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In the pol gene of bovine immunodeficiency virus (BIV) there is a sequence, located between the reverse-transcriptase and integrase (IN)-encoding sequences, that is not found in HIV-1 pol gene and encodes a 74-residue polypeptide with homology to dUTPases. We have expressed two BIV IN versions that differ in their amino termini. The longer version, containing the 74-residue sequence, did not show any detectable 3′-end processing and strand transfer IN activities and performed only the IN-associated disintegration. Consequently, the shorter version, lacking the dUTPase-related residues, performed all three activities and is most likely similar to the viral enzyme. A comparison between BIV IN and the well-studied HIV-1 IN, with substrates that mimic the U5 LTR sequences of BIV, HIV-1 and another bovine lentivirus, Jembrana disease virus, revealed that the extra 3′-end sequence beyond the conserved “CA” is probably less important for IN activities than the sequence upstream to the “CA".

Bovine immunodeficiency virus (BIV) is a lentivirus with no proven pathogenesis in infected cattle. Yet, in experimentally infected rabbits, it causes an AIDS-like disease. Consequently, we expressed two recombinant isoforms of BIV reverse transcriptase (RT), which differ in their C-termini, and studied their catalytic properties. Both isoforms prefer Mg+2 over Mn+2 with most DNA polymerase and ribonuclease-H substrates. The processivity of DNA synthesis by the BIV RTs is higher than that of HIV-1 RT, whereas the fidelity of synthesis is even lower than that of the HIV-1 enzyme. The ribonuclease-H cleavage pattern suggests that the spatial distance between the polymerase and ribonuclease-H active sites of the two BIV RT isoforms equals 20 nt, unlike the 17 nt distance observed in almost all other RTs. The longer BIV RT version is somewhat less active than the shorter version, suggesting that the extra 74 residues (with homology to dUTPases) might obstruct efficient catalysis.

Recent studies have shown that the integrase (IN) of HIV-1 is inhibited in vitro by HIV-1 reverse transcriptase (RT). We further investigated the specific protein sequences of RT that were involved in this inhibition by screening a complete library of RT-derived peptides for their inhibition of IN activities. Two 20-residue peptides, peptide 4286, derived from the RT DNA polymerase domain, and the one designated 4321, from the RT ribonuclease H domain, inhibit the enzymatic activities of IN in vitro. The former peptide inhibits all three IN-associated activities (3'-end processing, strand transfer, and disintegration), whereas the latter one inhibits primarily the first two functions. We showed the importance of the sequences and peptide length for the effective inhibition of IN activities. Binding assays of the peptides to IN (with no DNA substrate present) indicated that the two inhibitory peptides (as well as several non-inhibitory peptides) interact directly with IN. Moreover, the isolated catalytic core domain of IN also interacted directly with the two inhibitory peptides. Nevertheless, only peptide 4286 can inhibit the disintegration activity associated with the IN core domain, because this activity is the only one exhibited by this domain. This result was expected from the lack of inhibition of disintegration of full-length IN by peptide 4321. The data and the three-dimensional models presented suggested that the inhibition resulted from steric hindrance of the catalytic domain of IN. This information can substantially facilitate the development of novel drugs against HIV INs and thus contribute to the fight against AIDS.

The study of porcine endogenous retroviruses (PERVs) becomes increasingly important due to the potential use of pig cells, tissues, and organs as a source for xenogenic cell therapy and xenotransplantation into humans. Consequently, we have constructed a plasmid that induces in bacteria the synthesis of a soluble and highly active reverse transcriptase (RT) of PERV-B. The purified PERV RT was studied biochemically in comparison with the RT of murine leukemia virus (MLV), because of the high-sequence homology between these two RTs. The data show that in several properties the two enzymes are similar, particularly regarding the monomeric subunit composition of the proteins in solution, the high resistance to deoxynucleoside analogues, and the pattern of RNA cleavage by the ribonuclease H activity (RNase H) of the RTs. However, in several cases there are apparent differences between the two RTs, most notable the divalent cation preference (Mn+2 versus Mg+2) in the DNA polymerase reactions. As already shown for viral PERV RT, the novel recombinant PERV RT exhibits a relatively high resistance to several deoxynucleoside analogue inhibitors, suggesting that they might not be very efficient in inhibiting the replication of PERV virions. Therefore, the availability of large amounts of the recombinant RT can be useful for a wide screening of novel drugs against infectious PERV.

