Effects of monovalent and divalent cations on HIV
type 2 dimerization.
Marcela Majda
The University of Montana IBS-CORE Program
Mentor- Dr. J. Stephen
Lodmell
Introduction
HIV is a retrovirus containing a diploid genome formed by two single stranded RNA molecules. These RNA molecules physically link near their 5’ end in a region called the dimer linkage structure (DLS) (Paillart et al, 1996; Skripkin et al, 1994; Laughrea and Jette, 1994; Paillart et al., 1994; Muriaux et al., 1995). In the HIV type 1 virus, the more common form of HIV, the DLS corresponds to a stem and loop structure, or the kissing loop. This regions is highly conserved in all forms of the HIV virus which indicates dimerization may be an excellent target for antiviral drugs (Marquet et al., 1991).
What the results of this study may eventually lead to is a means of preventing the dimerization of the HIV type 2 virus. The dimerization step of the retroviral cycle is a crucial one which affects the ability of the virus to replication. For instance, dimerization may down regulate the translation of the gag gene (Baudin et al., 1993) and it may also provide a positive signal for the encapsidation machinery (Darlix et al., 1990; Gorelick et al., 1991). Should dimerization of the HIV virus be prevented than there is a possibility of stopping the virus from replication and further infection. Once the conditions for dimerization, which this study is attempting to determine, have been elucidated then the search for an antiviral drug therapy to prevent dimerization may begin.
Much research has been done into the HIV type 1 virus and many of the conditions required for its dimerization have been elucidated. These conditions include the types of cation, the temperature and the pH needed for dimerization (Marquet et al., 1991). While the HIV type 1 virus has been studied extensively, the HIV type 2 virus has not, and because of this many researchers have assumed it follows the same dimerization requirements as the HIV type 1 virus since it shows structural characteristics homologous to the HIV type 1 virus dimerization site (Jossinet et al., currently under publication). Despite these similar structural characteristics it has been found that the HIV type 2 virus does not dimerize at this site but links at a 500 base pair region before it. With this finding the question arises whether the HIV type 2 virus follows the same dimerization requirements as the HIV type 1 virus.
The purpose of this study was to determine the monovalent and divalent cation conditions under which the HIV type 2 virus. The divalent cations which were studied were: Mg++, Mn++, Ca++, Co++ and Cu++. The monovalent cations which were considered were: K+, Na+, Li+, Cs+ and Tl+. What this study has shown is that dimerization of the HIV type 2 virus occurs much more readily than the HIV type 1 virus. The HIV type 2 virus showed a significant amount of dimerization under almost all the monovalent cations whereas the HIV type 1 virus favored dimerization under conditions which contained smaller cations (Marquet et al., 1994). Also, the HIV type 2 virus demonstrated a significant amount of dimerization under all of the divalent cation conditions, the only exception being Cu++. Another important finding of this study is that the efficiency of dimerization may be highly dependent on the pH of the buffering solution the HIV type 2 RNA is incubated in.
Results
The elements which were altered throughout the study were the concentration and type of divalent cation in the dimerization buffer, the concentration and the type of monovalent cation in the dimerization buffer and the effect of the presence of magnesium in the gel. The standard dimerization conditions for the HIV type 1 viral RNA are 50 mM NaCac, 300 mM K+, 5 mM Mg++ (Cac50, K300, Mg5). These conditions were also used to ensure complete dimerization of the HIV type 2 RNA. RNA dimerized under these conditions was then partitioned on an agarose gel containing 45 mM tris-borate and 0.1 mM magnesium or alternatively on gels containing 45 mM tris-borate. The average dimerization of several gels run under these conditions was essentially complete dimer regardless of whether the gel contained magnesium or not (chart 1).
The low salt dimerization conditions which produce approximately equal amounts of dimer and monomer in HIV type 1 RNAs are 50 mM NaCac, 40 mM K+, and 0.1 mM Mg++ (Cac50, K40, Mg0.1). These conditions were also applied to HIV type 2 RNA in order to determine whether the type 2 virus followed similar requirements. RNA dimerized under these conditions was run in gels containing 45 mM tris-borate and 0.1 mM Mg++ and also in similar gels containing no magnesium. The average percent dimer of RNA run under the low salt dimerization conditions in a magnesium gel was approximately 80%, whereas the percentage when no magnesium was present in the gel was approximately 60% (chart 1). These results show a significant difference in the final amount of dimer and monomer in gels with and without magnesium when the RNA was dimerized in these low salt conditions.
