Courtney L. Merriam Week 3

Preparation for Week 4 Journal Club

Vocabulary

1. Make a list of at least 10 biological terms
   1. Coreceptors- any chemokine receptor that when expressed with a CD4 receptor on a cell’s surface allow for HIV entry into the cell interior. The major coreceptors that do this are CXCR4 and CCR5. ²
   2. PBMC- stands for peripheral blood mononuclear cell. A PBMC is a peripheral blood cell that has a round nucleus. Examples of PBMCs are T cells, B cells, and NK cells (lymphocytes), and simple white blood cells (monocytes). PBMC are distinct from red blood cells (erythrocytes) and platelets, which have no nucleus. ³
   3. Sanger Method- a technique for DNA sequencing developed by Frederick Sanger in 1977 that incorporates chain-elongating inhibitors known as dideoxynucleotides, which help in the identification of individual nucleotides ⁴
   4. PCR- Stands for polymerase chain reaction. A method used to create thousands of copies of a particular DNA strand from a single initial sequence. ⁵
   5. Substitution model- a description of the way that a length of nucleotides changes into another ⁶
   6. Tamura-Nei Distance measure- a special model used to correct genetic data to account for base composition and transition/transversion bias. There are twice as many possibilities for transversions, but transitions occur more often. This method helps account for this disparity. ⁷
   7. Jukes-Cantor correction- another substitution model for DNA, considered one of the simplest in that it assumes both equal base frequencies and equal mutation rates, leaving only overall substitution rate as the variable. ⁸
   8. Monophyletic- refers to a group of organisms that have descended from a common ancestor, particularly a group that is distinct from any other. Think of a single branch on a phylogenetic tree. ⁹
   9. (long term) Nonprogressors- In reference to HIV research, a nonprogressor is an individual who has tested positive for the HIV virus but has been able to maintain a relatively high CD4 number (an excess of 500) without the aid of extraneous antiretroviral therapy over a length of several months or years ¹⁰
   10. Seroconversion- seroconversion refers to the length of time between inception of the HIV virus within an individual and when HIV antibodies become prevalent enough to be detected. Often times takes only a few weeks, and may also involve fevers and rashes.¹
   11. Nonsynonymous mutations- When messenger RNA is making copies of DNA, either an insertion or a deletion occurs for a nucleotide. This single addition or deletion causes a shift on the entire side of the DNA, which shifts the nucleotide pairs and causes drastic differences in the codons. Obviously has a more substantial impact on the organism than a synonymous mutation. ¹¹

Outline

Main Result

In observed individuals with progressing amounts of the HIV-1 virus, CD4 T cells declined in relation to how many mutations an individual strain of the HIV virus acquired after splitting from a previous strain, as well as the specific kinds of mutations that occurred that enabled increased fitness within the environment. Essentially, increased genetic diversity of the HIV-1 virus is linked with quicker decline of CD4 protein producing T cells.

Introduction

HIV-1 mutate and replicate rapidly, meaning they can adapt quickly to a changing host environment.

• In an environment that changes very little, the strain of HIV-1 that is most adapted already would begin to dominate over less adapted strains, who wouldn’t be as well represented in the total gene pool
• In an environment that changes a lot, it is difficult to determine what might happen to the various strains of the virus.
  • Environment around HIV-1 virus disrupted by immune response’s selective forces of a variety of coreceptors (component that when in conjunction with CD4 protein allow HIV-1 virus into cell interior).
  • If the selective force of the coreceptor’s presence respond randomly, the current dominant strain experiences reduction in numbers; minority strains see a less severe reduction in their numbers. Known as frequency dependent selection, as was so far untested until this experiment was conducted.
    • As these minority strains multiply in the absence of the previous majority strain, the diversity of the pool expands
    • This diversity leads to the immune system’s inability to respond to the diversity of remaining threats, as was proposed by Nowak in 1991.

