•X-ray crystallographic studies of Fab fragments of various neutralizing antibodies in complex with V3 loop peptides have shown that the V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence (GPGR) at the tip of the loop (Figure 4). (2)

•The high degree of conservation of the GPGR motif, surrounded by regions of high sequence diversity suggests this structural conservation is related to biological function. (2)

•For Fab-V3 X-ray crystal structures it has been determined that a 12-residue section of the V3 loop (Table 1) including the GPGR tip, plays a critical role in chemokine receptor binding and macrophage recognition properties. (2)

•This peptide sequence, depending on the amino acid composition of the amino acids flanking the GPGR tip, can be represented by two possible conformations of the V3 loop. (2)

Evolution of the Conserved Region of the gp120 Loop and Its Affect on HIV Disease ProgressionDrew Foy, Rebecca Roberts. Departments of Biochemistry and Molecular Biology, Ursinus College; 601 E. Main St.; Collegeville, PA 19426-1000. 

References1. Kwong et al. (1998). Structure of an HIV gp120 envelope

glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393; 648-659.

2. Stanfield et al. (1999). Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs. Structure 7; 131-142.

3. Markham et al. (1998). Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. PNAS 95; 12568-12573.

INTRODUCTIONINTRODUCTION

General Background

HIV causes the destruction of CD4(+) lymphocytes in their respective hosts, resulting in the development of acquired immunodeficiency syndrome (AIDS). (1)

HIV entry into cells is mediated by the viral envelope glycoproteins, which are organized into trimeric spikes displayed on the surface of the virion. (1)

These envelope complexes are anchored in the viral membrane by the gp41 transmembrane envelope glycoprotein. The surface of the spike is composed primarily of the exterior envelope glycoprotein gp120. (1)

The conserved gp120 regions form discontinuous structures important for the interaction with the gp41 ectodomain and with the viral receptors on the target cell. (1)

Entry of HIV into the host cell involves binding of the gp120 envelope glycoprotein to the CD4 glycoprotein, which serves as the primary receptor. (1)

CD4 binding induces conformational changes in the gp120 glycoprotein that in turn allows binding of the virus to a specific subset of chemokine receptors on cell surface.

Once bound to the chemokine receptor the trimeric gp120 protein springs open, projecting three peptide fusion domains that “harpoon” the lipid bilayer of the target cell.

The fusion domains then form hairpin-like structures that draw the virus and cell membranes together to promote fusion, leading to the release of the viral core into the cell interior.

The V3 Loop

•The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity (Figure 3). (2)

•The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T-cells and macrophages. (2)

•Sequence changes in V3 can alter viral cell tropism, antibody neutralization, neutralization of soluble CD4, synctium formation and chemokine receptor usage. (2)

Intervisit variation of V3 peptide sequencesIntervisit variation of V3 peptide sequencesbetween rapid, moderate, and nonprogressorsbetween rapid, moderate, and nonprogressors

Goal of Present Study

•In this report, the resultant peptides corresponding to the env V3 sequences obtained from 4 patients in the Markham et al. study have been analyzed with the hypothesis that rapid and moderately progressing patients will initially contain or develop differences versus nonprogressors in the regions containing the critical 12-residue amino acid section crucial for chemokine receptor binding and macrophage recognition.

•It is also hypothesized that rapid progressors will contain a higher degree of intervisit diversity in their amino acid sequences corresponding to the critical 12-residue section of the V3 loop (Table 1) because its importance has been implicated in chemokine receptor binding and macrophage recognition properties.

Data collection procedure

•Nucleotide sequences from 4 patients in the Markham et al. study were randomly selected to obtain 2 clones from each visit.

•All nucleotide sequences were translated into peptide sequences using the EMBOSS Translate Sequence program (www.emboss.com).

•Each translated peptide sequence for a clone corresponding to a particular visit was separately compared to both clones corresponding to the subsequent visit using the EMBOSS Align Sequence program (www.emboss.com). This program aligns the amino acid sequences of the two peptides and calculates the degree of similarity between them.

•This procedure was performed in procession until each of the clones from one visit was compared to each of the clones from the subsequent visit

•This data collection procedure made it possible to compare the intervisit amino acid sequence diversity of the 12-residue section of the V3 loop (Table 1).