We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus. Both 3′-end processing and 3′-end joining (strand transfer) activities of IN were affected by the RTs. Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios. The disintegration activity of IN was also inhibited by RTs. Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform. On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs. We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities. We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction.

The AIDS-causing retroviruses, human immunodeficiency virus types 1 and type 2 (HIV-1 and HIV-2, respectively) undergo extensive genetic variations, which effect their pathogenesis and resistance to drug therapy. It was postulated that this genetic hypervariability results from high rates of viral replication in conjugation with a relatively low fidelity of DNA synthesis [typical to the reverse transcriptases (RT) of these retroviruses]. As part of studying structure/function relationship in HIV RT, mutational analyses were conducted to identify amino acid residues which are involved in affecting the fidelity of DNA synthesis. The formation of 3′-mispaired DNA due to nucleotide misinsertions, and the subsequent elongation of this mismatched DNA were shown to be major determinants in affecting those substitutions during DNA synthesis (exhibited in vitro by HIV RT). It was interesting to find a correlation between sensitivity to nucleoside analogs (due to the ability to incorporate or reject an incoming analog) and the fidelity of DNA synthesis (which depends on the capacity to incorporate and extend a wrong nucleotide). Such a connection has already been found for several drug-resistant mutants of HIV-1 RT, with an increased fidelity of DNA synthesis relative to the wild-type RT. In the present study we have examined the fidelity of DNA synthesis using the same parameters of misinsertion and mispair extension for five novel drug-resistant mutants of HIV-2 RT; i.e. the single mutants [Val74]RT, [Gly89]RT and [Tyr215]RT and the double mutants [Val74, Tyr215]RT and [Gly89, Tyr215]RT. This comparative study suggests that unlike the Val74 mutant of HIV-1 RT, which was shown earlier to display a substantially enhanced fidelity, the comparable mutant of HIV-2 RT has fidelity similar to that of the wild-type RT. Depending on the assay employed and the DNA sequences extended, most other mutants of HIV-2 RT display moderate effects on the enzyme, leading to mild increases in fidelity of DNA synthesis. This implies a more complex and less distinctive correlation between drug-resistance, misinsertion and mispair extension in HIV-2 RT in contrast to HIV-1 RT, providing evidence for potential biochemical differences between these two related RT.

The high error rates characteristic of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) are a presumptive source of the viral hypermutability that impedes prevention and therapy of acquired immunodeficiency syndrome (AIDS). We have analyzed two mutants of HIV-1 RT by conducting a comparative study of the accuracy of DNA synthesis. Each mutant bears a single amino acid substitution adjacent to the two aspartic acid residues at positions 185 and 186 in the highly conserved DNA polymerase active site. The first mutant, Met 184 →Leu (M184L), displays a marked reduction in both misinsertion and mispair extension, suggesting a fidelity of DNA synthesis significantly higher than that of the wild-type HIV-1 RT. The second mutant, Tyr 183→Phe (Y183F), shows a decrease in mispair extension with no significant change in misincorporation. Thus, the overall pattern of error-proneness of DNA synthesis is: wild-type HIV-1 RT > Y183F > M184L. Taken together, it is possible that residues 183 and 184 contribute to the low fidelity of DNA synthesis characteristic of the reverse transcriptases of HIV-1, HIV-2 and possibly, of other lentiviruses. Our observations may bear on the nature of potential mutations responsible for resistance to the nucleoside analogs used in chemotherapy of AIDS.

