Effects of Mutation on the Structure and Function of the HIV-1 Envelope Protein Transmembrane gp41 Subunit

Amanda Wilhelm

The University of Montana IBS-CORE Program

Mentor Dr. Jack Nunberg

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INTRODUCTION:

The spread of HIV world- wide has grown to epidemic proportions. Today, with nearly 35 million people infected, the need for an effective vaccine is of vital importance. Vaccines have previously proven effective in eradicating such diseases as small pox and polio. The research being performed in hopes of attaining an HIV-1 vaccine have focused on various aspects of the virus’ biological activity. The envelope of the virus has been recognized as showing great potential as a possible vaccine target. The envelope contains receptor binding proteins and transmembrane glycoproteins that are responsible for the fusion and infectivity of virus to target cells. These proteins are synthesized as a large precursor, gp160. This protein is then proteolytically cleaved by an enzyme to form the subunits, gp120 and gp41. Glycoprotein 120, which binds to receptors on target cells, remains associated through non-covalent interactions to gp 41, the transmembrane protein. (1,2,3,4,5,7,8)

Multiple studies have shown that conformational changes occur in the structures of the transmembrane proteins in the fusion process of envelope to human target cells. HIV-1 is believed to have a similar fusion process as the influenza virus hemagglutinin. In the (HA) virus, a low pH environment causes a conformational change from a loop to a coiled-coil structure near the fusion peptide. This change in structure, known as the “spring-loaded mechanism” places the fusion peptide closer to the target cell membrane making fusion possible. (1,3,5,6,7,8) It is believed that when gp120 of HIV binds to CD4, the primary receptor on human target cells, a similar conformation occurs in gp41 transforming it into a fusion ready conformation. In this state it is capable of fusing to the cell, where the viral genetic information enters to induce infection. (1,2,3,4,5,6,7,8,9)

The prefusogenic conformation of gp41 consists of two alpha helical domains, an amino(N) terminus and a carboxyl(C) terminus. The fusion ready conformation of gp41 forms from an interaction between the N and C terminuses.(Figure 1) The structure consists of a six-stranded alpha helical bundle. In the center, are the N-terminal helices, which associate to form a parallel trimeric coiled-coil. Packed in the hydrophobic grooves of the N-helices are three C-terminal helices arranged anti-parallel to the central N-helices. The helices are comprised of seven amino acid repeats, connoted as a-b-c-d-e-f-g. Positions a and d associate via hydrophobic interactions to give the helix its characteristic shape. Residues at positions e and g are predominately charged.(2,6,7,8,9)

Figure 1. Top and side views of the gp41 fusion-ready conformation. In the center are the three N alpha helices, with the three C helices wound in the grooves of the N trimeric coil.

Interfering with the process HIV-1 undergoes of evolving to a fusogenic state from a prefusogenic state has been shown to be potential vaccine approach. One method is to isolate the intermediate structure between the prefusogenic and fusogenic structures. It is believed that particular mutations inserted in the e and g sites have the potential to not only disrupt the structure of the N-C coiled-coil, thus “freezing” the protein at its intermediate structure, but also to abolish it’s function. This new non-functional protein could be a possible vaccine approach. In theory, this would work, by injecting complexes, including the defective gp41, into a human. The viral envelope protein would be incapable of fusing human cells, yet the body would still produce antibodies against the foreign intermediates. If the subject were later exposed to natural, wild-type virus, the antibodies present in their body would recognize the virions and eliminate them.

In this study, four separate mutations were introduced into the envelope protein in the glycoprotein 41 e and g positions of either the HIV-1 HXB2 or 168P S-Peptide envelope. The major difference between these envelopes is that in the 168P S-Peptide, an extra peptide has been cloned onto the end of the gp41 sequence that enables the protein to be more easily isolated. Each of these mutations changed one amino acid residue to an alanine. These altered envelopes were then tested to compare functionality among the mutants and a wild-type control in a cell-cell fusion assay test. When cells expressing envelope are mixed with cells expressing the target receptors, multinucleated cells, or syncytia or formed. The total amount of syncytia present in the mutants will be compared to the number present in the wild-type control to analyze how the amino acid substitution effect function. The mutants were also tested to determine the effect the amino acid substitution had on the biosynthesis, transport, and expression of the protein.