Figure 3: Structure of HIV-1 gp120 bound to CD4 T-cell receptor and 17b antibody. The V3 loop is sandwiched between the CD4 T-cell receptor and the top of the 17b antibody

Evolution of gp120

• Due to the importance of the V3 loop in virus infectivity its evolution has been the subject of intense study and it has been suggested that mutations in this region correlate with the degree of disease development in its hosts. (3)

• Markham et al. has found that different patterns of selection in the env V3 sequence are observed between nonprogressor and moderately or rapidly progressing subjects and that attainment of higher levels of genetic diversity was most frequently associated with more rapid CD4 T-cell decline (Figure 5). (3)

• However, despite the wealth of nucleotide sequence data in patients infected with HIV obtained from Markham, the corresponding peptide sequences have not been subject to critical analysis.

Mechanism of HIV entry into host T-cells

.

Representative Data of Intervisit Comparisons Between Clones from Rapid, Moderate, and

Nonprogressors

Table 2: This figure represents changes between visits for 4 subjects in the Markham et al. study. Blue highlighted residues represent residues that have been changed from the previous visit.

Data Analysis and Discussion

•As expected, the intervisit diversity between the amino acid sequences of the critical 12-residue sections varied significantly for rapid and moderate progressors versus nonprogressors.

•Subjects 10 and 7 represent fast progressors by measurement of their CD4 declines of -363 and -392/yr respectively. Each of these subjects initially contained 3 variations in the critical 12-residue section. However, the initial variations differed significantly. Subject 7 contained the variations R1S, H/S3P, and G12A while subject 10 contained the variations H/S3N, I4M, and G12T.

•The initial variations corresponding to H/S3P and H/S3N in subjects 7 and 10 respectively, could play a large role in altering the conformation of the V3 loop and hence, disease progression because both of these variations occur in the third amino acid residue in the conserved region and continue to show variations over the visitation period. Therefore, variation at the third site in the conserved region could play a large role in affecting the loop and hence disease progression. However, data from other subjects seems to nullify this observation.

•Subject 5 represents a moderate progressor with a CD4 decline of -41/yr. Like the rapid progressors, subject 5 contains 3 initial variations, which are R1S, H/S3L, and G12T. Similar to subject 7, subject 5 showed initial variation from the conserved sequence at residues 1, 3, and 12. However, subject 5’s CD4 decline was much less dramatic. Therefore, it is hard to characterize these sites as sites that significantly affect disease progression.

•It is also interesting to note that subjects 7 and 5 showed intervisit variation in amino acids that did not originally vary from the conserved sequence. However, with the current data it is not possible to draw a connection between these variations and their effect on disease progression.

•Subject 13 represents a nonprogressor with a CD4 increase of +53/yr. Subject 13’s initial variations are R1S, H/S3N, I4M, and G12A. Like the other subjects, subject 13 contains variations at sites 1, 3, and 12 in addition to variation at site 4. Unlike subjects 7 and 5, subject 13 does not show significant intervisit diversity.

•Although it is not possible to predict the conformation of the V3 loop in the subjects, there are differences between rapid and moderate progressors versus nonprogressors in intervisit diversity. Table 2 shows that in fast and moderate progressors, significant changes occur per visit in the amino acid sequences of the critical 12-residue section. However, it is not possible to characterize these changes into a pattern of how they contribute to the fast, moderate, or non-progression of disease. Therefore, further data analysis should be done in an attempt to characterize these changes and their effect, if there is one, on disease progression.

•Even though this study only focused on the critical 12-residue section of the V3 loop implicated in chemokine receptor binding and macrophage recognition, it is a possibility that changes distal to this 12-residue section had a major affect on disease progression (i.e.) it can be seen from the similarity data compiled in Table 2 that rapid progressor S7 had significantly lower similarities in subsequent amino acid sequences to S13 even though its critical 12 residue section stayed relatively constant.

Table 1: Section of the V3 loop critical in determining its conformation, which in general contains a β turn followed by a double bend

Figure 5: Comparison among different progressor groups of the mean slope per year of intravisit viral genetic diversity and the percent of nucleotides that diverged from the original postseroconversion consensus sequence (Markham et al., PNAS 1998).

Figure 2: The lesions of Kaposi's sarcoma are sometimes the first physical sign that a person with HIV infection has developed AIDS.

Figure 4: Dual conformations of the V3 loop (Stanfield et al. Structure 1999).

•Once an individual is infected with HIV he or she may live for up to ten years or more without any noticeable medical problems.

•The lesions of Kaposi's sarcoma are sometimes the first physical sign that a person with HIV infection has developed AIDS. There is no test to identify people who have KS before lesions develop (Figure 2).