depends on the interaction of susceptible alleles, methylation no longer depends on the presence of se- but the effect is not preserved after segregation of the in- quence duplications (Selker, 1990a). teracting alleles during meiosis. It is possible that the re- peat-induced inactivation systems in plants, fungi and Drosophila share common inactivation mechanisms at The Role of DNA Methylation in the chromosomal level, and that DMA methylation has Genome Evolution been evolved to stabilize changes in chromatin conforma- tion. A de novo methylation mechanism that specifically affects foreign DNA and repeated sequences should provide a significant advantage for the evolution of the genome. It Specific De Νονό enhances the tolerance of the genome to incorporate Methylation of Foreign Sequences foreign sequences, such as transposable elements or re- troelements, and it counterbalances the amplication of Besides the possibility that DNA methylation is induced by endogenous sequences. In this context, hypermethyla- conformational changes, it has been speculated that tion has at least three distinct functions: it prevents soma- foreign sequences provide specific targets for de novo tic recombination between homologous sequences, it methylation. In a petunia line that contained a single copy generates silent epigenetic states that may become reac- of the maize A1 gene, hypermethylation patterns were tivated under favourable environmental conditions, and it specifically imposed on the transgene region, while the provides a tool to induce sequence divergence when surrounding chromosomal sequences retained their methylated C residues are deaminated, which results in C hypomethylation pattern (Meyer and Heidmann, 1994). to T mutations. This result was in agreement with a hypothesis that DNA Support for the assumption that methylated sequences methylation has developed into a cellular defense are repressed in their potential to undergo homologous mechanism that allows the cell to specifically inactivate recombination comes from the observation that foreign DNA (Bestor, 1990). However, not all foreign se- hypomethylated mammalian cells show a high degree of quences that integrate into the plant genome become chromosome rearrangements (Almeida et al., 1993). Fur- methylated (Dehio and Schell, 1993), which raises the thermore, site-specific recombination leading to immuno- question how foreign sequences are recognized by or es- globulin gene maturation was blocked by C methylation. cape from the putative defense mechanism. One attrac- Evidence for the inhibitory effect of hypermethylation tive model that accounts for the differential recognition of patterns on transcription has mainly been accumulated in transgenes integrated at different chromosomal locations animal systems. For many viral or human cellular promot- is the isochore hypothesis. Plant genes have a very specif- ers, position-specific methylation has been shown to in- ic AT-content and are embedded into 200 kb large hibit or decrease promoter activity (Muiznieks and Doerf- chromosomal regions of a matching AT-content, term- ler, 1994). Most likely, the inhibition of recombination and ed isochores. Monocotyledonous and dicotyledonous transcription is mediated by DNA methylation inducing species contain distinct isochore compositions (Salinas et repressed chromatin structures (Keshet et al., 1986; An- al., 1988). Therefore, transgenes with deviating isochore tequera et al., 1990), an effect that is probably mediated by compositions may become specific targets for de novo proteins that bind to methylated C residues. In mammals, methylation. Endogenous genes should have a better methyl-CG-binding proteins have been identified that act chance to find appropriate integration regions with match- as mediators of repression and bind to heterochromatic ing isochore composition compared to sequences de- regions (Lewis etal., 1992). rived from unrelated species. In agreement with this as- An important feature of methylated sequences is the in- sumption, the A1 homologue from gerbera, a dicotyledon- stability of hypomethylation patterns, which, at least in ous plant, was less frequently methylated in dicotyledon- plants, can be highly dependent on environmental condi- ous petunia than the A1 -gene from monocotyledonous tions (Ten Lohuis etal., 1995). Methylated silent states of maize (Elomaa et a/., 1995). transposable elements can be reactivated by gamma ir- Alternatively, DNA methylation could be the default radiation or UV light (Walbot, 1988; Walbot, 1992) and en- state and endogenous sequences escape from the de- dogenous sequences show significant changes in DNA fault mechanism because they carry specific DNA signals methylation patterns after tissue culture (Brown, 1989; that prevent methylation, while foreign sequences which Kaeppler and Phillips, 1993). Interestingly, changing en- do not have these signals are prone to be methylated. It vironmental conditions not only modulate epigenetic has been suggested that the mutations generated by RIP states, but can also stimulate DNA amplification, as effectively transform unmethylated chromosomal regions shown for flax plants treated with high levels of fertilizers, into a methylatable state (Selker, 1990a). This model was which amplified specific genomic subsets (Cullis, 1986). It supported by the observation that foreign sequences has been proposed (Jablonka and Lamb, 1989) that transferred into Neurospora were much more prone to changes in heterochromatin content and DNA methyla- 92 R Meyer tion are also associated with this phenomenon. As in micro- and minisatellites which induces semi-stable ex- plants somatic and germline lineages are not strictly pression modulations on certain genes, often associated separated, a combination of environmentally-regulated with genetic diseases. Amplification of triplet repeats en- mechanisms of gene amplification and epimutation pro- hances the probability for this region to become methyl- vides the cell with an efficient system to adapt to changing ated and transcriptionally repressed (Künzler etal., 1995). external conditions. As in plants and fungi, repeat recognition and DMA meth- Finally, methylated sequences provide a potential to ylation appears to act as an antagonist to DNA ampli- generate point mutations which should also enhance the fication. development of sequence diversity. Homology-based Repeat-induced methylation can therefore be consi- silencing in plants is most likely not correlated with a muta- dered as part of a widely distributed mechanism that bal- tion mechanism as efficient as RIP. At least, no mutations ances two contradictory demands: the generation of se- could be found in a silenced transgene in Arabidopsis(Mit- quence divergence and the organization of the genome telsten Scheid et a/., 1994). However, it is conceivable that into domains that allow reliable gene expression. The methylated cytosines are preferred targets for deamina- similarities that have been detected in plants and filament- tion process which would contribute to the generation of ous fungi should stimulate cooperation among scientists sequence divergence. According to the deamination working in these areas, and may finally also provide useful theory, m5C residues can undergo deamination to information for researchers from the animal field. thymine, which leads to a depletion of CpG dinucleotides and an elevation of TpG and CpA dinucleotides (Coulondre etal.t 1978). In animals, CpG dinucleotides are Acknowledgements depleted to levels between 15% and 35% (Josse ef a/., 1961; Russell et a/., 1976), while dicot and monocot I would like to thank Dr. Celia Knight for critical reading of the genomes only show a depletion to levels of 68% and manuscript. 79%, respectively (Gardiner-Garden etal., 1992). The dif- ferent depletion levels might reflect a more efficient mis- match repair system in plants or a lower degree of DMA References methylation in plant cells that contribute to the germ cells (Gardiner-Garden ef a/., 1992). Allen, N.D., Morris, M.L, and Surani, M.A. (1990). Epigenetic con- An analysis of the distribution of the depletion of CG di- trol of transgene expression and imprinting by genoptype- nucleotides in mammalian genomes gave strong support specific modifiers. Cell 61, 853-861. for the assumption that repeat-specific methylation is not Almeida, A., Kokalj-Vokac, N., Lefrancois, D., Viegas-Pequignot, E., Jeanpierre, E., Dutrillaux, B., and Malfoy, B. (1993). limited to plants or fungi, but that mammals also contain Hypomethylation of classical satelite DNA and chromosome this mechanism (Kricker et a/., 1992). Assuming that CpG instability in lymphoblastoid cell lines. Human Genet. 91,538- depletion and TpG overrepresentation for a particular 546. genomic region indicates a high degree of previous DMA Amasino, R.M., Powell, A.LT., and Gordon, M.R (1984). Changes methylation, it was found that repetitive sequences in the in T-DNA methylation and expression are associated with mammalian genome were preferred targets for methyla- phenotypic variation and plant regeneration in a crown gall tion. Most repeated sequences contain substantial de- tumor line. Mol. Gen. Genet. 79^437-446. ficits in CpG dinucleotides compared with most unique Angenent, G.C., Franken, J., Busscher, M., Weiss, D., and van Tunen, A.J. (1994). Co-suppression of the petunia homeotic sequences. On average, ratios for the observed CpG gene fbp2 affects the identity of the generative meristem. values compared to the expected values (O/E values) are Plant. J. 5, 33-44. in the 20% range for LINE sequences, pseudogenes and Antequera, F., Boyes, J., and Bird, A. (1990). High levels of de novo members of functional gene families, while unique house methylation and altered chromatin structure at CpG islands in keeping genes and unique introns show values around cell lines. Cell 62, 503-514. 50%. In unmethylated genes, O/E CpG values reach an Assaad, F.F., and Signer, E.R. (1992). Somatic and germinal re- average of 82%, comparable to an average 83% found in combination of a direct repeat in Arabidopsis. Genetics 132, 553-566. repeated sequences in yeast and Drosophila, two sys- Assad, F.F., Tucker, K.L., and Signer, E.R. (1993). Epigenetic re- tems that lack sufficient cytosine methylation systems peat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol. (Kricker etal., 1992). These data suggest that sequence re- Biol. 22,1067-1085. peats are specifically recognized to become methylated. Bestor, T.H. (1990). DNA methylation: evolution of a bacterial This would not only affect highly repetitive DMA, but also immune function into a regulatoir of gene expression and homologous members of gene families and pseudogenes. genome structure in higher eukaryotes. Phil. Trans. R. Soc. It was proposed that the parental sequences are pro- Lond. B. 326,179-187. tected from the methylation mechanism, because the in- Boerjan, W., Bauw, G., Van Montagu, M., and Inze, D. (1994). Dis- tinct phenotypes generated by overexpression and suppres- sertion of introns into their sequence masks the sequence sion of S-adenosyl-L-methionine synthetase reveal develop- homology with their pseudogenes (Kricker et a/., 1992). mental patterns of gene silencing in tobacco. The Plant Cell 6, Another argument for the presence of repeat specific 1401-1414. methylation in mammals is the methylation of human Bollmann, J., Carpenter, R., and Coen, E.S. (1991). Allelic interac- Repeat-Induced Gene Silencing 93

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Biol. Chem. Hoppe-Seyler, Vol. 377, pp. 97 -120, February 1996 · Copyright © by Walter de Gruyter & Co · Berlin · New York