Effects of divalent atoms on dimerization
The divalent cations studied were Mg++, Mn++, Co++, Ca++ and Cu++. Each of these cations was incorporated into a buffer solution in either 5 mM concentrations or 0.1 mM concentrations. The standard dimerization and low salt dimerization buffer conditions were used to examine the effective dimerization in the HIV type 2 virus first using Mg++ and then replacing the Mg++ with each of the other divalent cations. In addition to these dimerization buffer conditions, the gel conditions were also varied between 45 mM tris-borate and 45 mM tris-borate containing 0.1 mM Mg++ to determine the effect of Mg++ in the gel.
RNA was dimerized under the optimal conditions and Mg++ was replaced by each of the divalent cations. The amount of dimer remained consistent between the Mg++, Mn++, and Ca++ cations with each of them showing almost complete dimer when no magnesium was present in the gel (chart 2). The dimer conditions which contained 5 mM Co++ had a slight decrease in the percentage of dimer (90.0% dimer) and the RNA incubated in 5 mM Cu++ had an even greater decrease in the amount of dimerization (50% dimer). When magnesium was present in the gel the results were the same as when no magnesium was present. The only difference between the two conditions was that the RNA incubated in the dimerization buffer containing 5 mM Cu++ exhibited a lack of RNA when the gel was run.
Gels were also run under conditions in which the dimerization buffer contained no divalent cation (chart 4). The resulting percent dimerization was much lower than that attained by comparable dimerization buffer conditions which contained a divalent cation. When RNA was dimerized in the standard buffer essentially all of it produced dimer regardless of whether the gel contained Mg++ or not. When there was an absence of divalent in the dimerization buffer and there was Mg++ present in the amount of dimer decreased slightly (approximately 90% dimer). When no magnesium was present in both the dimerization buffer and the gel the percent dimerization dropped significantly to approximately 70% dimer.
RNA was then dimerized under the low salt conditions with each of the divalent cations substituting for Mg++ in a gel which contained no Mg++. The divalent cation which resulted in the most dimer on average was Ca++ (70%), followed by Mg ++ (60%). The other three divalents (Mn++, Co++, Cu++) showed similar dimerization percentages of approximately 50% (chart 3). When RNA was run under similar conditions but on a gel which contained magnesium the amount of dimer in all the buffers was equal with each showing approximately equal amounts of monomer and dimer.
When RNA was incubated in a Cac50, K40 and no Mg++ buffer the amount of dimer was essentially equal in a gels which contained magnesium and those that did not. When the extent of dimerization of RNA incubated in these conditions and run on a magnesium containing gel is compared to the amount of dimerization in RNA incubated under the low salt conditions with magnesium in the gel, there is a significant difference with the low salt conditions showing more dimer (approximately 60% dimer in RNA dimerized with no magnesium vs. 80% in the low salt conditions). The percent dimerization of the RNA incubated in dimerization buffer not containing Mg++ in the non-magnesium gel was essentially equal to the percentage dimerization of the RNA in the marginal buffer run in a non-magnesium gel (approximately 60% in both).
Effect of monovalent atoms on dimerization
An evaluation of different monovalent cations at varying concentrations was also performed. The monovalent cations considered were K+, Li+, Cs+, Tl+. Each was present in a buffer at either 300 mM concentration or 40 mM concentration. Included in the dimerization buffers were concentrations of either 5 mM Mg++, 0.1 mM Mg++ or no Mg++.
The RNA was incubated in dimerization buffers containing Cac50, Mg0.1 and 300 mM concentrations of different monovalents. The resulting gels demonstrated high dimerization percentages for most buffer conditions. The percentage for the standard K+ ion was 90% and the percentages for Li+ and Tl+ were 100% regardless of whether there was magnesium present in the gel or not. Na+ also had 100% dimerization but only when magnesium was present in the gel. When magnesium was not present in the gel the percent of dimer dropped slightly to 90%. Cs+ had a dimerization percentage of 95% when the RNA was run in a magnesium gel but the percentage dropped to 75% with no magnesium in the gel (chart 5).