MATERIALS AND METHODS:

Construction of mutants. Mutations were introduced into either the HIV-1 (HXB2) env or the 168P S-Peptide env sequence by a site-directed mutatgenesis protocol and kit by Stratagene. Primers containing both the intended mutation and an added restriction site, to be used in determining which clones are carrying the intended mutation, were run in a QuikChange™ Site-Directed Mutagenesis (Stratagene) reaction with the env template. The product was then treated with Dpn I, a restriction enzyme which digests the unmethylated (parental) DNA. The remaining DNA was then transformed in supercompetent bacterial cells and plated on agar plates. Eight to nine colonies were then chosen and tested by the analysis of the incorporated restriction sites. The plasmids that displayed the apparently correct fragments were then sequenced at either the Murdock Molecular Biology Facility at the University of Montana, or at the Davis Sequencing-Automated Sequencing Facility at the University of California-Davis. Finally, two clones containing the intended mutations were chosen and prepared using a Qiagen midi prep protocol and kit.

Cell-Cell fusion assay. 1.3*10^5 COS 7 cells were seeded in 6 well plates and incubated overnight. The cells were then transfected with 1-2ug of DNA using a FuGene (Roche) transfection procedure in the afternoon of day two and incubated overnight at 37 degrees Celsius. In the morning of day three the cells were washed one time with PBS and refed with DMEM (Dulbecco’s minimum essential medium) containing 10% fetal bovine serum. In the afternoon of day three the cells were removed and mixed in a 96 well plate with U87 cells expressing the target cell’s receptors, and incubated for 16 hours at 37 degrees Celsius to allow cell-cell fusion to occur. After the 16 hour incubation the cells were fixed to the plate using a 1:1 acetone/methanol mixture and stained with an alkaline-phosphatase procedure to determine HIV expression using antibodies and the alkaline phosphatase. The plates were then scored to determine both transfection efficiency and fusion activity of the mutant envelope.

Envelope glycoprotein expression. The mutants incorporated into the 168P S-Peptide envelope and wild-type were tested to determine whether the proteins were transported to the cell surface and cleaved to form the gp120 and gp41 subunits for the respective envelope sequence. On day one 5*10^5 COS 7 cells per 6 cm dish were seeded. Day two, each dish was transfected with 3ug of envelope expressing plasmid DNA via a FuGene transfection procedure. In the morning of Day three the cells were washed one time with PBS and refed with DMEM plus 10% fetal bovine serum. Day four, the media from the plates was collected, spun, filtered, and frozen at –80 degrees Celsius for later analysis of the shed gp120. The cells were then washed twice on ice with ice cold PBS+0.1mM Calcium Chloride+0.1mM Magnesium Chloride. To identify the proteins present of the cell surface NHS-biotin (Pierce) was dissolved to 0.5mg/mL in PBS-Ca-Mg just before adding it to the cells. 4 mLs NHS-biotin was then added to the cells and incubated for 15 minutes on ice and repeated once more after the incubation. The NHS-Biotin was then “quenched” with 4mLs of 20mM Tris pH 7.4 +0.9% NaCl, in order to remove any unreacted NHS-Biotin. The cells were then washed once with 1x PBS and lysed with 0.5ml ice cold lysis buffer (TritonX100) containing protease inhibitors (A,L,P), and the cell lysate was collected.

BSA(bovine serum albumin) Agarose preclear: The BSA beads, which remove any non-specific binding proteins, were washed into the lysis buffer and mixed with the lysate at a 50% slurry. The mixture was then incubated for 30 minutes at 4 degrees Celsius while rocking. The mixture was then spun at 2000rpm and the “pre-cleared lysate” was collected to be analyzed with either SAG (S-Protein Agarose), which precipitate proteins with the S-Peptide attached (gp41 or gp160) or HIVIG. HIVIG is an immunoglobulin that has been isolated from HIV positive patients and will precipitate all gp41, gp120, and gp160 proteins.

