METHOD OF MONITORING HIV ASSEMBLY AND MATURATION

Abstract: The present invention provides methods to characterize the structure, stability, and intersubunit interfaces between the matrix, capsid, and nucleocapsid domains of the Gag polyprotein during HIV capsid assembly and maturation. A method of screening for compounds that promote or inhibit viral assembly and maturation is disclosed. A novel mass spectrometry based approach to measure hydrogen/deuterium exchange profiles is also disclosed. Quantitative data resulted from these studies may lead to well defined capsid assembly assays that can be adapted for rapid antiviral drug screening.

Description: METHOD OF MONITORING HIV ASSEMBLY AND MATURATION Cross-reference to Related Application This non-provisional patent application claims benefit of provisional patent application U.S. Serial number 60/ 1 86 , 98 1 filed March 6, 2000, now abandoned. BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the fields o f biophysics and virology. More specifically, the present invention relates to detailed biophysical studies which will lead to both a quantitative understanding of HIV assembly and maturation, a n d the development of in vitro assays that can be readily adapted for drug screening and evaluation. Description of the Related Art HIN- 1 (and retroviruses in general) assembles throu gh the controlled polymerization of the Gag polyprotein. The Gag polyprotein is transported to the plasma membrane and form s patches within which assembly occurs ( 1 ). Transport to th e plasma membrane acts to concentrate the Gag protein but is apparently not essential as Gag protein expressed at sufficiently high levels is capable of cytoplasmic assembly (2). During assembly, the patches enlarge and bud outward ultimately pinching off from the cell. During budding, the virion acquire s the envelope proteins necessary for receptor binding, as well as a lipid envelope. Morphologically, the released immature virion presents as an enveloped particle approximately 100 nm i n diameter, containing a spherical core (3). The viral protease i s incorporated into the virion as part of a Gag-Pol fusion protein , arising from a -1 translational frameshift. Budding activates th e protease, which cleaves the Gag polyprotein into the matrix (MA), capsid (CA), and nucleocapsid (NC) domains as well as th e "spacer" peptides P2, PI , and the C-terminal P6 domain. Th e immature virion is metastable, and cleavage of the polyprotein i s associated with a profound morphological change in the virion (4). The matrix domain stays associated with the m em bran e envelope, the capsid domain collapses to form a conical c ore , and the nucleocapsid domain condenses with the viral RNA in th e center of the conical capsid core. The structural rearrangements necessarily arise from the disruption of existing interdomain contacts and the formation of new ones (Figure 1 ). Despite a wealth of structural information about th e isolated HIV- 1 matrix, capsid, and nucleocapsid domains, little i s known about the structure and interactions of these elements within either the immature or mature virus. The structure of th e domains themselves may be different in the context of the full length Gag polyprotein than in isolation. The intersubunit interfaces between the domains which promote assembly an d their changes upon maturation have not been identified. Nothing is known of the nature of the large scale motions within the virion required for protease acces s , maturation, and uncoating. There are two reasons why this is so : the enveloped, non-icosahedral, pleomorphic nature of the virion makes cyrstallographic analysis of the intact virion nearly impossible, and techniques to study the dynamics of large protein complexes have not been readily available. The prior art is deficient in the lack of detailed quantitative information on the protein/protein interactions mediating HIV assembly and maturation. The present invention fulfills this long-standing need and desire in the art. SUMMARY OF THE INVENTION The present invention is drawn to methods t o characterize the structure, stability, and intersubunit interfaces between the matrix, capsid, and nucleocapsid domains of the Gag polyprotein in virus-like particles. The present invention feature s studies on HIV-1 in particular. Although high res olution structures are available for all three of these domains, th e structural determinations have been performed on i solated domains, and the relationship between these domains in the viru s is unknown and difficult to determine by traditional methods. The present invention provides a method to moni tor the kinetics of in vitro capsid protein (CA) assembly. In the se studies HIV-1 capsid protein was assembled by diluting i n concentrated NaCl to produce a final concentration of 1 M NaCl instead of dialyzing capsid protein into 1 M NaCl, thus allowing rapid transfer of the assembly reaction to the cuvette fo r analysis. The capsid protein assembly rate was dependent o n capsid concentration and the capsid protein C-domain interactions . Analyzing the kinetics of capsid assembly provide s a sensitive method for monitoring and screening mutations a n d inhibitory compounds that modulate capsid-capsid pro tei n interactions involved in assembly. The present invention also provides a novel ma s s spectrometry based approach to measure h ydro gen/deu terium exchange profiles is used to identify intersubunit interfaces a n d characterize domain stability. This is complemented with Raman spectroscopy-based studies of subunit structure and stability . Selected wild type and mutant proteins which have been shown t o polymerize into physiologically relevant forms are charac terized and then use this information to characterize mature a n d immature enveloped virus-like particles budded from cells grown in culture. These studies provide otherwise unobtain able information on the interactions between the structural dom ain s of HIV-1. The significance of this work is that these step s : assembly, protease activation, maturation, and uncoating a r e critical to the HIV lifecycle and represent either potential o r actual (i.e., protease inhibitors) therapeutic targets. Fully exploiting the potential of these interactions as targets requires a detailed understanding of their form, strength, stability and rate of formation. The present biophysical studies may also result i n the development of simple, rapid, and well defined assays fo r capsid assembly which can be then adapted for rapid antiviral drug screening. In one embodiment of the invention, there is provi ded a method of screening for compounds that promote or inhibit viral assembly and maturation comprising the steps of : maintaining viral structural protein in a soluble form; triggering assembly of said viral protein; contacting said buffer containing said viral protein with a candidate compound or a con tro l compound that does not inhibit viral assembly; and moni toring viral assembly. Other and further aspects, features, and advantages o f the present invention