The RNA was then incubated under dimerization conditions of 50 mM NaCac, 0.1 mM Mg++ and 40 mM concentrations of the various monovalents (chart 6). As stated previously the presence of magnesium in the gel significantly affected the percent dimerization under the marginal dimerization conditions. When the standard ion K+ (Cac50, K40, Mg0.1 in a magnesium gel, dimerization percentage of 80%) is replaced with Cs+, Na+, Li+ and Tl+ the percent dimerization is 75% in a magnesium gel. When the RNA was run on a gel with no magnesium all the dimerization percentages dropped to 50%, as compared to the 60% dimerization of the low salt buffer containing K+.
The HIV type 2 RNA was also incubated in dimerization buffers which contained no monovalent cations (chart 7). In buffers containing Cac50 and Mg5 the percent dimerization was fairly high (approximately 80% with magnesium in the gel and 90% with no magnesium in the gel). For the dimerization conditions of Cac50 and Mg0.1 the percent dimerization was significantly lower, 55% in a magnesium gel and 45% in a gel with no magnesium.
Affects of pH on dimerization
How the pH affected the amount of dimerization was also considered. When RNA was incubated in dimerization buffers which contained only Cac50, the purpose of which was to maintain the buffer at a pH of 7.4, on a magnesium gel the amount of dimer and monomer were equal. When magnesium was not present the amount of dimer dropped to approximately 30%. To determine whether this affect was due to the Na+ in the cacodylate buffer or to the pH buffering capacity itself. RNA was incubated in a dimerization buffer containing only NaCl. Under these conditions there was essentially no dimerization. When RNA was incubated in a buffer containing 50 mM Tris buffered to a pH of 7.4 the amount of dimer and monomer were equal in a gel containing magnesium. When there was no magnesium in the gel RNA incubated under the same conditions showed the same amount of dimer as that of RNA incubated in a dimerization buffer containing Cac50 and no magnesium.
Materials and Methods
In Vitro dimerization of HIV-2 RNA
The dependence of cations in HIV-2 dimerization was determined by incubating HIV 2 ROD in varying buffer conditions. The general procedures used are comparable to those used in similar research into the HIV type 1 virus (Marquet et al., 1994): RNA (2.5 mg) was diluted in 5.5 ml of nanopure (np) water to obtain a final volume of 8.0 ml. The sample was then heated for 2 minutes at 95C and then chilled on ice for 2 minutes. Two ml of the appropriate 5X buffer were added to the sample and it was then incubated at 37C for 15 minutes. The samples were then placed on ice for 2 minutes and 2 ml of 6X glycerol loading dye was added. Six ml of the sample were loaded onto an ethidium bromide 0.8% agarose gel which was run at 4C at 200V for 2 hours. The gels were run in either 45mM tris-borate buffer with 0.1mM MgCl2 or a buffer containing only 45mM tris-borate. The gels were then exposed to UV light and photographed using Polaroid film.
Statistical Analysis
The percent dimer and monomer were estimated based on the photographs taken on the Polariod film. The amount of dimer and monomer were estimated visually and expressed as a percent dimer. Experiments were repeated several time to ensure reproducibility and these percentages were then averaged over several experiments to determine the efficiency of dimerization with respect to each individual buffer used.
Discussion and Conclusion
The first tests were done in order to compare the efficiency of dimerization of HIV type 2 virus to the dimerization of HIV type 1. Under the standard dimerization conditions (Cac50, K300, Mg5) the type 2 virus showed similar results as the HIV type 1 virus in that it fully dimerized when run in a magnesium containing gel (Marquet et al, 1994). However, the type 2 virus did fully dimerized in gels that contained no magnesium, which is unlike the HIV type 1 virus since the type 1 require magnesium in the gel to fully dimerize (Marquet et al, 1994). Under the low salt (Cac50, K40, Mg0.1) dimerization conditions the type 2 virus showed slightly different amounts of dimer. When the type 2 virus was incubated under the low salt conditions and run in a gel containing magnesium the percent of dimer was approximately 80%. However, when the magnesium was not present in the gel the amount of RNA that dimerized decreased significantly (60%). This may indicate that when there is a lower concentration of magnesium in the incubation buffer, magnesium may need to be present in the gel in order to prevent dissociation of the dimerized RNA or the magnesium may cause the monomer to dimerize while the RNA is in the gel.