Review Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Michael Hottiger and Ulrich Hübscher* among prokaryotic and eukaryotic organisms as exempli- Institute of Veterinary Biochemistry, fied by: retroviruses, hepadnaviruses, retrotransposons, University of Zürich, Winterthurerstr. 190, retrons, group I introns and group II plasmids (Marquet et CH-8057 Zürich, Switzerland a/., 1995). In humans, not only exogenous retroviruses which are spread horizontally in their natural hosts are found, but * Corresponding author also endogenous retroviruses which are transmitted ge- netically and can therefore be detected in all cell genomes of the host species. A pathogenic potential of non defec- The Human Immunodeficiency Virus type 1 (HIV-1) is a tive endogenous retroviruses has so far only been dem- retrovirus and a causative agent of the Acquired Im- onstrated in mice, in which they may induce tumors and muno Deficiency Syndrome (AIDS). Retroviruses are immunological disorders (Coffin, 1982; Marrack et a/., distinct from other viruses in their ability to encode an 1991). In human DNA, endogenous retrovirus sequences enzyme called reverse transcriptase (RT). The RT is (HERV) are well known genetic elements. They all seem to the enzyme mainly involved in replication. It performs be defective due to multiple termination codons, deletions RNA- as well as DNA-dependent DNA synthesis in or- or the lack of a 5'long terminal repeat (LTR; Larson et al., der to convert the single-stranded viral RNA genome 1985; Leib-Mosch et al., 1990). Replication-competent into double-stranded DNA. The double-stranded DNA human endogenous retroviruses have not yet been iso- is stably integrated into the host cell genome and is lated. used as a template for the production of a new viral In 1983 Barre-Sinoussi etal. at the Pasteur Institute in generation. The HIV-1 RT is partially encoded by the Paris recovered an unknown virus containing a reverse POL open reading frame of the HIV-1 genome and con- transcriptase from the lymph node of a man with a per- sists of two subunits of 66 kDa (p66) and 51 kDa (p51). sistant lymphadenopathy syndrome (LAS) (Barre-Si- The p66 polypeptide encodes the reverse transcrip- noussi et al., 1983). This virus, later called lymphadeno- tase and the RNase H domain. Half of the p66 molecu- pathy-associated virus (LAV), grew to a substantial titer in les are further processed to generate the p51 protein CD4+ cells and killed these cellular targets (Montagnier et with an identical N-terminus, but lacking the C-termi- al., 1984). This was in contrast to the human T-cell leuke- nus which encodes the RNase H domain. In vivo both mia virus (HTLV) which maintained the cells in culture (for a polypeptides are found in equimolar amounts thus for- review see Sugamura and Hinuma, 1993). In early 1984, ming a heterodimer. This dimerization is critical for the Gallo and associates reported the characterization of enzymatic activity. In this review we summarize another human retrovirus distinct from HTLV I and II, (i) the replication cycle of HIV-1, which they called HTLV-III (Gallo etal., 1984; Popovic et (ii) the enzymatic properties of HIV-1 RT and al., 1984; Sarngadharan et a/., 1984; Schüpbach etal., (iii) the structure-function relationship of the HIV-1 RT 1984). In parallel Levy etal. reported the identification of a in view of the known three dimensional structure. retrovirus which they named AIDS-associated retrovirus Key words: Enzymatic activity / HIV-1 / Reverse tran- (ARV) (Levy et a/., 1984). This virus grew substantially in scriptase. peripheral blood mononuclear cells (PBMC), and killed CD4+ lymphocytes. These three newly identified retroviru- ses had similar characteristics. Within a short period of time, the three prototype viruses (LAV, HTLV-III and ARV) Introduction were recognized as members of the same group of retro- viruses, and their properties identified them as lentivirinae The discovery of the reverse transcriptase in 1970 by Te- (Chiu etal., 1985; Gonda et a/., 1985; Levy etal., 1985a; min and Mizutani (Temin and Mizutani, 1970) and inde- Rabson and Martin, 1985; Ratner etal., 1985a). Finally, in pendently by Baltimore (Baltimore, 1970) not only establi- 1986 the International Committee on Taxonomy of Viruses shed a genetic and biochemical basis for the classification recommended giving the AIDS virus a separate name: Hu- of retroviruses but also led to the discovery of different ge- man Immunodeficiency Virus (HIV) (Coffin et al., 1986). netic elements, all of which code for a reverse transcrip- The human immunodeficiency virus (HIV) is the causative tase. They have been shown to be widely dispersed agent of the Acquired Immuno Deficiency Syndrome 98 M. Hottiger and U. Hübscher

(AIDS) (Gallo and Montagnier, 1988). Soon after the disco- The HIV-1 genome displays a hitherto unprecedented very of HIV type 1 (HIV-1), a separate subtype, HIV type 2 economy in its coding potential, which is evident from the (HIV-2), was identified in Western Africa (Clavel et a/., presence of seven overlapping genes in the 3' half of the 1987). Both HIV subtypes can lead to AIDS, although the genome. The integrated proviral genome of HIV-1 has two pathogenic course with HIV-2 might be longer. These viru- long terminal repeat (LTR) regions, each 634 nucleotides ses are highly related in their genetical, morphological, long, flanking the genes which code for the three major and biological properties, and therefore must have recen- structural proteins, gag, pol and env. The LTRs are critical tly diverged from a common progenitor. HIV research has for the integration of the virus into the host genome. They also fruitfully influenced veterinary medicine since lentivi- contain promoter and enhancer elements with recognition ruses were discovered in primates (simian immunodefi- signals for cellular and possibly also viral transcription ciency virus (SIV), (Daniel et a/., 1985; Benveniste et al., factors. The ITRs are flanked by short internal repeats of 1986; Murphey-Corb et a/., 1986), cats [feline immunode- the conserved dinucleotideTG (Starcich etal., 1985). The ficiency virus (FIV), Pederson et al., 1987] and cattle [bo- tRNA primer binding site is located immediately down- vine immunodeficiency virus (BIV) Gonda, 1992]. stream of the 5'LTR. It contains 18 bp which are comple- It is estimated that a new HIV-1 infection takes place mentary to tRNALys<3. This RNA-RNA hybrid is the primer every 13 seconds and that a person dies from HIV-1 infec- for minusstrand DMA synthesis. Immediately upstream of tion every 9 minutes. By the year 2000, 30 to 100 million the 3'LTR is a perfect 15-16 bp polypurine rich region people will have been infected world-wide by HIV-1 or (called the PPT site) which is important for the initiation of HIV-2. Thereof 10 million are estimated to be children plusstrand DMA synthesis (Muesing etal., 1985; Ratner et (Mann et al., 1992). al., 1985b; Sanchez-Pescador et al., 1985; Wain-Hobson etal., 1985).

The Human Immunodeficiency Virus Type 1 Overview of the HIV-1 Life Cycle Electron microscope analysis indicated that HIV-1 has the characteristics of a lentivirus with a diameter of approxi- The life cycle of all retroviruses is similar (for a review see mately 110 nm. The viral surface is characteristically made Varmus et al., 1985). This chapter summerizes only the up of knobs containing trimers or tetramers of the enve- major steps in the viral life cycle (for further details see lope glycoprotein (Gelderblom et al., 1988; Ozel et al., Levy, 1993). In permissive activated T cells, HIV-1 under- 1988; Pinter et a/., 1989; Weiss et al., 1990; Earl et al., goes integration and replication within 24 h after infection 1992). The envelope glycoprotein is cleaved inside the cell (Kim etal., 1990). In macrophages, the process is similar from a 160 kDa precursor into a gp120 external surface but progeny production seems to occur after 48 h (Munis (SU) envelope glycoprotein and a gp41 transmembrane efa/.,1992). (TM) protein (McCune et al., 1988). The virion gp120 loca- ted on the virus surface contains the binding site for the Virus Entry cellular receptor(s). The inner portion of the viral mem- Entry occurs by adsorption and fusion of the virus and the brane is surrounded by a myristylated p17 core protein cell membrane. It is mediated by the envelope glycopro- that provides the matrix (MA) for the viral structure and is tein of the virus (Lifson etal., 1986; Kowalski etal., 1991). vital for the integrity of the virion (Gelderblom etal., 1987; Infectivity begins when a virus particle encounters a cell Gelderblom etal., 1988; Gelderblom etal., 1989). Recent with a high affinity receptor for the virus and presumably results suggested that the MA protein is required for incor- reflects the ability of a virus to fuse with a cell (Freed et al., poration of the envelope glycoprotein into the mature vi- 1991; Bergeron etal., 1992; De Jong etal., 1992; Freed et rions (Yu et a/., 1992). Finally, the capsid (or nucleoid), a al., 1992; Grimaila etal., 1992). Different rates and efficien- cone shaped core composed of the viral p25 Gag protein cies of entry are most probably linked to variations in the (Gonda etal., 1986), contains two identical RNA strands. envelope (Fernandez-Larsson etal., 1992; Shioda et al., The viral RNA-dependent DMA polymerase, commonly 1992). Only after penetration and uncoating of the virus termed reverse transcriptase (RT), and the nucleocapsid core do reverse transcription and steps leading to virus (NC) proteins are associated with the RNA. production take place. A big breakthrough in HIV-1 re- The HIV-1 genome is 9.7 kb in length and has the same search was the discovery of the major cellular receptor, general organization as other retroviruses with the polarity the CD4 molecule. The reason for preferential growth of of amRNA(Hahn etal., 1984; Luciw etal., 1984). However, HIV-1 in CD4+ lymphocytes was then explained by its at- molecular cloning (Hahn et a/., 1984) and sequencing tachment to the CD4 protein on the cell surface (for re- (Muesing et al., 1985; Ratner et al., 1985b; Sanchez- views see Peterson and Seed, 1988; Capon and Ward, Pescador etal., 1985; Wain-Hobson etal., 1985; Desai et 1991; Sattentau and Moore, 1991). The CD4 molecule is a/., 1986) of HIV-1 suggested that the genomic structure of found on a variety of cells of hematopoietic origin (Dal- HIV-1 is more complex than that of conventional retroviru- gleish etal., 1984; Klatzmann etal., 1984; McDougal etal., f ses (including HTLV-I and HTLV-II). 1985; Sattentau and Weiss, 1988). There is evidence that HIV-1 Reverse Transcriptase 99

HIV-1 may also enter cells that lack CD4 (Clapham et al., the virus envelope with the plasma membrane (Stein efa/., 1 989). Electron microscopy has revealed a direct fusion of 1 987).