We present a study of colony transformations during growth of Bacillus subtilis under adverse environmental conditions. It is a continuation of our pilot study of “Adaptive self-organization during growth of bacterial colonies” (Physica A 187 (1992) 378). First we identify and describe the transformations pathway, i.e. the excitation of the branching modes from Bacillus subtilis 168 (grown under diffusion limited conditions) and the phase transformations between the tip-splitting phase (phase T) and the chiral phase (phase C) which belong to the same mode. This pathway shows the evolution of complexity as the bacteria are exposed to adverse growth conditions. We present the morphology diagram of phases T and C as a function of agar concentration and pepton level. As expected, the growth of phase T is ramified (fractal-like or DLA-like) at low pepton level (about 1 g/1) and turns compact at high pepton level (about 10 g/1). The growth of phase C is also ramified at low pepton level and turns denser and finally compact as the pepton level increases. Generally speaking, the colonies develop more complex patterns and higher micro-level organization for more adverse environments. We use the growth velocity as a response function to describe the growth. At low agar concentration (and low pepton level) phase C grows faster than phase T, and for a high agar concentration (about 2%) phase T grows faster. We observe colony transformations between the two phases (phase transformations). They are found to be consistent with the “fastest growing morphology” selection principle adopted from azoic systems. The transformations are always from the slower phase to the faster one. Hence, we observe T→C transformations at low agar concentrations and C→T transformations at high agar concentrations. We have observed both localized and extended transformations. Usually, the transformations are localized for more adverse growth conditions, and extended for growth conditions close to the boundaries between morphologies. We have observed also transformations between different branching modes, as well as transformations via virtual states. Motivated by the contemporary knowledge about phages and plasmids, we postulate a theoretical framework to comply with our experimental findings. We explain our observations using these assumptions as well as our proposal of co-mutations and auto-catalytic mutations as presented in the above mentioned pilot paper. This theoretical framework is a part of the new evolving picture of genome cybernetics. We also discuss the concept of adaptive genome changes which are based on pre-existing knowledge as well as the concept of genetic learning. i.e. changes (in response to a new problem) which develop the potential for adaptive genome changes. These concepts follow naturally if the picture of genome cybernetics is accepted. We conclude with a discussion of the implications and with further predictions (to be tested experimentally) derived from our assumptions.

The tear film is the interface between the external environment and the ocular surface.1 It forms a smooth refractive surface over the corneal surface, lubricates the eyelids, and maintains the optimal extracellular environment for the epithelial cells of the cornea and conjunctiva.1 The tear film consists of three interacting layers: an inner mucous layer, a middle aqueous layer, and an outer lipid layer that floats on the middle layer. Different sets of glands are responsible for maintaining each specific layer. The lacrimal gland secretes the proteins, electrolytes, and water that make up the middle aqueous layer.2,3 The lacrimal gland is made up of acini, ducts, nerves, myoepithelial cells, and plasma cells. The majority, approximately 80%, of the gland are the acinar cells, which secrete electrolytes, water, and proteins.2,3 Dry eye syndrome is caused by inadequate quantity or quality of tears. Aqueous-deficient dry eye or keratoconjunctivitis sicca (KCS) is one of two major subtypes of dry eye syndrome, the other being evaporative dry eye.4–7 Although there are tear substitutes that can be used to temporarily lubricate the eye, there is no curative treatment for dry eye syndrome.4–6 Chronic inflammation of the lacrimal gland can lead to insufficient lacrimal gland secretion, leading to symptoms of dry eye.8–10 Pathologies associated with chronic inflammation, such as autoimmune diseases (Sjogren's syndrome, sarcoidosis, rheumatoid arthritis), organ transplantation, or viral infections (hepatitis, HIV), are often accompanied with symptoms of dry eye syndrome. Lacrimal gland inflammation is characterized by the presence of focal lymphocytic infiltrates, increased production of proinflammatory cytokines, and destruction of the tear-producing parenchymal cells.8–10 Development of the lacrimal gland occurs through a process known as branching morphogenesis.11,12 This process of morphogenesis is achieved through epithelial-mesenchymal interactions that include a highly coordinated spatio-temporal release of several growth factors and subsequent activation of critical transcription factors.11,12 Several growth and transcription factors have been shown to be crucial for branching morphogenesis of the lacrimal gland. Bone morphogenetic protein (BMP)7 plays a major role in tissue development and remodeling and was shown to play a pivotal role in murine lacrimal gland development.13 We have recently reported that the BMP7 pathway was not only activated but was upregulated in the lacrimal gland during the repair phase after injury.14 The presence of stem cells in adult tissues is a very active area of research because of the potential clinical benefits.15–18 Several studies have reported the self-regenerating capabilities of salivary glands and the pancreas, liver, intestine, and mammary gland.19–24 In particular, the regenerative capacity of the salivary glands has been well documented.22,23,25–30 Long-term (7–21 days) ligation of the main excretory ducts of the salivary glands leads to atrophy of the glands. The ligation of the salivary gland corresponds with inflammation, edema, and death of acinar cells. After the duct ligation is released, the salivary glands go through a phase of increased cellular proliferation and repair. Nestin-positive stem cells have been isolated and propagated in vitro from injured rat pancreas as well as salivary and mammary glands.22–24,31 We have recently reported that stem/progenitor cells were present in the murine lacrimal gland and that their number increased during the repair phase after experimentally induced inflammation.14 These cells were identified by their ability to express nestin, a stem cell marker. Some of the nestin-positive cells also expressed α-smooth muscle actin, a marker of myoepithelial cells.14 This suggested a common source of stem cells between myoepithelial cells and acinar cells in the lacrimal gland; that is, the same stem cell can give rise to both myoepithelial as well as acinar cells. The purpose of the present studies was to determine whether stem/progenitor cells can be isolated from the murine lacrimal gland undergoing repair and if they can be propagated in vitro.