will be apparent from the following description of the presently preferred embodiments of t h e invention given for the purpose of disclosure. BRIEF DESCRIPTION OF THE DRAWINGS So that the matter in which the above-recited features, advantages and objects of the invention, as well a s others which will become clear, are attained and can b e understood in detail, more particular descriptions of th e invention briefly summarized above may be had by reference t o certain embodiments thereof which are illustrated in th e appended drawings. These drawings form a part of th e specification. It is to be noted, however, that the appende d drawings illustrate preferred embodiments of the invention a n d therefore are not to be considered limiting in their scope. Figure 1 shows HIV capsid assembly. The Gag polyprotein precursor assembles at the cell membrane an d subsequently buds to release an immature viral particle. The Gag polyprotein is then cleaved by the protease releasing the matrix (MA), capsid (CA), nucleocapsid (NC) proteins and the P2, PI. and P6 peptides. The released capsid and nucleocapsid proteins condense forming a conical core while the MA protein remains associated with the membrane. Figure 2 shows thin section EM analysis of polymerized capsid (CA) protein formed with dilution technique. A stock solution of capsid protein was diluted to a final concentration of 350 μM capsid protein in 1 M NaCl. Thin section TEM showed that the polymerized capsid protein had formed cylinders similar to those previously observed. The inset show end on views of the approximately 33 nm diameter tubes. Thus, dilution into high salt produces biologically relevant structures. Figure 3 shows salt induced polymerization of capsid protein at 38 uM. Figure 4 shows the rate of assembly increases with salt concentrations. CA was diluted to final salt concentrations ranging from 1.8 to 2.4 M NaCl. There was a pronounced effect of salt on the rate of assembly. However, the final amount of turbidity achieved at extended period of time (140 min) was relatively independent of the salt concentrations, suggesting that salt increases the rate of assembly rather than the fraction of capsid protein capable of assembly. Figure 5 shows the measured assembly rates are reproducible. The data plotted in open circles and solid squares represents data collected in two different assembly reactions. The superimposed data points at 38 μM represent five different capsid assembly reactions, thus indicating that the assembly rates for capsid are reproducible. These data also indicate the s trong concentration dependence of capsid assembly. Figure 6 shows the C-domain inhibits c ap sid assembly. Addition of the C-terminal domain of the HIV- 1 Capsid to assembly reactions results in inhibition of assembly. This provides proof of principle for the ability of this assay to de tec t inhibitors of assembly. Figure 7 shows the C-domain inhibits capsi d assembly by forming CA-C-domain heterodimers . The effect o f added C-terminal domain on the rate of assembly was e valuated by plotting the rate of assembly versus C-terminal dom ai n concentration. The inhibition increased with increasing C- terminal domain in an exponential rather than linear manner, a n d no threshold concentration for inhibition was observed. Taken together these data suggest that the C-terminal domain inhibits assembly through the formation of a biologically inactive heterodimer . Figure 8 shows the C-domain decreases the effective concentration of capsid. The squares are the data for c apsi d assembly. The circles are the data for the effective c onc e ntration of capsid free to participate in assembly. The effective cap sid concentration was determined by subtracting the he terodimer concentration from the capsid concentration. The h eterodimer concentration was calculated based on the known as sociation constant. Figure 9 shows mutations alter the assembly rate . The assembly rates shown are for the wild type capsid (circles), the mutant capsid proteins E128A(triangles) and M l 85 A (squares) . The effects of mutations which either increase o r decrease subunit interaction can be detected. Similar analysis i s extendable to external modulators of subunit/subunit affinity. DETAILED DESCRIPTION OF THE INVENTION This invention encompasses a method for detailed biophysical characterization of HIV capsid assembly u sing spectroscopic and hydrogen/deuterium (H/D) exchange s tudie s . Hydrogen/deuterium exchange studies provide a powerful m e a n s of identifying structural elements in proteins and as sembli e s . The exchange rate of the amide protons of the polypeptide backbone can vary over five orders of magnitude depending u p o n whether the amide proton of interest is in a solvent expo s ed peptide, or buried in the folded core of a protein (5,6). In proteins and structured polypeptides, the amide protons a r e exchange protected by hydrogen bonding within α-helices and β- sheets. Further protection is afforded by the hydrophobic core i n globular proteins (7). As a result, the limiting step of hydrogen exchange is the formation of an open, exchange competent s tat e (8,9). The rate of open state formation depends on solvent accessibility and local and global stability. Protein/protein interactions within and between protein subunits can increase local and global stability and thereby retard the rate o f hydrogen/deuterium exchange. Regions which interact to form cooperative folding and association units can be determined b y identifying correlations between regions with regard to th e temperature dependence of exchange ( 10, 1 1 ). Because every amino acid (except proline and the N-terminal amino aci d ) carries an amide proton, hydrogen/deuterium exchange provide s a probe of the local environment of every amino acid within a protein . Two recently developed techniques can be used t o manipulate hydrogen/deuterium exchange as applied to viru s capsids. The first, a Raman spectroscopic probe o f hydrogen/deuterium exchange, takes advantage of the fact th a t the frequencies of the vibrational modes decrease when a deuteron replaces the amide proton ( 12). This results in a n easily monitored shift of the Raman bands, which can be as si gned to elements of secondary structure. This technique provide s information about overall exchange rates, and the elements o f secondary structure that are exchanging. A complementary technique is hydrogen/deuterium exchange as detected by ma s s spectrometry. The strategy in using mass spectrometry t o measure hydrogen/deuterium exchange is to exchange th e protein or protein complex of interest under a defined set o f conditions. During this interval, the exchange rate is determi ned by the protein structure and dynamic behavior. The protein i s then digested under acidic conditions to yield small ( 10- 15 ami n o acid) peptides whose mass and position within the primary sequence can be accurately determined ( 13- 17). Quantification of the increase in mass arising from replacement of the amide protons by deuterons yields the