SAG (S-Protein Agarose) pulldown: 40 ul of SAG beads were washed into lysis buffer and then spun down to pack the beads. 250 ul of the “pre-cleared” lysate was then added to the beads and allowed to incubate together for 2 hours at 4 degrees while rocking. The beads were then washed 5 times with lysis buffer before adding SDS-PAGE buffer with DDT. The samples were boiled for 3 minutes, the beads spun out of the mixture, and the lysate analyzed via SDS-PAGE (polyacrylamide gel electrophoresis).

HIVIG pulldown: 250 ul “pre-cleared” lysate was added to tubes containing 5 ul of 1 ug/ml HIVIG and incubated for one hour on ice. After the one hour 40 ul PAS (protein A-sepharose) beads, which bind HIVIG were added to the HIVIG/lysate mixture and incubated for 30 minutes at 4 degrees while rocking. The beads were then washed with lysis buffer, mixed with SDS-PAGE buffer with DDT, boiled for three minutes, the beads spun out and the lysate analyzed via SDS-PAGE.

Western Transfer: Following the SDS-PAGE separation, the proteins were transferred from the gel to nitrocellulose by a Western transfer method. The nitrocellulose was then placed in a 10% milk in wash buffer to block any non specific absorption to the nitrocellulose. This was incubated at 4 degrees over night.

Probing with Neutravidin: The nitrocellulose was washed three times for five minutes each time to remove the milk block from the nitrocellulose. The neutravidin (dilluted 1:20,000 in wash buffer) was added to the nitrocellulose and incubated for one half hour in a rotary apparatus. Neutravidin is used to detect any proteins bound to NHS-Biotin, as the Neutravidin strongly binds NHS-Biotin. The nitrocellulose was then washed and allowed to react with an ECL PLUS Detect solution and read on a phosphoimager.

Analysis of cell supernatants. On day one, 1 ul of the thawed supernatants were added to a tube containing 5 ul of 1mg/mL HIVIG. This was incubated one hour on ice. Protein A-Sepharose beads were then washed into lysis buffer and 40 ul of the beads were then added to the HIVIG/SUPE mixture. They were allowed to incubate together for 30 minutes at 4 degrees while rocking. The beads were then washed 4 times, mixed with equal volume SDS-PAGE buffer, boiled, and spun out. The resulting samples were loaded and separated by SDS-PAGE. After separation, the protein was transferred to nitrocellulose by western transfer. The nitrocellulose was then probed with the monoclonal antibody 50.1 (directed to the V3 loop of gp120) diluted 1:5000 in 5% milk for one hour in a room temperature rotary apparatus. The nitrocellulose was washed and then detected with the ECL-PLUS detect system and analyzed on a phosphoimager.

RESULTS:

Table 1.

MUTATION*	PARENTAL ENVELOPE USED	RESTRICTION ENZYME
L565A	168P S-PEPTIDE	Pst I
V570A	168P S-PEPTIDE	Dra III
G572A	HXB2	-
R579A	HXB2	Ssp I

*The letter at the beginning of the mutant name is the amino acid abbreviation for the original amino acid and the letter at the end is the abbreviation for the amino acid which was substituted in its place.

HIV-1 gp41 mutants. Four different mutations were introduced into the gp41 of HIV-1. A site-directed mutagenesis technique was utilized to switch an amino acid at a particular location. In three of the four mutations, a new restriction site was added without changing the amino acid sequence, to enable the clones carrying the mutation to be distinguished from clones expressing the wild-type sequence (table 1). Eight to nine clones were prepared and each was digested with its respective diagnostic enzyme and sequenced to determine which clones were carrying the intended mutations. In general, about eighty percent of the clones were carrying the intended mutation.