We have recently expressed in bacteria the enzymatically active reverse transcriptase (RT) of bovine leukemia virus (BLV) [Perach, M.Hizi, A. (1999) Virology 259, 176-189]. In the present study, we have studied in vitro two features of the DNA polymerase activity of BLV RT, the processivity of DNA synthesis and the fidelity of DNA synthesis. These properties were compared with those of the well-studied RTs of human immunodeficiency virus type 1 (HIV-1) and murine leukaemia virus (MLV). Both the elongation of the DNA template and the processivity of DNA synthesis exhibited by BLV RT are impaired relative to the other two RTs studied. Two parameters of fidelity were studied, the capacity to incorporate incorrect nucleotides at the 3' end of the nascent DNA strand and the ability to extend these 3' end mispairs. BLV RT shows a fidelity of misinsertion higher than that of HIV-1 RT and lower than that of MLV RT. The pattern of mispair elongation by BLV RT suggests that the in vitro error proneness of BLV RT is closer to that of HIV-1 RT. These fidelity properties are discussed in the context of the various retroviral RTs studied so far.

We have recently expressed in bacteria an enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV), a mammalian retrovirus with a typical B-type morphology [Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. (1998) Biochemical J. 329, 579−587]. The purified recombinant protein was shown to possess the catalytic activities characteristic of retroviral reverse transcriptases. In the present study, we have analyzed two basic parameters characteristic of the DNA polymerase activity of the novel MMTV RT, namely the processivity and the fidelity of DNA synthesis. Two features related to fidelity were studied, the capacity to misinsert wrong nucleotides at the 3′ end of the nascent DNA strand and the ability to extend 3′ mispairs. The studied properties of MMTV RT were compared with those of the RT purified from virions of avian myeloblastosis virus (AMV), since AMV RT shows a relatively high sequence similarity to MMTV RT. MMTV RT shows a relative processivity of DNA synthesis which is as high as the reference AMV RT. Regarding fidelity of DNA synthesis, MMTV RT shows a fidelity of misinsertion lower than that of AMV RT, whereas its capacity to elongate mispaired DNA is lower than that of AMV RT indicating a somewhat higher fidelity. These fidelity properties are discussed also in the context of the RTs of lentiviruses, especially those of HIV, which were reported to exhibit an exceptionally low fidelity of DNA synthesis. It is clear that MMTV RT has a fidelity higher than that of lentiviral RTs.

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.