When magnesium was replaced by the series of divalents at the optimal dimerization conditions there was little difference between Mg++, Mn++, and Ca++, with each showing almost complete dimerization. Co++ exhibited a slightly less dimer with more RNA moving into a monomer form. Cu++ demonstrated equal amounts of dimer and monomer though in general the majority of the lanes that contained Cu++ in the dimerization buffer were extremely poor and difficult to read. This may have been due to the Cu++ altering the pH of the dimerization buffer and therefore preventing the RNA from efficiently dimerizing. Or it may be due to the Cu++ either degrading the RNA or interacting with the agarose in a way which prohibited dimerization. When no divalent cation was present at all in the incubation buffer but the other aspects were retained (Cac50, K300) and magnesium was present in the gel the samples showed almost complete dimerization, though slightly more dimer moved into the monomer form. However, much more monomer was seen when there was a complete absence of divalent cation in both the dimerization buffer and gel. These results indicate that a divalent cation may be required for efficient dimerization.
The HIV type 2 RNA was also dimerized under low salt conditions with the magnesium being replaced by the divalents: Mn++, Ca++, Co++ and Cu++. The amount of dimer produced from incubation buffers containing Mg++ and Ca++ (60% dimerization with Mg++ and 70% dimerization with Ca++) were similar when no magnesium was present in the gel, with Ca++ showing a slightly increased amount of dimer. The other divalents, Mn++, Co++ and Cu++, all showed the same distribution of approximately equal amounts of monomer and dimer when no magnesium was present in the gel. This indicates that the divalents Mn++, Co++ and Cu++ do not promote dimerization in HIV type 2 as efficiently as Ca++ or Mg++. When there was a complete absence of divalent cation in the dimerization buffer and the gel the amount of dimer was comparable to that observed in the low salt conditions (both showed approximately 60% dimerization). When magnesium was present in the gel under the same conditions there were equal amounts of monomer and dimer. This is a decrease in the amount of dimerization than that seen in the low salt conditions with magnesium in the gel which produced approximately 80% dimer. These results indicate there may be another factor besides the presence of a divalent cation since dimerization is still seen even in the absence of a divalent.
When the HIV type 2 RNA was incubated in buffers containing Cac50, Mg0.1 and 300 mM concentrations of varying monovalent cations K+, Cs+, Li+, Tl+ the results were surprising. Both Li+ and Tl+ produced complete dimerization regardless of whether magnesium was present in the gel. K+ produced high amounts of dimer also, though showed a slight decrease when no magnesium was present in the gel. Na+ also produced complete dimerization but more RNA was present in the monomeric form when no magnesium was present in the gel. Cs+ showed significant dimerization with magnesium but then the amount of monomer increased when no magnesium was present. These results indicate an order of increasing dimerization as follows: Cs+ < K+ < Na+ < Tl+ = Li+. Since there was such a significant amount of dimerization in low divalent concentrations an increased concentration of monovalent cation may push the RNA towards dimerization.
Under the low salt conditions (Cac50, Mg0.1 and 40 mM of the different monovalents) the results indicate that the presence of magnesium in the running buffer significantly increases the amount of dimer. The amount of dimer produced from the cations Cs+, Na+, Tl+ and Li+ (in a gel containing magnesium) are similar and slightly lower than that of K+ in similar conditions. When the samples are run in a gel not containing magnesium the amount of monomer and dimer are equal in RNA incubated in buffers containing Cs+, Na+, Tl+ and Li+. This is slightly lower than RNA incubated in a buffer containing K+ and run on a gel not containing magnesium. These results indicate lower concentrations of monovalent cation require the presence of magnesium to increase the amount dimerization. This may be another indication of the presence of magnesium in the gel preventing dissociation of the dimer or causing the monomer to dimerize in the gel.
When the RNA was incubated in dimerization buffers containing no monovalent cation the amount of dimer depended on the concentration of magnesium present in the dimerization buffer. With a 5 mM Mg++ concentration the amount of dimer was significantly higher than with a 0.1 mM concentration. There was also a slight increase in the amount of dimer when magnesium was present in the gels. Both these concentrations showed a decrease in the amount of dimer than when a monovalent cation was present. This indicates that a monovalent cation may be required for efficient dimerization at low concentrations of divalent but dimerization may occur at higher concentrations of divalent without a monovalent cation.