(A)n R U5 PBS PPT U3 5'- 3' + strand RNA genome (two copies per particle) tRNA primer

R U5 PBS -strand DMA initiation RNaseH PPT U3 5'- and elongation

R U5 PBS PPT U3 5'

First Jump (intra-or intermodular) PBS PPT U3

R U5

PBS < RNaseH PPT PBS •i-strand RNA digestion -strand DNA elongation R US

PBS ^RNaseH PPT obligatory -»-strand DNA ~

PPT U3 R US PBS additional+strand DNA E> initiations and elongations "PPT U3 R US dJD

3PT RNaseH RNaseH | DDDS> ^\ U3 R US PBS strand displacement DNA 8 synthesis, PBS PPT U3 R U5| d

U3 R strand displacement DNA (f synthesis

U3 R US PBS PPT U3 R US -»-strand DNA 10 -strand DNA .U3 R U5.PBS PPT .U3 R U5, LTR LTR Fig. 1 The Reverse Transcription Process. PBS: primer binding site, PPT polypurin tract; (A)n: poly A tail, RNase H: activity of RNase H, RODS: RNA dependent DNA synthesis, DDDS: DNA dependent DNA synthesis. For details of steps 1 -10 see text. 100 M. Hottiger and U. Hübscher

Replication and Integration of the Viral Genome Thus, it is conceivable that the intracellular milieu can de- termine the relative dominance of viral regulatory proteins During its internalization, the virus is uncoated. The di- in an infected cell. Further evidence of this intracellular ploid viral RNA inside the capsid is converted to DMA by control is provided by biological studies showing varia- the action of a virus-specific RT. This reaction occurs tions in replication of HIV-1 strains in different cell lines, within the first six hours of infection and takes place in the macrophages, and PBMCs from various donors (Levy ef cytoplasm of the cell (Varmus and Swanstrom, 1985; Pan- al., 1985a; Castro etal., 1990; Cloyd and Moore, 1990; ganiban and Fiore, 1988; Kim et al., 1989; Farnet and Ha- von Briesen etal., 1990; Olafsson etal., 1991). The virus seltine, 1990). Viruses defective in the DMA polymerase furthermore specifies a protein called the transactivator and ribonuclease H activity do not complete proviral syn- (Tat), which prevents RNA polymerase II from falling off thesis (Linial and Blair, 1985). during transcription (Peterlin etal., 1986; Marciniak etal., After DMA synthesis, the HIV-1 nucleoprotein comple- 1990a). Several cellular proteins bind to the Tat protein xes, isolated from cytoplasmic extracts of CD4* cells, and to its recognition sequence, called TAR (Gaynor etal., were found to contain viral RNA and DMA in association 1989; Marciniak etal., 1990b; Gatignol etal., 1991). The with viral matrix (MA), integrase (IN), and reverse trans- roles of these interacting cellular proteins are still un- criptase (RT) but not capsid (CA) antigens. The newly known. synthesized proviral DNA migrates to the nucleus as a nu- In the absence of Rev protein, only small multiple spli- cleoprotein complex, the so called preintegration com- ced viral RNAs accumulate in the cytoplasm. The Rev pro- plex. Recent studies suggested that this preintegration tein is located primarily in the nucleus and nucleolus (Co- complex is rapidly transported to the nucleus by a pro- chrane etal., 1989; Venkatesh etal., 1990). It is responsi- cess requiring ATP but neither cell division nor a functional ble for the accumulation of unspliced and singly spliced viral integrase (Bukrinsky etal., 1992; LiG. etal., 1993). In- RNAs in the cytoplasm (Feinberg et al., 1986; Emerman et side the nucleus these complexes integrate with high effi- al., 1989; Felber etal., 1989; Malim etal., 1989; Farnet and ciency into the target DNA (Farnet and Haseltine, 1991). Haseltine, 1990; Steffy et al., 1992). In the cytoplasm This integration process is mediated by the virus encoded these RNAs are translated to make the capsid and the en- integrase IN (Varmus and Swanstrom, 1985). The integra- velope proteins of the virus. Free cytoplasmic ribosomes tion appears to be random and essential for the produc- use the full-length viral RNA as a template for the transla- tion of progeny virions. Once integrated, viral DNA re- tion of the capsid proteins and the replicative enzymes. mains permanently associated with the host cell genetic material. Cell-free integration proceeds to completion wit- The capsid proteins are made as polyprotein precursors hin 30 min. Exogenous energy sources, such as ATP, are (Ratner ei al., 1985b). The replicative enzymes are made not required for the integration reaction in vitro (Farnet and as a fusion protein with a capsid protein precursor (Wilson Haseltine, 1990). In addition, circularization may occur ei- etal., 1988; Jacks ef al., 1988). The Gag and Gag-Pol pro- ther via blunt-end joining of the full-length linear proviru- ducts are synthesized at a ratio of 20:1 (Oroszlan and Luf- ses to yield a circular molecule with two adjacent LTR se- tig, 1990). The envelope glycoprotein (Env) of HIV-1 is initi- quences, or by reciprocal recombination within the LTR ally synthesized as a gp160 precursor. It is cleaved in a yielding a circular molecule that contains a single LTR. non-lysosomal acidic compartment by some cellular pro- Whether HIV-1 can be produced without integration of the teases, and the mature envelope proteins (gp120 and provirus is still unknown. This phenomenon has been de- gp41) are transported to the cell surface (Willey et al., scribed for the Visna lentivirus (Harris et al., 1984). 1988; Kozarsky et al., 1989). In the absence of the Rev protein, only regulatory proteins are derived from the mul- Viral Gene Expression tiple spliced transcripts. Accessory HIV-1 viral proteins, Vif, Vpr and Vpu, appear to influence events such as as- Viral gene expression begins with the synthesis of a com- sembly and budding, DNA translocation, and infectivity plete RNA copy from the proviral DNA. The site of tran- (Greene, 1991; Vaishnav and Wong-Staal, 1992). scription initiation is located in the terminally redundant vi- ral sequences, the long terminal repeats (LTR). This se- Assembly and Budding quences are utilized by cellular RNA polymerase II. The transcription promoter of HIV-1 contains many sequences The capsid precursor protein and the capsid replicative that were recognized by cellular transcription proteins enzyme precursor proteins coassemble at the inner sur- (Garcia ef al., 1987; Nabel and Baltimore, 1987; Tong- face of the cell membrane by virtue of a myristic fatty acid Starksen et al., 1987; Franza et al., 1988; Jones et al., which is attached to the amino terminus of the capsid pre- 1988; Clouse etal., 1989; Wu etal., 1995, for reviews see cursor protein (Veronese ef a/., 1988). The frans-acting Tong-Starksen and Peterlin, 1990; Gaynor, 1992). During component required for viral RNA encapsidation is the RNA synthesis RNA polymerase II usually falls off before RNA-binding nucleocapsid (NC) protein derived from the completing transcription of all the viral genes. The HIV-1 Gag precursor (Berg, 1986). The NC protein contains a LTR promoter, however, can be switched from a weak to a histidine- and cystein-rich region. NC can bind zinc and strong promoter by changes in the concentrations of cel- forms two zinc fingers (Schiff et al., 1988; Green and Berg, lular factors that recognize the sequences of the promoter. 1989). The NC interacts with a sequence located near the HIV-1 Reverse Transcriptase 101

5' end of the RNA, called the packaging or Psi ( ) se- does not occur in protease-defective mutants (Kohl ef a/., quence (Lever et a/., 1989; Aldovini and Young, 1990). De- 1988; Gottlinger ef a/., 1989; Peng ef a/., 1989). letion of this Psi region prevents entry of viral RNA into the capsid protein (Lever et a/., 1989). The aggregation of Gag and Gag-Pol molecules under the plasma membrane is initiated and mediated by the Gag protein p17. This p17 Replication of the Viral Genome Gag protein also initiates the complex process of assem- bly. A complex of capsid protein, replicative enzyme pre- In the retroviral virion core, the genomic 70 S RNA is pre- cursor and viral RNA is assembled into a closed spherical sent as a dimer and is in close association with a large particle which then buds through the cell membrane (Gel- number of NC protein molecules (Fauci, 1993). The geno- derblom et a/., 1987). It was shown for HIV-1 and also for mic RNA is reverse transcribed to produce the double- other retroviruses that for cell-to-cell transmission of the stranded proviral DNA required for subsequent integra- virus, the previously mentioned precursor polyproteins, tion into the host genome (see Figure 1). Recent evidence Gag and Gag-Pol, are concentrated unidirectionally to the shows that complete proviral DNA synthesis occurs using tip of a cell pseudopod which is formed by an unidentified only one of the copackaged RNA templates (Jones ef a/., actin-isoform in an infected cell (Tarone et a/., 1985; Mor- 1994). Although there are two copies of genomic RNA tara and Koch, 1986; Busso ef a/., 1991; Pearce-Pratt et within each retrovirus particle thus enabling the possible a/., 1994). Different observations by Hottiger et a/., (1995) generation of two independent copies of preintegrated raise the possibility that eukaryotic ß-actin, by interacting proviral DNA, only a single provirus is generated per infec- with the Gag-Pol precursor, might be directly involved in tion (Panganiban and Fiore, 1988; Hu and Temin, 1990). HIV-1 virion assembly. These results are further supported The viral encoded RT plays a key role in this complex repli- by findings that an unidentified isoform of actin has been cation cycle. It is estimated that there are 50 to 100 RT mo- found in HIV-1 (Arthur et a/., 1992) and in a number of other lecules per virion, and it is not known whether one or seve- retroviruses (Vogt ei a/., 1975; Wang et a/., 1976; Damsky ral of these molecules contribute to produce a single DNA et a/., 1977; Lutz et a/., 1980; Stanislawsky et a/., 1984; copy. Synthesis of each retroviral DNA strand is initiated at Mortaraand Koch, 1986). a distinct site on the template by a specific primer. This pri- The final phase of the viral maturation is dependent mer defines each end of the linear, double-stranded retro- upon proteolytic cleavage. The Gag and Gag-Pol precur- viral DNA molecule (Varmus ef a/., 1985). The reverse tran- sors of HIV-1 are cleaved by the viral-coded protease (PR, scription process begins in the virion particle with the p10) either during or after budding to produce individual synthesis of the minus-strand as evidenced by the pre- proteins (Kohl et a/., 1988; Gottlinger ef a/., 1989; Peng et sence of minus-strand strong-stop DNA in virions (Darlix a/., 1989). This process is responsible for morphological ef a/., 1977; Lori efa/., 1992;Trono, 1992). maturation of virions in which the core condenses to form an electron-dense cylindrical structure. Since the HIV-1 Minus-Strand DNA Synthesis protease is active only as a dimer (for a review see Skalka, In accepted models of reverse transcription (Boone and 1989), the protease may not be able to dimerize until the Skalka, 1981; Panganiban and Fiore, 1988; Hu and Temin, Gag and Gag-Pol precursors have properly aligned them- 1990) the origin of the minus-strand is determined by a selves during the stages of the budding process. The con- tRNA molecule that is copackaged with the viral genomic densation process in the structure of the virus particle RNA and is hybridized near the 5' end (Figure 1, step 1).