degree of hydro gen/deuterium exchange. This technique yields information about the exchange rate of individual peptide fragments but cannot directly determine changes in secondary structure. The present invention features a method of s creening for a compound or viral protein mutant that promotes or inhibits viral assembly and maturation comprising the steps of: maintaining viral structural protein in a soluble form; triggering assembly of said viral protein; contacting said viral protein with a candidate compound or a control compound that does not inhibit viral assembly; and monitoring viral assembly. Preferably, the viral assembly and maturation is HIV-1 assembly and maturation. Representative viral structural protein include matrix protein, capsid protein, nucleocapsid protein and gag protein of HIV-1. Preferably, viral structural protein is maintained in a soluble form through the use of pertubant. In one embodiment, the perturbant is NaCl. The NaCl would be so used in a concentration of from about 1 M to about 4 M. In another embodiment, the perturbant is GuHCl. The GuHCl would be s o used in a concentration of from about 1 M to about 6M. Preferably, the assembly of viral protein is triggered by rapid removal of pertubant, e.g., by dilution. This method can be used to evaluate various candidate compounds such as protein, peptides derived from the HIV gag polyprotein and non-peptide mimics of those peptides. The viral assembly can be monitored by measuring turbidity, fluorescence or physical separation of the polymerized materials. Furthermore, one may use techniques for detecting subunit/subunit interactions suitable for high throughput screening. In another aspect, the invention features a method to optimize digestion conditions and assign peptides spanning the Gag polyprotein. In yet another aspect, the invention features a method to determine the alterations in the exchange protection profile of the Gag polyprotein and its domains accomp anying polymerization . The invention also features a method to determine th e dynamic stability of spherical and cylindrical polymers. The invention further features a method t o characterize the domain interactions within mature a n d immature budded viral particles. The following examples are given for the purpose o f illustrating various embodiments of the invention and are n o t meant to limit the present invention in any fashion. EXAMPLE 1 Purification of HIV- 1 structural proteins Coding sequences for capsid protein (CA), full length Gag protein, capsid protein with the four C-terminal matrix residues fused (MA 4 -CA), and a mutant of the capsid pro tein (M l 85 A), which cannot form the C-terminal dimeric in terface and therefore forms long strings rather than cylinders ( 18). w ere cloned into pET-based expression vectors and transformed E. coli BL21 (DE3). The Gag expressing cells also carried a pl asmid encoding dnaY gene which supplies a minor Arg tRNA and re sults in increased expression ( 19). The cells containing pl asmids expressing capsid and capsid variants were grown and i nduced with IPTG according to standard procedures. After three h o u r s the cells were lysed with the addition of lysozyme, the lysate sonicated and clarified by low speed centrifugation. The cap sid proteins were precipitated by the addition of ammonium sulfate, resuspended and dialyzed into 25 mM KMOPS pH 6.9 buffer a n d purified by Q-sepharose ion exchange chromatography. Cells expressing the his-tagged Gag protein were grown and induced as described above. The Gag protein form s inclusion bodies, and therefore had to be maintained in a soluble form for purification by the addition of denaturant. The cells were lysed in the presence of 6 M GuHCl and i ncubated overnight. Cell debris was pelleted with a low speed spin, and th e protein was bound to a charged nickel column (Qiagen) in 6 M GuHCl and extensively washed in 6 M GuHCl containing buffer . While the Gag protein remained bound to the column the GuHCl was exchanged by washing with 8 M urea. The bound Gag pro te i n was released from the column by acidification to pH 3.5 with sodium phosphate buffer. The released Gag, in the presence of 8 M urea, was neutralized by the addition of 1 M Tris-HCl pH 9 a n d applied to an SP-Sepharose ion exchange chromatography. The Gag protein passed through the column while contaminating proteins remained bound. Removal of the urea from purified Gag by dialysis into 50 mM NaCl, 25 mM Tris, pH 7.6 resulted i n aggregation. Therefore, the protein was stored in denaturing concentrations of urea. E. coli BL21 (DE3) cells carrying a pET based cl one expressing the CA-NC and CA-NC-p6 domains were induced a n d the protein purified as described (20). While the analyses of Gag structures assembled in vitro will yield significant new information on Gag protei n structure and assembly, such in vitro made structures may not b e accurate reproductions of those that are budded from cells. O n e of the advantages of the hydrogen/deuterium exchange technology is that the presence of lipids does not affect th e ability of the assay to examine proteins since membranes are permeable to D 2 0. Thus it is possible to directly probe t h e structure of enveloped particles produced from cells . Furthermore, since the HIN protease can be easily inactivated b y the introduction of a mutation into its active site sequence, it i s possible to produce and therefore examine both morphologically mature and immature particles . To produce such particles, the Tet-OfF M expres sion system (CLONTECH™) was used to express gag-pro in ti s sue culture cells. PCR primers PNL4GAGSacII and PNL4PRSTOP w ere used to amplify the sequence from the infectious molecular HIV clone pNL4-3 to generate wild-type gag-pro . For the introduction of the D25N mutation into the protease sequence two additional primers, PNLMFED25NPR and PNLMFED25NGAG, were used. EXAMPLE 2 Assembly of HIV-1 Capsid Protein The HIV-1 capsid (CA) protein plays a crucial role i n both assembly and maturation of the virion. The pre s en t invention develops a method for monitoring capsid assembly t o characterize capsid-capsid interactions involved in cap sid assembly. Monitoring the process of polymer assembly with kinetic analysis has been well established for many years. The in vitro assembly of the Tobaco Mosaic virus was the first to b e studied with kinetic analysis. Similar techniques have also b e e n applied to the assembly of P22 in order to elucidate key steps i n the assembly process. Analyzing the kinetics of capsid assembly provided a sensitive method for monitoring the effects of cap sid mutations and inhibitory compounds on capsid-capsid pro tein interactions involved in assembly. A dilution technique was developed to trigger capsid assembly instead of dialysis into 1 M NaCl in order to rapidly transfer the assembly reaction into the ultraviolet (UV) light spectrometer cuvettes for analysis. The proteins were dialyzed into 50 mM Na 2 HP0 4 at pH 8.0 and the c apsid protein w as concentrated to 1 mM. The capsid protein was then diluted