Cell-cell fusion assay. Clones carrying the correct mutation were chosen and analyzed in a cell-cell fusion assay. In each assay the clones were compared with both positive (wild-type) and negative (mock) controls. Both the transfection efficiency and the ability of the clones to form multi-nucleated cells, or syncytia, were observed (table 2 and 3). The amount of syncytia formed were scored on a scale with wild-type being the normal amount formed with a score of (+++). The control mock transfected, with zero syncytia is scored with a (-).

Table 2. clones created in 168P S-Peptide env.

SAMPLE	TRANSFECTION EFFECIENCY	SYNCYTIA FORMATION
MUTANT L565A CLONE #8	30%	-
MUTANT V570A CLONE #4	29%	-
168P S-PEPTIDE (w.t.) POS. CONTROL	24%	+++
MOCK NEG. CONTROL	-	-

Table 3. clones created in HXB2 env.

SAMPLE	TRANSFECTION EFFECIENCY	SYNCYTIA FORMATION
MUTANT G572A CLONE #3	32%	-
MUTANT G572A CLONE #5	40%	-
MUTANT R579A CLONE #5	41%	++
MUTANT R579A CLONE #6	36%	++
HXB2 (w.t.) POS. CONTROL	48%	+++
MOCK NEG. CONTROL	-	-

Envelope glycoprotein expression. In this experiment, cells were transfected to express the mutant envelope proteins. The proteins on the cell surfaces were biotinylated and the cells lysed. The proteins were then pulled down with either S-protein agarose, which would pull down all gp160 and gp41 proteins, or HIVIG, which would pull down gp160, gp120, and gp41. They were then probed with Neutravidin, which binds strongly to NHS-Biotin. The results showed that the proteins were transported to the cell surface, and cleavage of gp160 did occur.(Figure 2) It was evident that gp41 was present. In the range where gp160 and gp120 would be visualized, a large protein smear was present. Due to the ambiguous answer to whether gp120 was in fact present on the cell surface, further analysis was required.

Figure 2. Cell surface protein analysis. Proteins precipitated with HIVIG showing gp41, and the gp160/gp120 smear.

In order to answer the question of whether the gp160 cleaved to gp120 and it was present on the surface of the cell, the supernatants in which the cell had been grown in were analyzed for shed gp120. In the growth of infected cells, the cell regularly sheds gp120 from its surface. In this test, the supernatant proteins were pulled down with HIVIG, and probed with the antibody 50.1, which binds to the V3 loop of gp120. In this test, clear, distinct protein bands of 120 size were present.(Figure 3)

Figure 3. Cell supernatant analysis. Glycoprotein 120 present in the cell supernatant, precipitated by HIVIG.

DISCUSSION:

The transmembrane glycoprotein 41 plays a key role in the fusion of HIV-1 to target cells. As a result, this protein was chosen to test the effects an amino acid substitution would impart on the biosynthesis, transport, expression and functionality of the virus. Four separate mutations were introduced into either the 168P S-Peptide or HXB2 envelope in the N-C e and g positions. The two mutations inserted into the 168P S-Peptide envelope, L565A and V570A, resulted in an envelope that was fusogenically defective. The mutations inserted into the HXB2 envelope, G572A and R579A, displayed different results from each other. G572A was fusion defective and R579A’s ability to fuse was only partially impaired. R579A was still capable of producing approximately two-thirds of the number of syncytia produced by the wild-type HXB2 envelope.

There are explanations as to why the proteins are non-functional, or in the case of R579A, partially fusogenic. With R579A, we know that the essential proteins are on the surface due to the fact that cells expressing this envelope are still capable of fusing other cells. From the results of the cell surface protein experiments of the 168P S-Peptide envelope we also know that these proteins made it the cell surface. Cleavage also occurred as shown in the results. However, the amino acid substitution may have altered to conformation of gp41, this making it sterically hindered in the fusion process. Another possibility is that the gp160 reached the surface, cleaved to gp120/gp41 and the amino acid change was suble enough to preserve the structure yet, at the same time, inhibit the functionality of the protein.