R U5 .rev cvifu \\^tai —jüß— 3'L.TR vpr /· pol vpu env net

P66 || 32| PR RT IN

~\ 1 1— 1— I I I I I ) 1 2 3 4 5 6 7 8 9 (kb) Fig. 2 Genomic Structure of the HIV-1 Genome. LTR: long terminal repeat, Squares: open reading frames of the viral encoded proteins. 1 +: start of transcription. -: translation of pol, PR: protease, RT: reverse transcriptase, IN: integrase. For details see text. 102 M. Hottiger and U. Hübscher

This region defines the 3' boundary of the long terminal re- and 3). Subsequently the viral RNA of this RNA-DNA hy- peat (LTR) and is the called primer binding site (PBS). The brid is hydrolyzed by the second activity of RT, the RNase events leading to initiation of the minus-strand DMA syn- H resulting in a single-stranded DNA at the 3' end of the thesis include minus-strand (Das etal., 1995). After strong-stop minus- (1) specific incorporation of the host tRNA primer into the strand DNA synthesis template switching occurs. This viral particle, switch transfers the growing DNA minus-strand from the (2) partial unwinding of the tRNA structure to expose 18 or 5' end of the RNA molecule to the 31 end of the same, or a more nucleotides of the 31 end of the tRNA, second RNA molecule (Panganiban and Fiore, 1988; Hu (3) unwinding of the viral RNA template structure, and and Temin, 1990; Figure 1, step 4). This process is favou- (4) annealing of the tRNA to the PBS or to regions located red by the redundant LTR. Thus the 5' end can hybridize to close to the PBS (Aiyar et al., 1992; Weiss et a/.,1992). the 3' end of the viral RNA, allowing the RT to synthesize the full length minus-strand (Swanstrom etal., 1981). Si- These events are likely mediated by two viral proteins, NC multaneously the RNAse H hydolyzes the entire viral RNA and RT. Specifically, the annealing of the tRNA to the viral template with the exception of a small polypurin tract, cal- genome is stimulated by the NC protein (Prats et al., 1988; led PPT (Figure 1, step 5). Pullen et al. showed that HIV-1 Khan and Giedroc, 1992; Mely et al., 1995). Recent data RT also removes the tRNA primer, which is still intact after suggest that a small amount of viral DNA is synthesized the minus-strand strong-stop DNA synthesis except for within HIV-1 virions (Stein et al., 1987; Kim et al., 1989; the last ribonucleotide A, which is bound to the 5' end of Bukrinsky et al., 1993), a major extent of the full-length vi- the newly synthesized DNA strand (Pullen et al., 1992). ral DNA synthesis initiation, however, is detected immedi- Subsequent cleavage occurs at other internal sites of the ately after the virus is fused to the target cells within defi- tRNA molecule (Pullen et al., 1992; Smith and Roth, ned virion-like particles (Zhang et al., 1995). 1992). Recognition of its cognate tRNA by the RT may be ana- logous to tRNA recognition by the aminoacyl-tRNA syn- thetase and involves extensive surface contact, as exam- Plus-Strand DNA Synthesis plified by the structure of the complex between glutami- The origin of the plus-strand is determined by the PPT nyl-tRNA synthetase and tRNAGln (Rould et al., 1989). In which designates the 5' limit of the 3' LTR. It is assumed HIV-1, on the other hand, the sequence of the primer bind- that the PPT can create a RNA primer on the DNA minus- ing site (Ratner et al., 1985b; Sanchez-Pescador et al., strand by virtue of its resistance to the RNase H activity of 1985; Wain-Hobson et al., 1985) suggested that the the RT (Resnick etal., 1984; Smith etal., 1984; Figure 1, tRNALys·3 is used to prime DNA synthesis. HIV-1 RT has step 5). Processing of the PPT RNA primer for plus-strand been shown to preferentially bind to tRNALys>3 even in the DNA synthesis by HIV-1 RT requires generation of a PPT, presence of a 100-fold excess of other tRNAs (Barat et al., primer extension and primer removal. These three events 1989). Weiss et al. demonstrated that human tRNALys·3 can occur together but are not necessarily coupled (Huber and bovine tRNALys'3 can interact with HIV-1 RT (Weiss et and Richardson, 1990; Figure 1, step 6). A specific feature Lys 3 al., 1992). In vitro, HIV-1 RT and the tRNA · primer are of HIV-1, and other lentivirusess is the presence of a sec- sufficient for initiation of DNA synthesis at the PBS (Kohl- ond PPT at the pol gene, near the center of the genome staedt and Steitz, 1992; Weiss et al., 1992). cDNA product (Songio etal., 1985; Wain-Hobson etal., 1985; Charneau analysis of the viral genome indicated the presence of at and Clavel, 1991; Hungnes etal., 1991; Figure 1, step 7). least 18 nucleotides of the 3' ends of human and bovine This central PPT appears to be used as a second origin for tRNALys>3. The 51 ends of both human and bovine primer plus-strand DNA synthesis, since it determines a disconti- molecules are removed by the RNase H activity of HIV-1 nuity (or a gap) in that strand, and thus yields two discrete RT (Weiss et al., 1992). This suggests that RT is able to plus-strand segments of unintegrated linear DNA (Char- bind and unwind its tRNA primer. The addition of the NC neau etal., 1992). The presence of the central PPT confers protein enhanced unwinding (Barat etal., 1993). The bind- a replication advantage but does not significantly contri- ing of the tRNALys·3 induces significant structural changes bute to the absolute requirement for viral replication. As in HIV-1 RT (Robert et al., 1990). Cross-linking studies in- soon as the 3' end is replicated beyond the tRNA primer, dicated that recognition of tRNALys·3 by HIV-1 RT occurs the RNase H removes the rest of the tRNA at the 5' end of through interactions of the anticodon loop with the en- the minus-strand (Figure 1, step 8). A second switch trans- zyme (Barat et al., 1989). In a competition experiment, fers the growing plus-strand DNA from the 5' end to the 3' tRNALys·3 inhibited polymerization by the HIV-1 RT, thus in- end of the same molecule of newly synthesized minus- dicating the strength of this binding (Sallafranque-An- strand DNA (Panganiban and Fiore, 1988; Hu and Temin, dreola etal., 1989; Bordier et al., 1990; Richter-Cook et al., 1990). This intramolecular process leads to the generation 1992). This further supported the analogy to the amino- of a circular molecule (Withcomb et al., 1990; Figure 1, acyl-tRNA synthetase-tRNA recognition. step 9). Plus-strand strong-stop DNAs which represent The viral RT can utilize tRNALys'a as a primer and replica- the partially completed, newly made DNA prior to the sec- tes to the 5' end of the genome, thus producing the ond template switch, have been shown to accumulate strong-stop minus-strand intermediate (Figure 1, steps 2 during reverse transcription (Varmus and Shank, 1976; HIV-1 Reverse Transcriptase 103

Coffin and Haseltine, 1977; Li R et al., 1993). The plus- RNA and DNA templates. This raised the question of strand is then fully replicated from the upstream PPT pri- whether retroviruses arose directly from components of mer through a strand displacement DMA synthesis mech- the host cell or from an RNA virus whose genome replica- anism (Huber eta/., 1989; Hottiger et al., 1994). Thus in- ted through an RNA-RNA intermediate (Poch et al., 1989). tramolecular strand switching and strand displacement Another interesting possibility involved the replication of guarantee that already synthesized downstream plus- host chromosomes telomeres and the enzyme telome- strands are hybridized (Boone and Skalka, 1993) and that rase, which appears to be able to copy RNA into DNA. Te- the LTRs are duplicated (Boone and Skalka, 1981). The lomerases contain an RNA component that serves as the end product of reverse transcription, called proviral DNA, template for the enzyme to copy the DNA repeats found in is double stranded and longer at both ends than the origi- telomeres (Greider et al., 1989; Shippen-Lentz and Black- nal template viral RNA (Gilboa et al., 1979; Figure 1, step burn, 1990; Yu et al., 1990). Sequence comparisons of 10). It appears in the cytoplasm four hours after infection EST-1, the yeast polymerase component of the telome- (Kim etal., 1989; Sato etal., 1992) and undergoes integra- rase, with the polymerase domain of retroviral and with vi- tion after 24 hours (Robinson and Zinkus, 1990). ral RNA-dependent RNA polymerases are quite sugges- tive (Lundblad and Blackburn, 1990). It may well be that both of these families of RNA-dependent polymerases Cell Activation and Reverse Transcription descend from a host telomerase that had lost its intrinsic RNA template. Thus one can propose the idea that the line Several reports indicated that the level of cell activation of evolutionary descent is from telomerase, through re- can influence the extent of viral reverse transcription and verse transcriptase, to the RNA-dependent RNA polyme- consequently infectious virus release from the cell. Cells rases found in many RNA viruses not belonging to the re- that express CD4 molecules but are blocked in DNA repli- troviridae. cation and remain inactivated (at G0) could not be infected by HIV-1 (Gowda et al., 1989). In contrast to this observa- tion other investigators have reported that non-dividing Expression of HIV-1 Reverse Transcriptase cells such as CD4+ lymphocytes can be infected in vitro The HIV-1 RT is encoded by the open reading frame pol but replication of the viral genome proceeds only to the which is located downstream from the open reading frame stage where limited extension of the minus-strand- as well gag, and codes for the virus capsid proteins. The gag and as the plus-strand are synthesized (see Figure 1, step 6 pol genes overlap by 241 nucleotides. A-1 ribosomal fra- and Zack ef al., 1990). From a few days to two weeks after meshift relative to the gag open reading frame (Ratner ef initial infection, unintegrated viral cDNA can be detected a/., 1985b) allows for expression of the pol gene (Jacks in T cells by PCR (Stevenson ef al., 1990; Zack ef al., ef a/.,1988; Wilson ef al., 1988; see Figure 2). This frame- 1990). The cause of the failure to complete the reverse shift event, which occurs on an UUA leucin codon at a low transcription process in quiescent cells is not known. It frequency of 5%, is directed by a short homopolymeric could be due to a limiting nucleotide pool or to other co- sequence (slippage sequence) at the gag-po/overlapping factors that decrease the activity of reverse transcriptase region followed by a hairpin loop structure (for a review itself. Specific cellular factors only found in activated T see Coffin, 1990). In contrast to other retroviruses, which cells may also be required to complete reverse transcrip- also have ribosomal frameshifts, the slippage sequence tion. In another report, completed cDNA viral forms could alone appears to be sufficient to induce ribosomal frame- be detected but were not integrated, and therefore unpro- shift (Jacks etal., 1988; Wilson ef al., 1988). This unique ductive for viral infection (Stevenson ef al., 1990). Thus it strategy of gene expression guarantees that while the seems that a partially or fully synthesized, but unintegra- structural Gag proteins are produced in large amounts, ted, viral DNA structure in a quiescent cell is a labile, latent the catalytic proteins (Pol) are made in relatively small intermediate, which when the cell is stimulated, can still amounts. Pol is expressed as a Gag-Pol fusion protein be activated for up to 2 to 4 weeks after the initial infection from a full-length genomic mRNA. The Pol precursor is (Stevenson ef al., 1990; Zack ef al., 1990). Taken together autocatalytically cleaved by the viral protease (PR, p10) viral entry and expression appear to be influenced by the to produce the RT (p66) and the integrase (p32). state of cell activation. The time frame that a cell carries Reverse transcriptases have originally been characteri- the unintegrated viral DNA also reflects the level of its acti- zed as naturally occurring monomers [murine leukemia vi- vation (Stevenson ef al., 1990; Zack et al., 1990; Bukrinsky rus (MLV RT) or dimers (HIV-1 RT, HIV-2 RT, FIV RT and etal., 1991). Rous sarcoma virus RT)]. HIV-1 RT contains all the se- quence information needed for activity, implying that mo- nomeric p66 should be a fully active RT. However, in both HIV-1 Reverse Transcriptase virions (diMarzo Veronese ef al., 1986; Wondrak et al., 1986) and infected cells (Lightfoote ef al., 1986), HIV-1 RT The origin of the polymerase domain of RT is unclear. One has been characterized as a heterodimer consisting of 66 simple hypothesis would be that it was derived from a and 51 kDa chains. The p66/p51 HIV-1 RT is recognized DNA polymerase that had acquired the ability to copy by antibodies in about 80% of HIV-1 positive individuals 104 M. Hottiger and U. H bscher