with 50 mM Na 2 HP0 4 at pH 8.0 to give the final concen trati on necessary for the assembly reaction at a volume of 197 uL. Th e cap si d protein was triggered to polymerize by the addition of 1 97 uL of 50 mM Na 2 HP0 4 4 M NaCl at pH 8.0 to produce a final 2.25 M NaCl concentration. Initial assembly reactions were done using a final 1 M NaCl concentration. Following the addition of NaCl the assembly reaction was rapidly mixed and place into a cuvette. Approximately 20 sec elapsed between when cap sid protein was triggered to assemble and the first time point w as monitored. The assembly of cap sid protein into larger polymers was followed as an increase in optical density. The large polymers formed during capsid assembly caused an increase in light scattering that was observed as an increase in the solutions turbidity (optical density). The increase in optical density w as monitored on a spectrometer at 350 nm every 20 sec for 0.5 sec . The data points representing the largest increase in optical density were linear fit using the origin fitting program to ob tai n the slope of the line tangent to these data points. For th e assembly reactions with either the N-domain or C-domain pre s en t they were mixed with the full-length capsid protein prior t o triggering assembly. CA protein was assembled at a final 350 uM capsid protein concentration and final 1M NaCl concentration. The capsid assembled at 350 μM was collected by centrifugation a n d observed by thin-section transmission electron microscopy (TEM) to have formed long hollow cylinders similar to the po lymers observed previously (Figure 2). The end on view shows that th e cylinders had a diameter similar to that reported by Schwedler e t ai (ca. 33 nm ) . Capsid could also be triggered to as semble polymers at lower protein concentrations when a higher NaCl concentration (2.25 M NaCl) was used. The oligomers formed a t lower capsid concentrations were bunched together making i t difficult to observe individual cylinders with thin-section TEM. The cylinders observed in the micrographs for capsid assembly a t 38 μM also had a similar diameter to the capsid protein assembled at 350 μM. This suggested the capsid-capsid interactions formed during cap sid assembly at 350 μM w ere similar to the interactions formed at 38 μM and the latter concentration could also be used for studying interacti ons involved in capsid assembly (Figure 3). The assembly rate of cap sid protein was affected b y the salt concentration used. As shown in Figure 4, there was a pronounced effect of salt on the rate of assembly. However, a t extended period of time ( 140 min), the final amount of turbidity achieved was relatively independent of the salt concentration s , indicating that the salt increases the rate of assembly rather th an the fraction of cap sid protein capable of assembly. A final 2.25 M NaCl concentration was used to trigger assembly of cap sid mutant M185A (150 μM) to assemble on a time scale of minutes and a lower capsid (38 μM) concentration could be used to keep the spectrometei fiom being saturated The capsid protein mutant M185A required a concentration approximately three times higher then capsid to assemble on the same time scale (data not shown) Similar to the change in morphology caused by Ml 85 A mutation, this decreased rate of assembly reflected the loss of capsid-capsid interactions necessaiy foi assembly Capsid was assembled at different protein concentrations to determine the effect concentration had on assembly lates A small concentration range of 56 μM to 26 μM showed the assembly rate is stiongly dependent on capsid concentration The region of large increase in turbidity was linearly fit to obtain the assembly rate The slope foi this line represents the rate for assembly The rate verse the capsid concentration demonstrates the dependence on capsid concentiation The data plotted in open cncles and solid squares in Figure 5 represent data collected in two different assembly reactions The superimposed data points at 38 μM represent five different capsid assembly reactions, indicating that the assembly rates for capsid are reproducible These data indicate that the present invention is able to monitor capsid polymerization kinetics and the rates reflect capsid-capsid interactions The known capsid C-domain dimeπzation inteiface was an obvious choice to test if blocking CA-capsid mtei actions would disrupt assembly Capsid and C-domam were mixed prior to assembly, and the assembly rate was decieased (Figure 6) The decreased rate of capsid assembly was concentration dependent on C-domain. The capsid rates verse the C-domain concentration shows an exponential dependence on C-domain concentration (Figure 7). The most probable mechanism for C-domain inhibition is through caps id inactivation by formation of cap sid and C-domain heterodimers. The c apsid C-domain heterodimer c onc entration present in solution prior to assembly was determined using th e capsi d dissociation constant ( 10 mM) and C-domain dissociation constant (18 mM) average ( 14 mM). The capsid conc entration minus the heterodimer concentration (effective cap si d concentration) was taken to be the capsid concentration free i n solution to participate in assembly. When the capsid as sembly rates were plotted verses their effective capsid concentration , they aligned very close to the assembly rates in the presence o f capsid alone. These data are in agreement with the notion that the C-domain inhibits capsid assembly by the formation o f heterodimers . Another capsid interaction necessary for assembly i s the proposed N-domain interactions responsible for formation o f the capsid hexamers. The N-domain and capsid were mi xed together prior to assembly to determine if the N-domain would affect capsid assembly. Although the N-domain interactions would be expected to be essential to capsid polymer formation , capsid assembly was not affected by the addition of N-domain (data not shown). To determine whether the C-domain and N-domain interact to change the capsid assembly rates, C-domain and N- domain were mixed with capsid prior to assembly. The N-domain was found to partially alleviate the C-domain inhibition. Presumably the N-domain interacts with the C-domain to interfere with the interactions necessary for C-domain inhibition . Assembly with the N-domain and C-domain added separately o r combined together showed no increase in turbidity. Thus th e change in optical density with time was not due to an inh eren t effect of the N- and C-domain but instead was due to an effect they have on capsid assembly. These data suggest that the N-domain interacts wi th capsid or the C-domain. Amine to amine cross-linking was u s ed to determine if the N-domain was interacting with the C-domain . The N-domain and C-domain (45 μM for both) were mixed together with a final 2.25 M NaCl concentration. In addition t o the C-domain and N-domain homodimers that were detected b y SDS-PAGE, a C-domain and N-domain heterodimer was al s o observed, supporting the idea that the N- and C-domain w ere