Question

HIV virions wrap themselves with a lipid bilayer membrane as they bud off from infected cells; in this viral membrane envelope are "spikes" composed of two different proteins (actually glycoproteins): \(8 p 41\) and \(8 p 120\) The \(g p\) denotes glycoprotein, and the number thates their molecular weight in kilodaltons. \(\mathrm{BP} 120\) and \(\mathrm{gp} 41\) together form the trimeric envelope spike on the surface of HIV that functions in viral entry into a host cell. The primary receptor for \(g p 120\) is \(C D 4,\) a protein found mainly on the white blood cells known as T lympho. cytes. \(8 p 120\) avoids detection by the host immune system through a number of strategies, including rapid changes in sequence due to mutations. In this exercise you will grab a sequence for \(8 p 120\) and "blast" it to find related proteins. Use the "Search database" on the LANL HIV Sequence Database site (http://www.hiv.lanl.gov/) to find a sequence for gp120: the complete gp120 molecule has about 500 amino acids so a complete DNA sequence will have roughly 1500 base pairs. With the BLAST website (http.//www.ncbl.nim.nih.gov/ blast) open a new window and select "blastx" under the "Basic BLAST heading. Copy and paste your approximately 1500 nucleotide sequence of \(\mathrm{gp} 120\) into the top box under "Enter Query Sequence," For the database select "Protein Data Bank proteins (pdb)." What we are doing is having BLAST translate the \(8 p 120\) nucleotide sequence into an amino acid sequence and then compare it with amino acid sequences of proteins in the PDB. Note that there are some proteins which appear multiple times In the PDB because their structures have been analyzed and determined more than once or in different contexts. Finally, push the "BLAST" button and wait for your results to appear on a new page. (a) How does BLAST determine the ranking of the results from your search? (b) For your top search result, identify the percentage of sequence identity with your query sequence. Explain how this number is determined. (c) You will notice that BLAST has used gaps in many of your alignments. What is the evolutionary significance of these gaps? (d) For your top hit, how many alignments with this high a score or better would have been expected by chance? (e) Looking at your ranked list of results and using only the "E value," which hits do you expect to (possibly) have a genuine evolutionary relationship with your gp 120 sequence and why?

Short answer

BLAST ranks results based on scores which represent the measure of alignment between sequences. The percentage of sequence identity relates to the number of exact matches in sequence. Gaps in alignments suggest evolutionary changes. The number of alignments with a high score expected by chance can be determined from the BLAST results. Using the 'E value', sequences with a small E value could possibly have a genuine evolutionary relationship with gp120.

Step-by-step solution

Perform gp120 Sequence Search

Visit the LANL HIV Sequence Database and use the 'Search database' option to find a sequence for gp120. Note this sequence, which will be approximately 1500 nucleotides in length and will serve as the 'query sequence'.

Sequence Comparison with BLAST

Visit the BLAST website, open a window and select 'blastx' under the 'Basic BLAST' heading. Paste the gp120 sequence into the 'Enter Query Sequence' box and select 'Protein Data Bank proteins (pdb)' from the database choices. Activate the search and wait for the results.

Interpret BLAST Results

BLAST ranks results based on a scoring system which weighs certain factors, such as region length and number of matches, mismatches and gaps. Higher ranking represents a closer match to the query sequence. The percentage of sequence identity is determined by calculating the number of exact matches divided by the total length of the sequence. Gaps in alignments suggest evolutionary differences and changes. 'E value' or Expectation Value predicts the number of hits that can be expected to occur by chance. The smaller the E value, the more significant the match. The top hits with the smallest E values have likely shared a common ancestry with gp120.

Examine Top Result

Examine the top search result from BLAST and note the percentage of sequence identity with the query sequence. Also, note the number of alignments with a high score or better that could occur by chance.

Evaluate Potential Evolutionary Relationships

Based on the 'E value', identify the sequences that may have a genuine evolutionary relationship with gp120. An 'E-value' close to 0 indicates high sequence similarity, suggesting shared evolutionary history.

Key concepts

Understanding gp120

The protein gp120 plays a crucial role in the lifecycle of the Human Immunodeficiency Virus (HIV). It is a glycoprotein located on the outer surface of the virus, known for its role in HIV's ability to infect host cells. It does so by binding to the CD4 receptor on the surface of T lymphocytes, which are white blood cells that play a key role in the immune response.

The gp120 protein is characterized by its high level of variability, which allows the virus to evade the host's immune system. This variability is the result of rapid changes in its amino acid sequence caused by frequent mutations. This adaptability contributes significantly to the challenges faced in developing effective vaccines against HIV.

Students exploring the structure and function of gp120 learn about its role as a target for potential HIV therapies and vaccines, as well as its importance in understanding the mechanisms behind viral entry into host cells. This knowledge underpins much of modern virology and immunology, making gp120 a keystone topic in the study of viral protein evolution.

Viral Protein Evolution

The concept of viral protein evolution is at the core of understanding how viruses like HIV adapt and persist in the face of host immune defenses. Proteins like gp120 exhibit high mutation rates, leading to a variety of strains that can challenge immune recognition and treatment efforts.

Evolutionary pressures, including immune system attacks and antiretroviral therapies, select for viruses that have mutations conferring a survival advantage. These changes may involve the introduction of gaps, as seen in BLAST sequence alignments. Such gaps represent insertions or deletions in the protein sequence, which are significant indicators of evolutionary divergence.

It's essential for students to appreciate how these mutations impact protein function and viral fitness, as this understanding informs the ongoing battle between evolving viruses and medical science. The dynamics of viral protein evolution continue to be a hotbed of research, influencing the strategies used to combat viral diseases, including the development of vaccines and antiviral drugs.

Applying Bioinformatics Techniques

Bioinformatics techniques are powerful tools in modern biology, allowing researchers and students alike to analyze the genetic makeup of organisms at an unprecedented scale. BLAST (Basic Local Alignment Search Tool) is one such technique that is used to compare nucleotide or protein sequences to databases of known sequences.

When using BLAST for sequence analysis, the search results are ranked by a scoring system that takes into account the length of the matching region and the number of similarities, differences, and gaps between sequences. The Expectation Value or 'E value' is particularly important as it indicates the number of hits one can expect to see by chance, with lower values signifying more statistically significant matches.

The use of these bioinformatics techniques in the study of gp120 allows students to identify potential evolutionary relationships between the virus and other organisms, observe the evolutionary impact of sequence variation, and gain insights into the molecular mechanics of HIV infection. Understanding how to interpret BLAST results is essential for anyone aiming to engage with the field of genomics or any area of biological research that relies on sequence comparison. By mastering these tools, students are empowered to perform and understand cutting-edge research that can lead to groundbreaking findings in virology and beyond.