(diMarzo Veronese et al., 1986). Mapping by monoclonal nus by proteolytic processing of the p66 chain by the antibodies of the p66 subunit of HIV-1 with C-terminally HIV-1 protease. The RT activity site is localized in the truncated mutants revealed that p51 was derived from the N-terminal and is 156 amino acids of p66 and p51. Restle p66 polypeptide (Tisdale et al., 1988) and that p66 and et al. (1990) showed that both subunits, p66 and p51, are p51 possess identical amino termini (Lightfoote et al., active as heterodimers and as homodimers and are thus 1986). The tightly associated p66/p51 heterodimer is gen- able to perform RNA dependent DNA synthesis. Both erated from a HIV-1 p66/p66 homodimer (Farmerie et al., subunits are inactive as monomers; thus different con- 1987; Mous et al., 1988) in the presence of the HIV-1 PR tents of the homodimers may account for the different (diMarzo Veronese et al., 1986; Lightfoote et al., 1986; conclusions mentioned hi other publications (Hizi ef al., McHenry, 1989), thus generating an additional polypep- 1988; Tisdale et al., 1988; Starnes ef al., 1988; Bathurst et tide of 15 kDa from the C-terminus. Once the heterodimer al., 1990; Restle etal., 1990; el Dirani-Diab ef al., 1992; is formed, it is not susceptible to further cleavage. Expres- Thimmig and McHenry, 1993; summarized in Richter- sion of the HIV-1 RT gene in bacteria yields both an active Cook etal., 1992). Additionally Hottiger etal. showed that p66/p66 homodimer and an active p66/p51 heterodimer the heterodimer as well as the p66 homodimer are able to that was apparently processed by bacterial proteases (Ja- perform DNA dependent DNA synthesis whereas the p51 cobo-Molina and Arnold, 1991). The HIV-1 PR generates homodimer is completely inactive in DNA dependent DNA F440 as the carboxy end of the p51 subunit (Mizrahi et al., synthesis (Hottiger etal., 1994). 1989; Bathurst etal., 1990; Graves etal., 1990). Bacteri- The role of each subunit within the heterodimer has ally expressed HIV-1 RT, on the other hand, has a hetero- been investigated by reconstituting HIV-1 RT. In three in- geneous C-terminus centered around P433 (Lowe et al., dependent reports, analysis of selectively mutated hete- 1988). The crystal structure of the HIV-1 PR with and with- rodimer HIV-1 RT showed that p51 cannot compensate out substrate analogs (Miller et al., 1989) shows that it is for inactivating mutations that were introduced into the highly unlikely that the protease could reach the cleavage p66 subunit, whereas the same mutation has no effect site unless the RNase H domain of HIV-1 RT p66 is parti- when introduced into the p51 subunits (Le Grice ef al., ally or totally unfolded upon dimerization (Jacobo-Molina 1991; Boyer, PL ef al., 1992; Hostomsky etal., 1992). Ex- and Arnold, 1991). In addition, it appears that energy is re- periments using chimeric HIV-1/HIV-2 heterodimers also quired to unfold the RT RNase H domain and that energy support the conclusion that p66 is the catalytically active might be provided by the dimerization process. Davies et subunit (Howard ef al., 1991). In addition, competition stu- al. concluded that an unfolded RNase H domain is likely to dies of HIV-1 RT p66/p51 heterodimers with different pri- be the substrate for the proteolysis of HIV-1 RT p66 to p51 mer-template complexes indicated the presence of only by HIV-1 PR (Davies et al., 1991). This supports a model one binding site (Painter etal., 1990). Activity gel analysis for proteolytic processing in which the p66 homodimer is (Starnes ef al., 1988) and cross-linking studies of p66/p51 structurally asymmetric, with the RNAse H domain unfol- with substrate analogues have shown that only the p66 ded in the subunit destined to give rise to p51 and thus ex- subunit is labelled (Cheng etal., 1991). Furthermore, the posing the cleavage site. Formation of the p66 homodi- non-nucleoside-analogue HIV-1 RT inhibitor BI-RG-587 mer may force unfolding of the RNase H domain in one su- selectively binds to the p66 chain in the p66/p51 heterodi- bunit (Davies ef a/., 1991; Hostomska et al., 1991). This mer (Wu etal., 1991). Thus, the catalytic-site residues pre- would account first for the asymmetric cleavage by PR sent on p51 do not seem to contribute directly to activity. and second for the observation that the p15 RNase H re- Hostomsky ef al. showed that p51 in the heterodimer has leased from the p66/p66 homodimer is unstable (Hostom- an altered conformation in comparison to the p51 ho- sky ef a/., 1991). modimer (Hostomsky ef al., 1992). However, chemical Additionally it has been reported that enzymatic activity cross-linking studies indicated that the tRNALys>3 forms is almost exclusively confined to the dimeric forms of the contacts with both the p66 and p51 subunit of HIV-1 RT proteins (Restle etal., 1990; Goel etal., 1993). The hetero- (Barat ef al., 1989). Recent studies suggested that p51 dimer is the most stable dimer, with an equilibrium disso- might have a role in altering the processivity of the p66 9 cation constant (Êύ) of approx. 1 χ 10" M (Restle et al., subunit (Huang ef al., 1992) and in facilitating strand dis- 1990; Divita ef al., 1993a). p66 and p51 homodimers have placement DNA synthesis (Hottiger ef al., 1994). Finally, it been observed in vitro but they have a weaker association could be involved in tRNA binding (Jacques ef al., 1994; (Divita ef a/., 1993a). Mishimaand Steitz, 1995). For HIV-1 RT a functional interdependence of the two domains (RT/RNase H) in the p66 subunit has been sug- Roles of the HIV-1 Reverse Transcriptase Subunits gested. With HIV-1 RT p66, most insertions in the predic- p66 and p51 ted polymerase domain inactivated the polymerase func- The HIV-1 RT heterodimer, as do all other retroviral RTs, tion (Prasad and Goff, 1989; Hiti ef al., 1990). However, has DNA polymerase and RNase H activities (Hansen et some insertions also inactivated RNase H, suggesting ei- al., 1987; Larder et al., 1987a). The amino termini of the ther a global effect, or a disruption of specific interactions two chains (p66 and p51) are identical although the smal- between the domains mentioned above. Similarly, some ler subunit of the heterodimer is derived from the C-termi- insertions in the RNase H region of p66 had a significant HIV-1 Reverse Transcriptase 105 effect on polymerase activity (Hizi et a/., 1989; Prasad and p66 and p51 subunits are arranged differently (Arnold et Goff, 1989; Hizi et a/., 1990). The RNAse H domain ex- a/., 1992; Kohlstaedt et a/., 1992), despite identical amino pressed in bacteria is inactive by itself, although an addi- acid sequences up to F440. The polymerase regions of tion of the p51 polypeptide leads to enzymatic activity p66 and p51 are divided into four subdomains denoted (Hostomsky et a/., 1991). The RNase H domain is present 'finger', 'palm', 'thumb' and 'connection' (see Figure 3; on both the p66 and the p15 fragment. The p15 fragment Wang et a/., 1994). appears to be structurally unstable and several studies in- The major contacts between the p66 and p51 subunits dicated that it had little (Hansen et a/., 1988) or no RNase H occur within the connection domains. The extended activity. The biologically relevant form of RNase H proba- thumb of p51 contacts the RNase H domain of p66, and bly resides on the p66 subunit (Hostomsky et a/., 1991), this interaction may be required for RNase H activity (Ho- although a role for the p15 fragment in the viral life cycle stomsky et a/., 1991). In the p51 subunit, the connection cannot be totally ruled out (Schulze et a/., 1991). domain is rotated such that it occupies the palm and bu- ries the active site residues. The fact that the p51 subunit displays a distinct structure (and consequently function) Structure-Function Relationship of HIV-1 Reverse from the p66 subunit appears to be another exciting Transcriptase example of the economical use of a limited viral coding The overall structure of the HIV-1 RTp66/p51 heterodimer capacity; a single DNA coding sequence produces poly- is highly asymmetric as the polymerase domains of the peptides with the same amino acid sequence that are

A 50 100 150 200 250 300 350 400 450 500 550 N Fingers Fingers Thumb Connection RNase Ç Palm Palm