interacting. The assembly assay described above can also be u s e d to examine the effects of capsid mutations on capsid assembly . As shown in Figure 9, the effects of mutations which increase o r decrease subunit interaction can be detected. Similarly, the as s ay can be extended to examine the effects of external modul ators that modulate subunit interactions. Di scussi on Cryo-electron microscopy showed the cap sid cylinders were constructed with a hexamer lattice, wh ere hexamer rings were connected to adjacent rings by density regions protruding from the rings. In these density maps th e crystal structure of the capsid N-domain packed best into th e density region forming the hexamer ring. A N-domain w ould form interactions with the N-domains adjacent to it thro u gh helices one and two. Repetition of these N-domain interac ti ons formed a hexamer ring, and these rings were tethered together b y the C-domain that packed best to the density region b e tw een these hexamer rings. This C-domain interaction was observed i n the crystal structure where the C-domain crystallized as a di mer . A methionine to alanine mutation at this dimerization interface (residue 185) caused the capsid protein to form no detec table dimers when observed with equilibrium analytical ultracentrifugation. Both the N-domain and C-domain interactions described above are then propagated to form th e hexamer lattice of the cylinder. The same hexamer l attices observed in the cryo-EM reconstructions were modeled to fo rm conical cones with angles similar to those observed in the viral conical core. These data suggest the in vitro assembled c ap sid cylinders are valid models for studying the CA-capsid interac tions involved in conical core formation. In addition the cryo-EM data above, the in vitro assembled capsid makes a good system for studying the capsid- capsid interactions involved in forming the conical core bec aus e the recombinant capsid protein can be produced in large quantities, the capsid protein can be triggered to assemble in vitro , and the in vitro assembled capsid polymers are m or e amenable to structural studies. Most studies done with in vitro assembled capsid had focused on the effects mutations had o n the morphology of the polymer or the solution state of the cap sid protein. These data had provided insight into both the cap sid starting state and the assembly end product. However, no studi e s have focused on the process of assembly. CA polymerization into cylinders involves many complex interactions that can be studied through as sembly kinetics. The present invention provides a method of cap sid assembly that represents capsid-capsid interactions similar t o those observed in the virus. During the assembly process, th e increase in NaCl concentration causes capsid to form the pr op er interactions necessary to form hexamers and these interaction s are propagated to form cylinders. However, not all of the capsid protein assembled i n to cylinders. Some capsid remained in solution, and capsid that w as in oligomers smaller then 24S was not pelleted, suggesting th a t there is a critical concentration for capsid to assemble. Another portion of the capsid formed clumps of aggregates of some fo rm that could be observed by TEM. These clumps probably are n o t nonspecific aggregates. Nonspecific aggregates are usually associated with a change in secondary structure; however, th ere was no difference in secondary structure between soluble cap si d and assembled capsid when analyzed with Raman spectroscopv (data not shown). The N-domain and C-domain either separately o r mixed together did not assemble into oligomers when moni tored with turbidity, suggesting the NaCl-induced assembly of capsid oligomers requires specific interaction of both the N-domain a n d the C-domain in the intact capsid protein. This indicates that t h e capsid protein was assembling through only specific interactions and not nonspecific aggregates. The requirement for specific interactions of both domains suggests the assembled capsid consists of cylinders and aberrant species that are formed with similar capsid-capsid interactions as the cylinders. The pathway of assembly could be considered similar to that of protein folding. At the start of assembly the capsid proteins primarily exists as a dimer or monomer in equilibrium. Following th e addition of NaCl, the capsid protein forms intermediates that will progress to form larger polymers. Similar to protein folding th e assembly falls off during the formation of intermediates . Th e capsid protein primarily aggregates to form three final species : the conical core, the cylinders or the aberrants . Slight varian ces in the global conformation of these intermediates could b e reflected in the formation of aberrants, cylinders, or coni cal cores . The curvature at different positions in the h ex amer lattice in the cylinders will be different depending on how th e capsid protein aligns with the lattice. Adopting a curvature th a t is slightly unfavorable could destine the two sides of the cylinder to not meet and progress into an aberrant as more capsid pro tein was bound to the intermediate. The capsid protein must also form slightly different angles within the hexamer lattice to form the conical core. The inherent flexibility capsid protein relative to each other allows it to use a similar lattice to form b o th cylinders and tubes. The aberrants probably form when th e intermediates are forming angles specific to cylinders and s o m e of the capsid proteins form angles involved in forming the c ore . The incompatibility of the two forms causes the formation of t h e aberrant species. The kinetic analysis of capsid assembly can be used t o study changes in capsid-capsid interactions through mutations o r through inhibitors. The effects of mutations on the assembly rate were observed with the M185A mutation which required a concentration approximately three times higher than wild typ e capsid to produce a similar rate for assembly. This dramati c decrease in assembly reflects the decreased interaction at t h e capsid dimerization interface. However, the decreased but n o t absence of assembly suggests that some of the interactions necessary for assembly are still present in M 185A. Further studies of M185A assembly may reveal differences in t h e assembly process for capsid and M 185A. Sundquist a n d coworkers have suggested the C-domain interaction functions t o tether together the hexamer units formed from the N-domain. If the C-domain functions just as a tether, it should not affect morphology unless the capsid dimers are limiting to the as sembly proces s . The C-domain was used to test the ability of th e kinetic analysis to detect inhibition. The present invention demonstrated that the inhibition of capsid assembly by the C- domain is related to the ability of the capsid and C-domain t o form heterodimers. The high Kd for capsid dimerization is not i n the desired range for typical inhibitors. The sensitivity of th e assembly reaction to high Kd suggests this kinetic