Methods

15 people picked from a group of individuals who participated in injection drug use in a study in Maryland

• The participants were sorted into three groups
  • Rapid Progressors- individuals measured 200 or less CD4 T cells within 2 years of seroconversion
  • Moderate Progressors- individuals having between 200 and 650 CD4 T cells during 4 years of observation
  • Nonprogressors- individuals who kept an excess of 650 CD4 T cells during the observed period
• A 285 base pair region of the env gene (enables retroviruses to attach to host cells) multiplied using Polymerase chain reaction
  • Continued cycles of PCR were run at varying temperatures for varying times to determine viral multiplication and mutation
  • The viral clones produced by PCR were found to originate from an initial viral genome template
  • Plasma Viral Load determined by reverse transcription PCR
  • Phylogenetic tree of results show viral clones from the different participants diverged independently between one participant and another, subject 1 and 2 were known to be genetically related individuals
    • Each branch was colored differently depending on the time the data was collected
    • Correlation Analysis
    • Two variables analyzed to understand the relation of time point and genetic diversity
      • X0 (divergence of mutation)
      • Y1 (count of CD4 T cells)
• dS/dN Ratios
  • Differences in strains were classified synonymous or non synonymous
  • Values of dS/dN averaged to account for unequal sample sizes from different sampling visits
• Examination of Source of Greater Initial Diversity in S9 and S15
  • Significant genetic variation observed in S9 and S15
    • May be infected with two different viruses
    • Found that their respective viruses were monophyletic
• Comparison of Rate of Change in Divergence and Diversity
  • Each participant received a regression line comparing divergence and diversity over time
  • Slopes of these regression lines were compared to random effects models

Results

• Variable CD4 T cell decline across all participants
• Large difference in viral load between moderate/rapid progress ors and nonprogressors
  • little difference between moderate and rapid
• Median diversity observed as changing from -2.94 to 5.10 nucleotides per clone per year
  • Both diversity and divergence were observed to increase with time
• Rapid and moderate progress ors seemed to experience roughly the same divergence and diversity rates
  • Nonprogressors had noticeably lower rates
• Increased diversity/divergence correlate with reduced CD4 T cell counts
• dS/dN ratio for rapid and moderate progress ors indicate nonsynonymous mutation
  • Nonprogressors seemed to select against nonsynonymous mutations
• Studies of phylogenetic tree data showed larger presence of a single strain
  • Subjects 10 and 15 were outliers

Discussion

• Increased genetic diversity and divergence correlated to reduced CD4 T cell count
• Moderate and rapid progress ors appeared to be unable to select against amino acid changes
  • Nonprogressors were able to
• McDonald et all study found differing data
  • Similar data- correlation between divergence and drop in CD4 T cell number
  • Differing data- slow progress ors had higher diversity than rapid progress ors
    • May be linked to incomplete data, lack of enough participants
• Wolinsky et all study found differing data
  • Less diversity was recorded in both rapid and slow progress ors
  • Individuals whose data most substantially supported this may have been exceptions
• This study supports Nowak Study
  • Host immune response cannot handle the variety of viral clones’ respective mutated resistances to the T cell’s capabilities
  • Increased genetic diversity and divergence of virus leads to decreased CD4 T cell count
  • Decrease in viral genetic diversity once T cells are overwhelmed

Figures and Tables

• Fig. 1
  • A graphical examination of CD4 T cell trajectory compared with diversity and divergence rates. Rapid progressors had a characteristic drop in CD4 T cells as time passed.
• Table 1
  • A layout of the statistical data for each individual test subject. At the time the study began, all individuals had an adequate CD4T cell count. Their rate of decline from further observation classified them into rapid, moderate, or nonprogressor.
• Fig. 2
  • Graph of slope of divergence and slope of diversity in all three progresso categories. Rapid progressors showed significantly higher rates of diversity than moderate and nonprogressors, and divergence that was far higher than nonprogressors but only slightly higher than moderate progressors
• Fig. 3
  • A phylogenetic tree of subject 9’s HIV-1 mutation progression. The data of the single mutation reflects a denial of the idea that S9 may have been infected with two different viral strains.
• Fig. 4
  • A phylogenetic tree of 4 random subjects from the initial 15 participants. Data shows that the participants had single mutations in between samplings, and the branching patterns were diverse between subjects.

Figure 2A: "Comparison among different progresso groups of the mean slope per year (