Fig. 3 Schematic Drawing of the Polypeptide Backbone of the HIV-1 RT Heterodimer. (A) Scheme of the DNA polymerase domains of HIV-1 RT. The colours correspond to the structure shown in Â. (Â) á-helices and -strands are represented by tubes and arrows, respectively. The p66 (upper) and p51 (lower) subunit are pulled apart in the vertical direction to make the interaction surface clear. For details see text. Reproduced from Wang et a/., 1994, Proc. Natl. Acad. Sei. USA, 91,7242-7246. 106 M. Hottiger and U. Hübscher structurally and functionally distinct (Kohlstaedt et al., towards the 3* end of the template strand of the double- 1992). In the HIV-1 RT p66/p51 heterodimer, there are sig- stranded DNA extended beyond the RNase H active site nificant contacts between the RNase H domain, the con- (Wöhrl et al., 1995). nection subdomains of both p51 and p66, and the p51 The amino acid side chains in close proximity to an in- thumb subdomain (Kohlstaedt et al., 1992; Ding et al., coming dNTP include the following amino acids residues 1994). Structural elements of the palm and fingers subdo- of 110-117, 160-161, 183-186 and 219-221, respec- mains of HIV-1 RT were found to form a clamp-like struc- tively. These residues, which are well conserved within the ture that holds the template-primer in precise orientation pol genes of many retroviruses (motifs A and C, Larder et relative to the polymerase active site. These elements al., 1987b; Poch et al., 1989; Delarue et al., 1990), are in- were named 'primer grip' and 'template grip' (Jacobo- volved in forming the topology of the nucleotide-binding Molina et al., 1993), and include sequences from conser- site, which is located in the palm subdomain of the p66 su- ved motifs E and B, respectively (Poch et al., 1989; Dela- bunit of HIV-1 RT. The most conserved element of this re- rue et al., 1990). Most of the contacts between enzyme gion in the HIV-1 RT and other lentiviruses have the YMDD and template-primer involve the sugar-phosphate back- motif (Tyr-Met-Asp-Asp amino acid residues 183-186) bone of the DMA and are therefore not sequence specific. (Kohlstaedt et al., 1992; Jacobo-Molina et al., 1993). The RT-DNA interaction involves elements primarily from HIV-1 RTs in which these residues are mutated are inactive the thumb, fingers and palm subdomains of the p66 sub- (Larder et al., 1989; Boyer, PL. et al., 1992). During the unit. Other regions that are located near the template-pri- dNTP-binding step and prior to nucleotide incorporation, mer include the connection subdomain and the RNase H the polymerase must select the complementary base. domain of p66 (Jacobo-Molina et al., 1993; Ding et al., Along with binding complementary dNTPs, the polyme- 1994). The monomeric forms of both p66 and p51 are pro- rase active site must also position the growing primer and posed to have the same closed structure as seen in the the template chains. The amino acids that interact with the p51 subunit of the heterodimer. The free energy required 31 nucleotides of the primer strand include the residues to convert the p66 from a closed p51 -like structure to the within the primer grip (i.e. M230 and G231) and the Tyr- observed open p66 polymerase domain structure is gene- Met-Asp residues of the YMDD motif. The amino acid resi- rated upon formation of the heterodimer (Wang et al., dues near the polymerase active site that position the 1994). It is likely that the only kind of dimer that can be for- template strand include those in the template grip (resi- med is an asymmetric one like that seen in the heterodi- dues L74 to R78, E89 and Q151 to W152). mer structure, since one dimer interaction surface exists only in p51 and the other in p66. Pathway of DNA Polymerization In the HIV-1 RT-DNA complex, the polymerase and RNase H active sites are separated by approximately 17 During replication of the retroviral RNA genome, HIV-1 RT to 18 nucleotides (Wöhrl and Moelling, 1990; Furfine and must polymerize approximately 20000 nucleotides, 50% Reardon, 1991 a; Gopalakrishnan et al., 1992; Jacobo- of which are RNA-templated, in order to generate dou- Molina et al., 1993). The double-stranded DNA bound to ble-stranded DNA (for a review see Coffin, 1990). The HIV-1 RT has an unusual and unexpected geometry. The steps involved in nucleotide addition and the rates at majority of the 18 base pair duplex region is B-form, but in which each step occurs have been determined (Majumdar the vicinity of the polymerase active site there are about 6 etal., 1988; Kati etal., 1992; Reardon, 1992; Hsieh et al., to 7 base pairs of DNA which are in the A conformation 1993). A general mechanism for a single nucleotide addi- (Jacobo-Molina et al., 1993). At the junction between the tion during DNA synthesis includes four steps: -form and B-form regions of the template-primer, there is (1) RT binds with its template-primer, a 40 to 45° bend that widens the minor groove. The inter- (2) the appropriate dNTP binds to the RT-nucleic acid actions between the residues of the thumb, palm, and fin- complex, ger subdomains with the first 6 base pairs may displace (3) a nucleophilic attack results in phosphodiester bond key water molecules. Since the DNA hydration can affect formation, and the B to A transition, the loss of the water could influence (4) the pyrophosphate is released. the propensity of the DNA near the polymerase active site Biochemical experiments showed that the rate limiting to assume an A form. The rest of the molecule may simply step is either phosphodiester bond formation or the puta- adapt the B form, since in the absence of a strong DNA- tive RT conformational change preceeding nucleotide in- protein interaction, this is the preferred conformation. A corporation (Kati etal., 1992; Hsieh etal., 1993). Overall, combination of biochemical data and molecular modell- HIV-1 RT can complete these four steps 20 times per sec- ing suggested that side chains of the amino acids 65 to 74, ond (Kati et al., 1992; Reardon, 1992). If the reaction is within the p66 finger subdomain, interact with the single- continued in a distributive fashion, the rate of enzyme stranded template and thus extend it approximately six turnover, specifically the rate of RT dissociation, becomes nucleotides (Boyer et al., 1994). Additionally enzymatic rate limiting. The faithful insertion of nucleotides during footprinting in combination with molecular modelling of polymerization involves a dynamic interaction between HIV-1 RT-DNA complexes indicated that the p66 subunit HIV-1 RT, the nucleic acid and dNTP substrates. During of the p66/p51 heterodimer protects about 6 base pairs these interactions it has been proposed that RT under- HIV-1 Reverse Transcriptase 107 goes at least three conformational changes. Divita etal. sence of the next complementary dNTP. All results sho- suggested that HIV-1 RT, in the presence of DNA, changes wed similar values and indicated that binding of a single conformation upon binding to nucleic acids (Divita et al., dNTP stabilizes the RT-DNA-dNTP complex (Muller ef al., 1993b). The polymerase is thought to undergo a second 1991 a; Kati ef al., 1992; Patel et al., 1995). Just as HIV-1 conformational change subsequent to dNTP binding im- RT undergoes a conformational change upon binding to mediately prior to the chemical catalysis step. This puta- DNA, it is thought that the enzyme performs a second tive conformation change is thought to facilitate the inter- structural change following dNTP binding. Although action of the incoming nucleotide with the 3'-OH of the constraints imposed by Watson-Crick base pairing are primer (Patel et al., 1991; Kati et al., 1992; Hsieh et al., presumably major factors in selection of the correct nu- 1993). The final conformational change involves translo- cleotide, RTsfrom different sources (e.g. HIV-1, MLV) have cation toward the new 3'-OH primer terminus after addi- fidelities varying over 100-fold with identical DNA templa- tion of the dNMR tes (Preston and Garvey, 1992), suggesting that the struc- ture of the polymerase active site must also be involved in (i) Template-Primer Binding The first step of polymeri- the selection of the proper base. Specifically, Y115 could zation involves the physical association of the polymerase affect the topology of the dNTP-binding site and could be with its nucleic acid substrate. HIV-1 RT tightly binds dou- directly involved in fidelity. This residue is likely to be anal- ble-stranded nucleic acids with a K^ of 5-38ΠΜ. Structu- ogous to Y865 of mammalian DNA polymerase á (Dong ef ral and biochemical data as well as fluorescence quench- a/., 1993) and to Y766 of E. coll DNA polymerase Klenow ing studies suggested that RT binds to DNA by a two step fragment (Carroll efa/., 1991), both of which are thought to mechanism (Divita et al., 1993b; Kruhoffer et al., 1993). be involved in fidelity (Wong efa/., 1991; Johnson, 1993). The structure of HIV-1 RT, in the absence of DNA, shows However, by using kinetic techniques, it has not been pos- the p66 thumb subdomain folded over the p66 palm sible to dissect the conformational change from the subdomain, and coming into contact with the finger sub- chemistry step. Because a high-resolution structure of domain (Raag et al., 1994). In this formation, the nucleic HIV-1 RT complexed with DNA and a dNTP does not yet acid binding cleft is 15A in diameter, which is large enough exist, it would be premature to give a detailed account of to accommodate single-stranded but not double-stran- how the conformation of RT might change after dNTP ded DNA. This may have biological significance since it is binding. possible that prior to polymerization HIV-1 RT may first Horlacher efa/. have recently reported that HIV-1 RT, in bind to single-stranded RNA or DNA and then slide until it contrast to eukaryotic DNA polymerases, uses also non- encounters the primer 3'-OH. In this two step model, the standard Watson-Crick bases during its DNA-dependent rate of the first step is dependent on RT concentration and DNA synthesis. Such bases, e.g. 5-(2,4-diaminopyrimi- the rate of the second step is independent of RT concen- dine) heterocycle (dK) opposite deoxyxanthosine (dX), are tration (Divita ef al., 1993b). After RT binds double-stran- included in the synthesized strand (Horlacher efa/., 1995). ded DNA, the tip of the p66 thumb is revealed approxima- In addition, the nucleoside triphosphates modified on tely 30 away from the finger subdomain (Smerdon et al., the sugar appear to be accepted by HIV-1 RT (Schinazi, 1994; Ding et al., 1995). This alteration of the thumb posi- 1993). There is no a priori reason why a tolerance of modi- tion presumably also affects the mobility of the thumb fied sugars should foreshadow a tolerance of non-stand- subdomain and could also inhibit, for example, enzymatic ard base pairs. However, HIV-1 's toleration of the variation turnover (Gopalakrishnan and Benkovic, 1994) or could in both sugar and bases is consistent with the common lower the processivity of the enzyme by influencing trans- view that the active site in HIV-1 RT is 'looser' than location (see below). that in cellular DNA polymerases. Subsequent to the All known activities, as well as biochemical measure- dNTP binding, the polymerase (if in the proper conforma- ments of the nucleic acid binding affinity of the polyme- tion) can catalyze formation of the phosphodiester bond. rase with RNA and DNA templates (Kati et al., 1992; Yu In atwo-metal mechanism model proposed by Patel efa/., and Goodman, 1992), indicated that HIV-1 RT binds both the amino acids residues D110 and D186 chelates one of RNA/DNA and DNA/DNA within the same cleft in the the two Mg2+ ions while D185 could chelate the second orientation described by Jacobo-Molina ef al. (1993). Mg2* ion. The second metal is in position to directly parti- High-resolution structures of RT complexes with relevant cipate in the nucleophilic attack step by contributing to intermediates may help to understand this enzymatic the electropositive character of the á-phosphate facilita- mechanism. ting a nucleophilic attack by the oxygen atom of the 3'-OH (ii) dNTP Binding After binding HIV-1 RT to nucleic primer terminus. The resulting PPj may transiently bind to acids, the next steps of polymerization include the binding D110 and D186 via Mg2+ coordination prior to its release of the appropriate dNTP and the nucleotidyl-transfer reac- (Patel efa/., 1995). tion which leads to incorporation of the nucleotide. To test whether the binding of several successive dNTPs kineti- (iii) Translocation After the RT has incorporated a nu- cally favours polymerization, different groups determined cleotide, it can either dissociate from the DNA or translo- the rate of dideoxythymidine nucleotide incorporation cate to the new 3'-primer terminus. The majority of con- onto a double-stranded DNA in the presence and ab- tacts with RT involve the sugar-phosphate backbone of 108 M. Hottiger and U. Hübscher the DMA. Thus, the resulting overall binding affinity re- 1989; Hottiger et al., 1994). The enzyme furthermore exhi- mains approximately constant. Since the double helix is a bits poor fidelity (Preston etal., 1988; Roberts etal., 1988; spiral, the motion of the protein relative to the DMA must Ji and Loeb, 1992; Yu and Goodman, 1992; Ji and Loeb, also be a spiral moving down the nucleic acid in steps of 1994; Patel and Preston, 1994) and low processivity (Hu- 3.4Ä (along the axis of the DMA), and this movement invol- ber et al., 1989; Abbotts et al., 1993; Klarmann et al., ves a ratchet-type mechanism (Patel et al., 1995). 