assembly assay could be used to detect weak interacting compounds or mu tant s that cause only small changes in capsid-capsid interactions. The other possibility for capsid-capsid interactions i s the N-domain interactions that should only be present at high concentrations of NaCl because they are the driving forces for assembly. The C-domain interactions are not dependent upon th e addition of NaCl because they are already present in solutions with low NaCl concentrations. and there is little differenc e between association constants in high NaCl concentrations a n d low NaCl concentrations. If it is assumed that the C-domam interactions formed in solution are similar to the interactions i n the hexamer lattice of the tubes, then the interactions that m u s t be formed are the N-domain interactions. This means the N- domain interaction is the necessary interaction to form cylinders . However experimental evidences for this interaction have b e e n difficult to obtain. The capsid assembly rate was not changed i n the presence of the N-domain, indicating that there is n o observable interaction between the N-domain and the N-domain of the capsid protein. This is not surprising because as sugge ste d by the difficulty to observe this interaction, the N-domain interactions are probably weak at best. However, the N-domain was observed to affect t h e assembly rate when the C-domain is present. The N-domain alleviated the C-domain inhibition on capsid assembly. With t h e known data about the capsid assembly it is difficult to ascertain a mechanism for this alleviation caused by the N-domain. On e possible mechanism is the N-domain interacts with the N-domain of capsid protein to increase the rate of nucleating assembly b y increasing the concentration of the limiting component. This i s probably unlikely because the N-domain does not affect cap si d assembly unless C-domain is present. This suggests the N-domain is interacting with the C-domain during alleviation of cap sid assembly . EXAMPLE S Optimize digestion conditions and assign peptides spanning _______ Gag polyprotein Since hydrogen/deuterium exchange experiments require the analysis of peptides generated from the targe t molecules, a library of assigned peptides for the Gag polyprotein (that is, peptides of known mass whose location in the primary sequence has been determined) need to be generated. Gag polyprotein is used because it contains all of the s tru c tural elements such as matrix, capsid, nucleocapsid and the sp ac er regions . A key issue during digestion is the generation o f fragments which span the entire protein. For regions in which n o peptide can be identified no exchange information can b e obtained. Pepsin is the enzyme of choice because it displays high activity at pH 2.5 where hydrogen/deuterium exchange i n minimized, and is relatively non-specific (21 ). Under the acidic conditions at which digestion is performed, protein are largely unfolded rendering a large number of sites available. Digestion with pepsin typically yields fragments covering 50-80% of th e protein subunit ( 14, 15). The digestion time that yielded a reasonably large number of fragments was ~6 min. Reduction o f the digestion time could reduce back exchange. To increase the amount of digestion, the relative amount of enzyme provided can be varied and the number o f fragments produced examined. Standard digestion conditions i s established as the lowest concentration of enzyme required t o generate all fragments. Once optimal digestion conditions i s determined, and reproducibility ascertained, the peptide digests are analyzed by MALDI-TOF mass spectrometry. Three strategies can be used for the assignment of the peptides produced. The simplest strategy is to determine if the peptide can be examined by exact mass matching. In cases where mass searching yields more than one possible peptide within a 0.3 Da mass unit window, the peptides can be identified by either postsource decay sequencing or electrospray MS/MS. In rare cases, two peptides of overlapping mass may be present in the digest, and not be resolved in the first stage MS. If required, these peptide can be separated by reverse phase HPLC prior t o mass analysis. As peptides are assigned, they are mapped onto the primary sequence. EXAMPLE 4 Determine the alterations in the exchange protection profile of the Gag polyprotein and ils domains accompanying polymerization A comparative analysis of the hydrogen/deuterium exchange protection profiles of individual structural domains of the Gag polyprotein in various oligomerization states, as well as in intact monomeric Gag can be made. Wild type and mutant proteins which can be induced to polymerize into a variety of physiologically relevant morphological forms are used for comparisons. Among these comparisons are monomeric and dimeric capsid protein, spherical and cylindrical capsid polymers, CA-NC monomers and CA-NC polymers with RNA, and individual Gag domains compared to the intact polyprotein. EXAMPLES Identification of intersubunit interfaces involved in L __ dimerizntion of capsid protein in solution Monomeric wild type and mutant capsid proteins can be compared to dimeric capsid protein to characterize the dimer interface location and stability. Under low salt conditions, the capsid protein is in a monomer/dimer equilibrium with an estimated K d for dimerization of between 10-30 μM (22-24). Based on this K d , at concentrations above ~ 5mg/ml the protein will be predominately dimeric. H/D exchange of capsid protein is therefore performed at 5-10 mg/ml protein concentration in 25 mM sodium phosphate (pH 6.5), 100 mM NaCl buffer (23). For the exchange experiments, the first step is to establish an appropriate time scale for the CA(M185) monomer. Wild type capsid protein and the mutant CA(M185A) are exchanged at 4 °C for 5, 20, 40, 120 min and the extent of D 2 0 exchange is examined. This straightforward experiment allows discrimination between rapidly exchanging and protected protons and map them onto the three dimensional structure. Amide H/D exchange rates at solvent accessible sites are variable but should be on the order of 0.05 - 4 sec " ' (15). In the second step, peptides which exchanged rapidly in solution and those whose exchange required large scale breathing motions are determined. The experiment described above for measuring the rate of exchange is repeated at 35°C where breathing motions is enhanced. Distinguishing between these peptides allows one to focus on peptides which exchange without breathing motions, and therefore presumably lie on the subunit surface. The third step is to determine the exchange rates fo r dimeric capsid and compare the exchange rates to those obtaine d for the monomer, focusing on exchange conditions chosen t o favor exchange of surface residues (i.e., 4 °C). Regions which display exchange protection are identified as candidates fo r intersubunit interfaces. Alterations in exchange protection c ou l d also arise from conformational changes, or decreased breathing motions induced by dimerization. If