The reverse transcriptase (RT) of human immunodeficiency virus (HIV) undergoes rapid mutagenesis due to selective pressure by RT inhibitors which renders the mutated RT variants resistant to these inhibitors. Resistance to nucleoside analogs during drug therapy results from point mutations that lead to specific variations in the RT sequences. It was recently shown that several well-defined drug-resistant variants of HIV-1 RT (i.e. Leu74Val, Glu89Gly, Tyr183Phe, Met184Lue, Met184Val and Met184Ile) show enhanced accuracy of DNA synthesis relative to wild-type HIV-1 RT (as evident from a reduction in the capacity to introduce mispairs and to elongate them). Since the last two Met184 variants were shown also to possess decreased processivity of DNA synthesis, it was recently suggested that there might be an inverse correlation between the apparent in vitro fidelity and processivity of DNA synthesis in drug-resistant HIV-1 RT mutants. In the present study we have conducted a comparative analysis of the processivity of DNA synthesis on both DNA and RNA templates of the Leu74Val, Glu89Gly, Tyr183Phe and Met184Leu drug-resistant mutants of HIV-1 RT in comparison with wild-type RT. Apart from the Met184 mutant, which shows reduced relative processivity (similar to the other mutants of residue 184 already studied), the other three variants have relative processivity at least as high as that of wild-type RT. This suggests that the inverse correlation between reduced processivity and increased fidelity is restricted only to mutants with modifications of Met184. The results presented may bear on potential mechanistic and structural differences in the involvement of the various mutated residues studied in processivity, fidelity and sensitivity to nucleoside analogs.

Treatment of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with N-ethylmaleimide (NEM) selectively inhibits the RNase H activity. The cysteine residue at position 280 (C280) is the target for NEM; HIV-1 RT carrying the mutation C280S is resistant to NEM. Since HIV-1 RT is composed of two related subunits (p66 and p51) that play distinct roles, we asked whether the C280 in p51 or the C280 in p66 is responsible for the sensitivity of the enzyme to NEM. HIV-1 RT versions were prepared that had one mutant and one wild-type subunit. When these chimeric enzymes were tested, both the p51 and p66 subunits were found to contribute to the sensitivity of the enzyme to NEM. The implications of these results are discussed in the context of the structure of the enzyme.

The relatively low fidelity of DNA synthesis characteristic to the reverse transcriptases (RTs) of the AIDS-causing viruses, human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) was implicated as a dominant factor that contributes to the genetic hypervariability of these viruses. The formation of 3’-mispaired DNA and the subsequent extension of this DNA were shown to be key determinants that lead to the error proneness of these RTs. As part of our goal to study the structure/function relationship in HIV-1 RT, we have conducted mutational studies aimed at identifying amino-acid residues involved in affecting the fidelity of DNA synthesis by the enzyme. We have recently found that two niutantc of HIV-1 RT, which show resistance to nucleoside analog inhibitors ([Leul84)RT and [Phe183]RT), exhibit in vitro error proneness of DNA synthesis lower than that of wild-type enzyme [Bakhanshvili, M., Avidan, 0. & Hizi, A. (1996) Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis, FEBS Lett. 391, 257-2621. Using both criteria, the current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart. In the current study we have analyzed two additional drug-resistant mutants of HIV-1 RT, [Va174]RT and [Gly89]RT, for their in vitro fidelity of DNA synthesis using two parameters of DNA synthesis : 3’ mispair formation and elongation of 3’-mismatched DNA. The current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart, using both criteria. Analysis of the relative frequencies of misinsertion and mispair extension indicates that the overall error proneness of DNA synthesis in HIV-1 RT is wildtype > [Va174]RT > [Gly89]RT mutant. The results further support the possible linkage between the capacity of an enzyme to incorporate a nucleoside analog instead of the correct dNTP (leading to drug sensitivity) and the ability to incorporate and extend a wrong nucleotide (resulting in mutagenesis). Our results may bear on the potential use of selecting and maintaining HIV virions with high fidelity and drug-resistant RTs to suppress the subsequent appearance of virions resistant to other drugs.

Our efforts to express in bacteria the enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV) have shown that the RT is active only after adding 27 amino acid residues, which are derived from the end of the pro gene, to the amino-terminus of the RT (Biochem, J. (1998) 329, 579–587). In the present study we have tested whether the mature RT found in virions is also fused to protease-derived sequences. To this end, we have analyzed the RT molecules in virions of MMTV by using two antisera directed against peptides, derived from either the carboxyl-terminus of MMTV protease or the middle of MMTV RT. The data suggest that the mature RT, located in virions, contains at its amino-terminus sequences from the carboxyl-terminus of the protease protein. This finding supports previous suggestions that MMTV RT is a transframe protein (derived from both pro and pol reading frames of MMTV) and that amino acid residues located at the carboxyl-terminus of the protease have a dual usage as integral parts of both the protease and the RT enzymes.