1993). This section reviews these activities. Biochemical data showed that some DMA polymerases are more processive and can catalyze the addition of RNA and DNA Dependent DNA Synthesis many nucleotides before dissociating from the nucleic Although reverse transcriptase uses both DNA and RNA acid substrate. HIV-1 RT and E. CO//DNA polymerase Kle- templates in its natural replicative reaction, the enzyme now fragment are examples of enzymes with moderate to exhibits a marked preference for the RNA template in low processivity (Eckert and Kunkel, 1993; Klarmann ei vitro. Synthesis on poly (rA) templates is processive with an al., 1993), while T7 RNA polymerase exhibits high proces- incorporation rate of 10 to 15 nucleotides per second at sivity. Comparing sizes of thumb and finger subdomains, 37 °C (Huber et al., 1989). This elongation rate is similar to HIV-1 RT and Klenow have finger and thumb subdomains that of other eukaryotic DNA polymerases (for a review of approximately the same length, while the finger and see Kornberg and Baker, 1992). Reverse transcriptase thumb subdomains of the processive T7 RNA polymerase forms a stable complex with poly (rA)/oligo (dT) primer- are nearly twice as long (Sousa et al., 1993). The distance templates and has in the absence of Mg2* and dTTP an between the tips of the thumb and finger subdomains in equilibrium dissociation constant of 3 nM. Preformed the HIV-1 RT-DNA complex is 30Ä, as compared to only complexes decay with a maximal half-life of 2 to 3 min 20A in the T7 RNA polymerase DNA complex (Sousa et (Huber et al., 1989). Synthesis from these preformed com- al., 1993). This means that while the opening between fin- plexes can be initiated and restricted to a single proces- 2 ger and thumb subdomains in the HIV-1 RT-DNA complex sive cycle by the simultaneous addition of Mg * and dTTP. is wide enough to allow for dissociation of DNA, in the T7 The catalytic properties of HIV-1 RT show no dependence RNA polymerase this opening is smaller than the diameter for monovalent cations, but require a divalent cation, with 2 2 of the DNA double helix (25A), thereby allowing the T7 a preference for Mg * over Mn * (Le Grice etal., 1988). The RNA polymerase to wrap around DNA and to prevent its elongation rate on natural DNA templates is processive dissociation (Sousa etal., 1993). If the DNA polymerases with an incorporation of 0.6 to 0.7 nucleotides per second of an organism are not inherently processive, and a high at 37 °C (Reardon, 1993). Subsequent turnovers are limi- processivity is required in vivo, other proteins which pro- ted by the rate of dissociation of the primer/template from mote polymerase-nucleic acid binding may be recruited. the enzyme at rates of 0.18 and 0.06 per second for du- This is the mechanism by which proliferating cell nuclear plex DNA and RNA/DNA heteroduplexes, respectively antigen (PCNA) and replication factor C (RF-C) augment (Katiefa/., 1992). processivity of eukaryotic DNA polymerases and e (Hüb- Specific terminations of the processive synthesis on ei- scher et al., 1996). Since HIV-1 RT does not possess pro- ther native DNA or RNA templates occur most frequently cessivity (see below) and the virus seemingly needs pro- at positions where the RT pauses during synthesis. These cessive polymerization for efficient replication, HIV-1 RT positions, which are known as termination sites, correlate may also recruit other proteins to increase the efficiency of with the template sequence 3'-(A/U)(A/U)(G/C)-5', partic- its RNA- and DNA-templated polymerization activities. ularly when this sequence is predicted to be base paired These proteins could be of either cellular or viral origin and with another region of the template in a secondary struc- would likely function with the polymerase in such a way ture (DeStefano etal., 1992a). The termination sites could that the binding cleft opening is reduced, interfering with be associated with template secondary structures in the dissociation of the RT-DNA complex (Patel et al., some cases, but many termination sites appear to be tem- 1995). plate sequence-related rather than secondary structure- related (Abbotts et al., 1993). These data indicated that template runs containing a strong termination site are Enzymatic Properties of HIV-1 Reverse greatly slowed (up to 15 min per termination site) and are Transcriptase hot spots for frameshift mutations by the HIV-1 RT (Klar- HIV-1 reverse transcribes its RNA genome into double- mann ef al., 1993). Taken together, several data suggest stranded DNA by a complex mechanism involving RNA- that for the completion of proviral DNA synthesis, one or and DNA-templated DNA polymerase and RNase H activ- more accessory factors are required in vivo (Klarmann et ity (Larder etal., 1987a; Hizi etal., 1988; Muller et al., 1989; al., 1993). These accessory factors could be either virus- LeGriceandGruninger-Leitch, 1990; Mullerei a/., 1991b, or cell-encoded and may include HIV-1 RT itself, since RT for reviews see Varmus, 1987; Coffin, 1990; Goff, 1990). is present in more than 50 copies per virus. During reverse transcription of the viral genome, HIV-1 RT also catalyzes strand transfer (Luo and Taylor, 1990); Peli- Processivity ska and Benkovic, 1992; Peliska and Benkovic, 1994a) For productive infection HIV-1 RT must catalyze the effi- and strand displacement DNA synthesis (Huber et al., cient incorporation of 20000 bases. Processivity varies HIV-1 Reverse Transcriptase 109 widely with the template used, increasing from a few to strand displacement was dependent on DNA synthesis over 300 nucleotides in the order of: poly (dA) < double- and that the HIV-RT p66, but not the HIV-1 RT p51 homo- stranded DMA < single-stranded DMA < single-stranded dimer, was able to perform strand displacement. Further- RNA < poly (rA) (see also above). Processivity, on natural more kinetic experiments after complementation of HIV-1 templates and homopolymers other than poly (rA), is low RTp66 with HIV-1 RTp51 indicated that HIV-1 RTp51 can as compared with other replicative DMA polymerases (Hu- restore the rate and also the extent of strand displacement ber et a/., 1989). On native or homopolymer templates, the DNA synthesis activity by HIV-1 RT p66 compared to that overall length distribution of processively synthesized of the HIV-1 RT heterodimer p66/p51. Amacker et al. re- products is increased by a higher temperature, the deoxy- cently obtained very similar data for the FIV RT p66/p51 ribonucleoside triphosphate concentration, or a decrea- and for the same interactions between the two subunits of sed ionic strength (DeStefano et a/., 1992a). The probabi- FIV RT, p66 and p51 (Amacker ef a/., 1995). lity of termination decreases considerably after addition of the first nucleotide, suggesting that initiation and proces- Strand Transfer sive synthesis are distinct steps. Finally, the RNase H do- The current model for reverse transcription predicts two main may also contribute to the processivity of the poly- 'jumps' between the ends of templates (for a review see merase (Telesnitsky and Goff, 1993a). Telesnitsky and Goff, 1993b). The first and second jump are from RNA and DNA templates, respectively, and are Fidelity thought to be essential for replication. The jump from an Because of the lack of proofreading function, all RT are RNA template may depend on RNase H activity to expose notoriously error-prone (Coffin, 1990). HIV-1 RT seems to the end of the product strand DNA such that it can hybri- be significantly more error-prone than other RTs, with fre- dize to the acceptor template (Tanese ef a/., 1991; Teles- quencies of misincorporation ranging from 1:1700 to nitsky ef a/., 1992). In vitro studies with model substrates 1:4000 (Preston et al., 1988; Roberts et al., 1988), which is suggested that RT is pausing due to template sequences about ten times higher than that for AMV RT. This rate ap- or the reduced concentration of dNTP can enhance strand pears to partly account for the high mutation rate of HIV-1 transfer (DeStefano ef al., 1992b; Klaver and Berkhout, in vivo (Preston and Garvey, 1992). In vitro, RT errors in- 1994). The jump may not require the dissociation of the RT clude misinsertion and rearrangements. Their frequencies from the primer and may be the reason for a high processi- are dependent on the source of RT, the template se- vity (Buiser ef a/., 1991; Peliska and Benkovic, 1992). The quence, the local sequence context, and the composition annealing reaction of the two complementary strands was of the mismatch (Bebenek ef a/., 1993). For example, mis- shown in vitro to be accelerated up to 3000 fold by the incorporation by HIV-1 RT can be as high as 1:30 at some addition of NC under optimal conditions (Chang and positions, while at other positions it can be as low as McHenry, 1994). However, this activity of NCp7 is not suf- 1:106. The high error rate may be due to the loose associa- ficient to explain all of the kinetic data, which indicates tion of RT with the template, which is a necessity for that NCp7 enhances the RNAse H activity of the reverse strand transfer (Pathak and Temin, 1990; Temin, 1993). transcriptase (Peliska ef a/., 1994). The RT incorporates Furthermore, the mutations appear to be nonrandom and additional bases beyond the 51 end of the RNA template, can include substitutions, insertions and deletions (Pre- resulting in base misincorporations upon DNA strand ston et a/., 1988; Roberts et al., 1988). Fidelity is several transfer. Such a process occurring in vivo during retroviral fold higher with RNA than with DMA, suggesting that most homologous recombination could contribute to the hy- mutations occur during the second DMA strand synthesis permutability of the HIV-1 genome (Peliska and Benkovic, with the DMA template-DNA primer (Boyer, J.C. et al., 1992). 1992). Based on the cumulative frequencies of base sub- stitutions, frameshifts and deletions/insertions, about 10 Template Switching/Genetic Recombination to 100% of replicating viruses will produce mutant pro- Genetic recombination in retroviruses requires the pack- geny after each replication cycle. In mixed infections that aging of two genetically distinct genomic RNAs, and takes permit formation of heterozygous virions, 1 out of every 2 place during the reverse transcription process (Hu and to 3 replication viruses will produce recombinants (see Temin, 1990). One model for recombination (the so called below). Thus, altogether about 50% of replication viruses forced copy choice model) proposes template switching will produce mutant progeny during each cycle (Preston during minus-strand DNA synthesis (Coffin, 1979). This and Garvey, 1992). model predicts that DNA synthesis must stop when RT encounters an interruption in one viral RNA template and Strand Displacement DNA Synthesis presumably a blunt RNA-DNA hybrid terminus would be More recent studies with HIV-1 reverse transcriptase he- formed. RNase H activity at this point could help to ex- terodimer and heterologous templates have clearly dem- pose a single-stranded DNA terminus that might anneal to onstrated that this enzyme can catalyze strand displace- a homologous location on the second viral RNA; this ment DNA synthesis (Huber ef a/., 1989; Hottiger et al., would be analogous to the 'first jump' in reverse tran- 1994). Hottiger et al. (1994) additionally showed that scription, in which RNase H has been shown to play a role. 110 M. Hottiger and U. Hübscher