regions outside of t h e crystallographic interface display increased exchange protec tion , it is likely due to conformational change. Surface amide p ro t o n s should exchange on the time scale of minutes, whereas th o s e which are buried should display slower exchange. If there is a n increased protection of a peptide whose exchange rate is on t h e order of an hour in the monomer, it is likely that this is in t h e folded core of the protein. Protons with exchange rates on t h e order of minutes are considered good candidates for interfacial protons ( 15) . EXAMPLE 6 Determination of the effect of the matrix capsid junction on t h e conformation of the capsid protein The M A 4 -capsid dimer can be compared to dimeric capsid protein to characterize the contribution of N-terminal hairpin to capsid structure and stability. Biologically active MA 4 - capsid protein can be obtained in sufficient quantities and at high enough concentration. These experiments are done essentially a s described for the wild type capsid protein (22-24). The exchange experiments are performed essentially as described above. EXAMPLE 7 Identification of the differences in capsid protein pa cki n g between spherical and cylindrical polymers To identify the differences in subunit packing which accompany polymerization, the hydrogen/deuterium exchange protection profiles of spherical polymers assembled from MA 4 - capsid and cylindrical polymers assembled from capsid can b e compared to each other and to their constituent subunits . Comparisons of the hydrogen/deuterium exchange protec ti on pattern within the polymer to that of the constituent s ubun i t identify the interfacial regions formed during assembly . Comparisons of the exchange protection profile for the spheri cal and cylindrical forms identify the differences in the interfacial regions between the mature and immature capsid core. The studies on spherical and cylindrical polymers c a n also be extended to studies of the polymerized M 185A cap sid mutant. This capsid mutant provides a unique opportunity t o gain insight into the role of the structure of the C-terminal c ap sid domain in determining the morphology of the viral core. Th e M185A mutation disrupts the C-terminal dimer interface ; however, the protein still forms polymers presumably throu gh interactions involving only the N-terminal domain ( 18). Unlike the cylindrical polymers formed from the wild type cap si d protein, the mutant protein polymerizes into large string-like polymers which appear to not have been able to wind up into a cylinder. Comparison of the hydrogen/deuterium exchange protection profile of these polymers to those assembled from wild type capsid protein allow separation of the contribution o f the N-terminal domain towards polymerization from that of t h e C-terminal domain. Capsid proteins (CA, MA 4 -CA, and CA(M 1 85A)) can b e polymerized into tubular structures by dialysis against 1 M NaCl ( 18, 25, 26), and the polymerized structures are harvested b y centrifugation. Exchange of the polymerized capsid can b e initiated by resuspending the pelleted polymer in 1 M NaCl buffer in D 2 0. Following exchange, the polymer is quenched a n d analyzed in an identical manner to that of the dimeric c ap sid protein. It has been reported that polymerized capsid m ay dissociate upon dilution (25, 26). If dissociation is slow, relative to the time of exchange, it can be safely ignored. If dissoci ation occurs on a time scale comparable to the time required fo r exchange, the rate of dissociation can be decreased with increasing salt concentration over the 1 -4M NaCl range. EXAMPLE 8 Determination of whether the polymers produced by ra p i d dilution are structurally related to those produced by di alysis High salt promotes the polymerization of c ap si d protein. It is well established that increasing the s alt concentration rapidly (by dilution of the protein into high s alt) leads to the formation of ill-formed polymers, while increasing i t slowly (by dialysis) leads to relatively well formed cylindrical o r spherical particles. For practical purposes, it would b e advantageous to be able to trigger assembly simply by dilution o f the protein into high salt. The ability to rapidly and simply trigger assembly is a prerequisite for high throughput screening of compounds designed to block key interactions duri n g assembly. To test the hypothesis that the intersubuni t interactions in polymers produced by rapid dilution may b e similar to those produced by dialysis, the h ydrogen/deuteri um exchange protection of CA, MA4-CA, and CA(M 185) in polymers produced by rapid dilution can be compared to tho s e polymerized by dialysis. EXAMPLE 9 Determination of the effect of RNA dependent polymeri zation o n the structure of the CA-NC protein The hydrogen/deuterium exchange profile f o r monomeric CA-NC, CA-NC-p6 protein and the same pro tei n s polymerized in the presence and absence of RNA can b e determined to study the stabilization of intersubunit interacti ons . Raman spectroscopy can be used to detect changes in secondary structure which accompany polymerization with an eye tow ard s identifying folding of the N- and C-terminal regions of NC u p o n polymerization. Biologically active CA-NC, and CA-NC-p6 ar e purified by the method of Campbell and Vogt (20). CA-NC i s reported to remain monomeric in 1M NaCl at concentrations ~ 1 mg/ml in the absence of RNA (26), and at substantially higher concentration (~9 mg/ml) below 0.5 M NaCl (27). For hydrogen/deuterium exchange experiments, solvent exchange into D 2 0 is accomplished by "spin column" desalting a n d followed by digestions as described above. EXAMPLE 10 Identification of the i nteracti ons between MA, CA, and NC domains in the Gag polyprotein During the lifecycle of HIV, the domains of Gag (MA, CA, and NC) exist in two distinct states, l e as part of th e polyprotein and individually following proteolysis Although high resolution structures exist for each of these domains individually, there is currently no data available on whether these dom ain s interact in the intact protein and influence each others ' structure, or exist as isolated domains connected by the sp ac er regions Thus, the exchange protection of the capsid region o f mtact Gag piotein can be compared to that of monomeric a n d dimeric capsid Increases and decreases in the degree o f protection are interpreted as evidence of eithei stabilizing o r destabilizing interactions respectively EXAMPLE 11 Determination oi the dynamic stability of spherical a_n_d cylindri cal polymers Dynamic transformations, such as protease acces s , maturation, and uncoating require flexibility within the viral capsid Interfaces which stabilize the immature virion must b e disrupted and new inteifaces formed during maturation Those new interfaces must subsequently dissociate during uncoating Recent evidence using mass spectrometry and protease di gestion suggests that there are substantial breathing motions within viral particles (29. 