Due to the fact that the HIV type 2 RNA appeared to be dimerizing under many cationic conditions the affect of pH on the amount of dimerization was explored. The trials are in the preliminary stages but results thus far have shown that an important aspect of the efficiency of HIV type 2 RNA dimerization may be the pH. When RNA was incubated in buffers containing only sodium cacodylate and no other cations there was still significant amounts of dimer when the samples were run on a magnesium gel. To determine if this effect was due to the presence of sodium RNA was incubated in buffers containing no buffering ability (NaCl50) and in buffers containing 50 mM tris. The results showed that when the pH was not buffered no dimer formed. However, when the tris was present the amount of dimer was the same as that seen in samples incubated in sodium cacodylate when run in gels containing magnesium. However, when the samples were run under similar conditions but in gels which contained no magnesium there was no dimer in the RNA incubated in the tris buffer as compared to approximately 30% dimerization in the RNA incubated in sodium cacodylate. This indicates that pH may be extremely important in promoting dimerization in the HIV type 2 RNA but that the presence of a cation is also required.
As these studies have shown the efficiency of dimerization of the HIV type 2 RNA depends on many factors. To begin with, the presence of magnesium in the gel always promoted more dimer than when it was not present with respect to each dimerization buffer condition. The reason for this may be due to the magnesium in the gel being able to maintain the dimeric form or it may cause monomeric RNA to dimerize while the samples are in the gel. Also, these experiments have shown that the presence of both a monovalent and divalent cation are important in the dimerization of HIV type 2 RNA. While the absence of only one of these in a dimerization buffer still produces some dimer that amount of dimer significantly increases when both are present. The only cation which exhibits significant negative affects on HIV type 2 RNA dimerization has been found to be Cu++. Whether these affects are due to an altering of the pH of the buffer and thereby preventing dimerizatin, or the degradation of the RNA itself or the alteration of the itself gel is yet to be determined. Finally, and perhaps the most significant finding in this study, is the apparent dependence of the HIV type 2 RNA on pH. While almost all conditions of dimerization buffer produced some amount of dimer when the pH of the buffer was not maintained no dimer was produced. Further studies are currently being performed in order to determine how significantly the pH affects the efficiency of dimerization of HIV type 2 RNA.
Chart 1. Percent dimerization under standard conditions
Chart 2. Comparison of varying 5 mM divalents
Chart 3. Comparison of varying 0.1 mM divalents
Chart. 4 Absence of divalents
Chart 5. Comparison of 300 mM monovalents
Chart 6. Comparison of 40 mM monovalents
Chart 7. Absence of monovalents
Chart 8. pH dependence
Literature Cited
Baudin, F., Marquet R., Isel, C., Barlix, J.L., Ehresmann, B., Ehresmann, C. (1993) Journal of Molecular Biology, 229, 382-397.
Darlix, J.L., Gabus, C., Nugeyre, M.T., Clavel, F., Barre-Sinoussi, F. (1990) Journal of Molecular Biology, 216, 689-699,
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Laughrea, M., Jette, L. (1994) Biochemistry, 33, 13464-74.
Marquet R., Baudin, F., Gabus, C., Darlix, J., Mougel, M., Ehresmann, C., Ehresmann, B. (1991) Nucleic Acid Research, 19, 2349-2357.
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Currently under publication:
Jossinet, F., Lodmell, S., Ehresmann, C., Ehresmann, B., Marquet, R. Identification of the HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1.
Executive
Summary
HIV is a retrovirus, a virus which contains two single stranded RNA molecules. As a retrovirus the two strands must come together, or dimerize, in order for the virus to continue its viral cycle. This dimerization requires certain conditions such as the type of cation, monovalant or divalent, and a specific pH in order to efficiently dimerize. This study examined how those conditions affected the dimerization of the HIV type 2 virus, the less common form of HIV. The results demonstrate that the HIV type 2 RNA exhibits significant dimer when both a monovalent and divalent cation are present and has a slight decrease when one is absent or in a decreased concentration. The results have also shown that the efficiency of dimerization may depend on the pH of the dimerization buffer. This aspect of the research has only recently come under investigation and further studies on the significance of pH are currently being performed.
Acknowledgements
I would like to thank my mentor, Dr. J. Stephen Lodmell, for the opportunity to work in his laboratory and his guidance and support throughout this project.
I would like to thank the Howard Hughes Medical Institute for giving me the opportunity to conduct research as an undergraduate student.
I would like to thank all those involved in the IBS-CORE project here at The University of Montana for all the support and assistance they have provided me during my fellowship.