In vivo, RNase H activity may facilitate this by creating sin- val of RNA primers (Levin ef a/., 1988; Oyama ef a/., gle-stranded DMA, but in vitro it is not essential for jum- 1989). ping between templates (Luo and Taylor, 1990; Buiser et RNase H would thereby function in a processive, or poly- a/., 1991). It is possible that the DMA terminus can be ex- merase-coupled mode. Several groups have studied the posed by melting as well as by RNase H. RT catalyzes ho- coordination of DNA polymerase and RNase H activities mologous recombination in wVoand in vitro with high effi- (Oyama ef a/., 1989; Luo and Taylor, 1990; Wöhrl and ciency (Srinivasan et a/., 1989; Goodrich and Duesberg, Moelling, 1990; Furfine and Reardon, 1991 a; Furfine and 1990; Hu and Temin, 1990; Luo and Taylor, 1990). Whether Reardon, 1991b; Gopalakrishnan ef a/., 1992; Kati ef a/., genetic recombination plays a role in generating diversity 1992). The two activity site are separated by 10 to 19 nu- in vivo is not known. cleotides (Gopalakrishnan ef a/., 1992; Kati ef a/., 1992). This separation is consistent with the modelling of an -form DNA-RNA template product in the putative bin- ding cleft (Kohlstaedt ef a/., 1992). Although there is consi- RNase H derable support for coupling of DNA polymerase and RNAse H activities, there are both biochemical (Huber ef RNase H enzymes degrade the RNA portion of a RNA- a/., 1989; DeStefano ef a/., 1991b) and genetical data(Te- DNA hybrid. The RNAse H activity plays a crucial role in lesnitsky and Goff, 1993c) that suggested uncoupled ac- proviral DNA synthesis. Tanese et a/, showed that virus tivities. One way to rationalize these results is to imagine particles containing mutations in the RNase H are compe- that RNase H can function in two modes, one dependent tent for initiation and DNA synthesis, but not for subse- upon and the other independent of polymerization (Furfine quent elongation of the minus strong-stop DNA (Tanese and Reardon, 1991b; Gopalakrishnan ef a/., 1992). Gene- and Goff, 1988). The defect of HIV-1 with mutations in the tic studies indicated that the uncoupled mode may be suf- RNase H to elongate DNA synthesis is presumed to be in ficient for low level viral replication (Telesnitsky and Goff, translocation (Prasad and Goff, 1989; Hizi et a/., 1990). In 1993c). Recent in vitro studies have indicated that during vitro, HIV-1 RNase H has been shown to have both endo- the first jump, the template RNA may be removed in two and 3'-> 5' exonuclease activities (directional processing) steps, using the two modes (Peliskaand Benkovic, 1992). (Krug and Berger, 1989; Oyama ef a/., 1989; Schatz et a/., Finally, it was found that NCp7 enhances the RNase H ac- 1990; DeStefano et a/., 1991 a) which limits digestion pro- tivity of HIV-1 RT and changes the specificity of RNA hy- ducts ranging in size from 5 to 20 nucleotides (Vaishnav drolysis (Peliska ef a/., 1994). and Wong-Staal, 1992). In the presence of Mn2+, directio- nal processing activity predominates, while both activities were catalyzed with equal efficiency in the presence of Virus Diversity and Reverse Transcriptase Mg2"1" (Zahn ef a/., 1994). Recent data with RT from human retroviruses indicate that RNase H activity is not restricted One of the more striking characteristics of HIV-1 is its ex- to RNA/DNA hybrids. Initially Ben-Artzi ef a/, presented treme genetic variability that is not only manifest in strains evidence that the RNAse H domain of HIV-1 RT was capa- isolated from individual to individual but also in those from ble of hydrolyzing double-stranded RNA in the presence a single individual. The rapid genetic change of the virus in of Mn2+ (Ben-Artzi ef a/., 1992a). Although the biological any individual host may contribute to the prolonged and significance of this activity [RNase D (Ben-Artzi ef a/., progressive nature of the infection by allowing the virus to 1992b), now designated RNAse H*] remains unclear, escape immune destruction (Desai ef a/., 1986). The con- several observation provide strong evidence that hydroly- siderable genetic diversity of HIV-1 was realized soon sis of RNA/DNA hybrids and RNA/RNA duplexes are me- after the first viruses were isolated and was based on re- diated by the same active site in the RNase H domain. This striction enzyme analyses of cloned isolates as well as of is in conflict with the accepted view that RT-associated fresh uncultured HIV-1 isolates (Alizon ef a/., 1984; Luciw RNase H is able to discriminate between such duplexes ef a/., 1984; Shaw ef a/., 1984; Benn ef a/., 1985; Hahn ef (Ben-Artzi ef a/., 1992a; Blain and Goff, 1993). It seems a/., 1985; Wong-Staal ef a/., 1985). Additionally, biological that the duplexes are bound similarly to the enzyme re- variants of the virus have been measured by cell tropism, gardless of its precise composition and that discrimina- replication characteristics and cytopathicity. The evalua- tion between RNA/DNA and RNA/RNA is accomplished tion and measurements of genetic and biological diversity locally near the RNase H active site, rather than globally of viral isolates, however, is further complicated by the ob- through the general RT interaction (Gotte ef a/., 1995). servation that any in vitro manipulation such as a passage Retroviral RNases H (for a review see Champoux, 1993) in tissue culture can alter the dominant genotype and phe- are thought to perform two important functions during re- notype of the virus (Meyerhans ef a/., 1989). However, re- verse transcription, striction enzyme polymorphism ranges from single site (1) the removal of the RNA template strand to prepare the variations to changes in > 50% of sites. The rate of chan- DNA intermediate for plus-strand DNA synthesis and ges has been calculated to be 10"3 nucleotide substitu- for both template jumps, and tions per site per year in EM/and 10~4 nucleotides in Pol (2) the specific cleavage involved in formation and remo- (Hahn ef a/., 1986). How these differences in the genetic HIV-1 Reverse Transcriptase 111 sequences and the diverse strains of HIV-1 arise is not lular proteins bound to immunodeficiency viruses: implications clear, but the viral RT itself is very error prone and thus ap- for pathogenesis and vaccines. Science 258,1935-1938. pears to rise readily to changes in the genome (Preston et Baltimore, D. (1970). RNA-dependent DNA polymerase in virions a/., 1988; Takeuchi et a/., 1988) (see below). It was estima- of RNA tumour viruses. Nature 226,1209-1211. Barat, C., Lullien, V, Schatz, ., Keith, G., Nugeyre, M.T., Grunin- ted that about 10 base changes in the HIV-1 genome per ger-Leitch, F., Barre-Sinoussi, F., Le Grice, S.F., and Darlix, J.L. replicative cycle occur. Yet an asymptomatic patient can 6 (1989). HIV-1 reverse transcriptase specifically interacts with harbour at least 10 genetically distinct variants of HIV-1 the anticodon domain of its cognate primer tRNA. EMBO J. 8, 8 and for an AIDS patient the figure is more than 10 , among 3279-3285. which one may find drug-resistant mutations (e.g. AZT) Barat, C., Schatz, O., Le Grice, S., and Darlix, J.L (1993). Analysis even in the absence of therapy (Najera et a/., 1995; of the interactions of HIV-1 replication primer tRNA(Lys,3) with Wain-Hobson, 1995). Despite the evidence that supports nucleocapsid protein and reverse transcriptase. J. Mol. Biol. the existence of the biological variants of the virus it has 237,185-190. Barre-Sinoussi, F, Chermann, J.-C., Rey, F., Nugeyre, M.T., Cha- been difficult to link these characteristics directly with maret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, distinct genetic profiles and with the development of the F., Rouzioux, C., Rozenbaum, W., and Montagnier, L. (1983). disease in the host. Nevertheless, it is reasonable to con- Isolation of a T-lymphotropic retrovirus from a patient at risk for clude that genetic diversity contributes to biological vari- acquired immune deficiency syndrome (AIDS). Science 220, ations, and that such variations have important implica- 868-871. tions concerning the pathogenic behaviour of the virus Bathurst, I.C., Moen, L.K., Lujan, M.A., Gibson, H.L, Feucht, P.H., within an individual host (Tersmette etal., 1988; Tersmette Pichuantes, S., Craik, C.S., Santi, D.V., and Barr, P.J. (1990). etal., 1989). Characterization of the human immunodeficiency virus type-1 reverse transcriptase enzyme produced in yeast. Biochem. 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