30) Destabilized regions undergoing breathing motions would appear to be good targets for antivirals The stability and breathing motions within spherical and cylindrical polymers of capsid protein can be examined by determining the thermal stability of the capsid protein using circular dichroism and Raman spectroscopy. and by measuring the increase in the hydrogen/deuterium exchange rates of the slowly exchanging regions of the protein with increasing temperature and GuHCl. This latter approach is an adaptation of that of Bai (10, 11). These studies will define the relative strengths of the interactions within the core of the virus, and suggest a molecular mechanism for destabilization. EXAMPLE 12 Characterization of the domain interactions within mature and immature budded viral particles The environment within the budded virus is difficult to mimic in vitro. To gain structural information about the immature and mature forms of the virion, hydrogen/deuterium exchange studies on viral particles budded from cells are performed. H/D exchange studies can be applied to enveloped viruses because the presence of the envelope itself does not interfere with hydrogen/deuterium exchange (31). Wild type and protease mutant versions of gag-pro, plasmids pTREGPCTE and pTREGP*CTE respectively, are cloned and used to produce both budded particles that are proteolytically mature or morphologically immature for comparative studies. Stable cell lines for particle production can be created within the neomycin-resistant HeLa Tet-Off cells obtained from Clonetech. Briefly, the pTRE-based expression constructs are co- transfected into HeLa Tet-Off cells with a plasmid carrying th e hygromycin resistance gene. The ratio of pTRE:Hygro plasmids i s 20: 1 thus ensuring that the majority of hygro-resistant clone s contained the pTRE expression plasmid. Clones are selected and then evaluated for Gag production by commercially available p24 CA ELISA kits. B oth during the hygro selection process and for culture m aintenanc e these cells are maintained in medium containing doxycycline i n order to repress expression of gag-pro . Expression of gag-pro i s induced by the exchange of the culture medium with the s am e medium without doxycycline and containing a tet-free certifi ed fetal bovine serum (Clonetech). Twenty-four hours p o s t induction the culture medium is collected, filtered through a 0.45 μm filter and virus-like particles harvested by centrifugation o n t o a 40% w/w sucrose cushion. The medium/sucrose interface i s collected, multiple samples pooled, and the pooled material m ad e 50% w/w in sucrose. This particle/sucrose material is then u s ed , along with a 25% w/w sucrose solution to construct a linear 25 - 50% sucrose gradients in a BioComp™ gradient maker. After 1 6 hours of centrifugation the region of the gradients containing sucrose at a density of - 1.16 g/ml is pooled, diluted, and th e virus particles pelleted. Pelleted particles are then re su spended in the appropriate buffer for H/D exchange. The following references were cited herein: 1. Weldon, R. A., Jr., and E. Hunter. 1997. Molecular requirements for retrovirus assembly. In Structural Biology of Viruses. Oxford University Press, New York. 381 -410. 2. Boulanger and Jones. 1996. Current Topics in Microbiology and Immunology. 214:237-260. 3. Nermut and Hockley. 1996. Current Topics in Microbiology and Immunology. 214:1-24. 4. Vogt. 1996. Current Topics in Microbiology and Immunology. 214:95-131. 5. Englander et al. 1979. Analytical Biochemistry. 92:517-524. 6. Woodward. 1994. Current Opinion in Structural Biology. 4:112-116. 7. Dill. 1990. Biochemistry. 29:7133-7155. 8. Englander and Mayne. 1992. Annu. Rev. Biophys. Biomol. Struct. 21:243-265. 9. Englander et al. 1996. Current Opinion in Structural Biology. 6:18-23. 10. Bai et al. 1994. Proteins: Structure, Function, and Genetics. 20:4-14. 11. Bai et al. 1995. Science. 269:192-197. 12. Tuma and Thomas, Jr. 1996. Biophysical Journal. 71:3454- 3466. 13. Dharmasiri and Smith. 1996. Analytical Chemistry. 68:2340- 4. 14. Mandell et al. 1998. Anal Chem. 70:3987-95. 15. Mandell et al. 1998. Proc Natl Acad Sci U S A. 95:14705-10. 16. Ohguro et al. 1994. Protein Science. 3:2428-34. 17. Smith et al. 1997. Journal of Mass Spectrometry. 32:135-46. 18. von Schwedler et al. 1998. EMBO J. 17:1555-1568. 19. Brinkmann et al. 1989. Gene. 85:109-14. 20. Campbell and Vogt. 1995. J Virol. 69:6487-6497. 21. Juhasz and Martin. 1997. Int. J. Mass Spectrom. Ion Processes. 169:217-230. 22. Rose et al. 1992. Proteins. 13:112-119. 23. Yoo et al. 1997. J Mol Biol. 269:780-795. 24. Brooks et al. 1994. Methods Enzymol. 240:459-478. 25. Ehrlich et al. 1992. J Virol. 66:4874-83. 26. Gross et al. 1997. Eur J Biochem. 249:592-600. 28. Ganser et al. 1999. Science. 283:80-3. 29. Bothner et al. 1998. J Biol Chem. 273:673-676. 30. Lewis et al. 1998. Proc Natl Acad Sci U S A. 95:6774-8. 31. Tuma et al. 1996. Journal of Molecular Biology. 257:102-115. Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

Claims: WHAT IS CLAIMED IS: 1 . A method of screening for a compound th a t modulates viral assembly and maturation comprising the steps of: maintaining viral structural protein in a soluble form; triggering assembly of said viral structural protein; contacting said viral structural protein with a candidate compound or a control compound that does not inhibit viral assembly; and monitoring viral assembly, wherein an increase o r decrease of viral assembly in the presence of said candi date compound compared to control compound indicates s ai d candidate compound promotes or inhibits viral as sembly respectively . 2 . The method of claim 1 , wherein said viral assembly and maturation is HIV- 1 assembly and maturation. 3 . The method of claim 2, wherein said viral structural protein is selected from the group consisting of matri x protein, capsid protein, nucleocapsid protein and gag protein o f HIV- 1. 4 . The method of claim 1 , wherein said viral structural protein is maintained in a soluble form through the u s e of pertubant. 5. The method of claim 4, wherein said perturbant is NaCl. 6. The method of claim 5, wherein said NaCl is in a concentration of from about 1 M to about 4 M. 7. The method of claim 4, wherein said perturbant is GuHCl. 8. The method of claim 7, wherein said GuHCl is in a concentration of from about 1 M to about 6M. 9. The method of claim 1, wherein said assembly of viral protein is triggered by rapid removal of pertubant. 10. The method of claim 9, wherein said rapid removal of pertubant is by dilution. 11. The method of claim 3, wherein said candidate compound is selected from the group consisting of protein, peptide derived from the HIV-1 Gag polyprotein and a non- peptide small molecule. 12. The method of claim 1, wherein said monitoring of viral assembly is by a method selected from the group consisting of measuring turbidity, measuring fluorescence and physical separation of the polymerized viral protein.

Inventors: PREVELIGE PETER E, PREVELIGE, PETER, E.
Applicants: UAB RESEARCH FOUNDATION
Application year: 2001
Main classification: C12Q1
IPC codes: G01N33