The goal of inserting these mutations is that the last of the above possibilities would occur. In the search for a possible vaccine, one would need a viral envelope similar to the envelope structure of the wild-type virus, yet at the same time, remain non-functional. In order to determine whether or not the proteins did in fact make it to the surface and are being expressed as the correct subunits, cell surface analysis was performed on the L565A and V570A mutations. In order to determine whether gp160 cleaved to gp120, a further test was performed to analyze the media in which the cells were grown. When cells are infected with HIV-1 and expressing proteins on their surface, they regularly shed gp120 into their surroundings. If the mutants were in fact producing cleaved gp120/gp41 subunits, one would expect to find gp120 in the media. The results indicated that there was in fact gp120 present in the media from both mutants.

Due to the promising results of the L565A and V570A mutants, they have been further tested. They are currently in immunogen preparations tests where they are present in complexes with target receptors. These complexes are placed in mice, which then elicit an antibody response. Blood is drawn and the sera is tested to analyze its ability to neutralize virus from various clades of HIV-1. To date, data is pending.

ACKNOWLEDGMENTS:

This work was funded in part by an IBS-CORE Undergraduate Research Fellowship to Amanda Wilhelm through a grant from the Howard Hughes Medical Institute to the University of Montana. We also acknowledge members of the Montana Biotechnology Center, especially Dr. Meg Trahey, Scott Larson, and Marisa Stoller for their guidance and much appreciated assistance on this project. Special thanks to our collaborator, Dr. Min Lu of Cornell Medical School and to Dr. Jack Nunberg for his guidance and support as a mentor.

LITERATURE CITED:

1. Cao, J., L. Bergeron, E. Helseth, M. Thali, H. Repke, and J. Sodroski. 1993. Effects of Amino Acid Changes in the Extracellular Domain of the Human Immunodeficiency Virus Type 1 gp41 Envelope Glycoprotein. J. Virol. 67: 2747-2755.

2. Chan, D., D. Fass, J.M. Berger, and P.S. Kim. 1997. Core Structure of gp41 from the HIV Envelope Glycoprotein. Cell 89: 263-273.

3. Chan, D., and P.S. Kim. 1998. HIV Entry and Its Inhibition. Cell. 93: 681-684.

4. Chang, D., S. Cheng, and V.D. Trivedi. 1999. Biophysical Characterization of the Structure of the Amino-terminal Region of gp41 of HIV-1. J. Biol Chem. 274: 5299-5309.

5. Freed, E.O., and M.A. Martin. 1995. The Role of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins in Virus Infection. J. Biol Chem. 270: 23883-23886.

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EXECUTIVE SUMMARY:

The spread of HIV world-wide has grown to epidemic proportions. The need for an effective vaccine is of vital importance. There are many different approaches being studied in the hopes of attaining this goal. One such area focuses on the envelope of the virus as a potential target. The envelope contains a transmembrane protein, gp41 which is non-covalently bound to the gp120 protein. Glycoprotein 41 has been shown to play a pivotal role in the viral fusion to target cells. It is due to this fact that gp41 has been extensively studied, as it shows great potential for vaccine research.

The study of gp41 has yielded a great deal of results. Just before viral fusion, the structure of gp41 changes conformations. This conformational change enables the virus to fuse to the target cell. It is believed that if the intermediate of this structural change could be isolated, it may play a critical role in the search for a vaccine. The hope is that an intermediate structure which looks similar to natural, wild-type, virus, yet at the same time is non-function (incapable of binding cells) would, when inserted into a subject, elicit an antibody response. These antibodies would then be capable of neutralizing any natural HIV-1 virions they might come in contact with.

In this study, four separate amino acid substitutions were incorporated into the envelope protein gp41. The results showed that three of the four mutations were non-functional, while the fourth showed a decrease in activity. Two of the four mutations were further studied to analyze the surface proteins of cells expressing these mutant envelope proteins. In both cases the proteins present on the cell surface were similar to those present on the surface of natural, wild-type envelopes. This research shows that the mutations produced, that are non-functional, yet structurally similar to wild-type show great potential to be elements in the search for a vaccine.

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