# Angela A. Garibaldi Week 3

## Prior knowledge of the HIV virus:

• Currently I don't know very much about the biological aspects of the HIV virus. I only know that it makes the immune system very weak and is a RNA retro virus.
1. What is the overall molecular mechanism of the disease?
2. What type of virus is it specifically and what does this imply?
3. What are the mechanisms of some of the methods of treatment currently used?

## "Becoming an Expert in Pubmed"

Note: Version of Pubmed used is The following are the searching processes used to find review articles relevant to answering the above questions:

1. My first search query was: HIV [TI] evolution Limits:links to free full text, abstracts review, english.
• [TI] specifies that only papers with "HIV" in the title of any papers.

• I've highlighted the PubMed ID number of the article that I chose. This is what is used to complete the bibliography code and used to easily find the paper again.

• The following review seemed most relevant and clear as an introductory paper
1. Cohen MS, Hellmann N, Levy JA, DeCock K, and Lange J. The spread, treatment, and prevention of HIV-1: evolution of a global pandemic. J Clin Invest. 2008 Apr;118(4):1244-54. DOI:10.1172/JCI34706 | [Paper1]

### Further Articles for Investigation:

1. Williams KC and Burdo TH. HIV and SIV infection: the role of cellular restriction and immune responses in viral replication and pathogenesis. APMIS. 2009 May;117(5-6):400-12. DOI:10.1111/j.1600-0463.2009.02450.x | [Paper1]

# Markham Article

### 10 Biological Terms

1. serotype- The genotype of a unicellular organism as defined by antisera against antigenic determinants expressed on the surface. Therefore; the conversion of the serotype.Dictionary of Cell and Molecular Biology
2. non-synonymous mutation- a.k.a. a missense mutation -A form of point mutation resulting in a codon that codes for a different amino acid, and thus, causes the synthesis of a protein with an altered amino acid sequence during translation. biology-online.org dictionary
3. CD4 T-Cells- A form of T lymphocyte with CD4 receptor on the cell surface that recognizes antigens of a virus-infected cell. This type of T lymphocyte releases lymphokines upon stimulation of its recognition of antigens of a virus-infected cell. Upon its antigen recognition it is activated, stimulating, in turn, B lymphocytes and killer T lymphocytes during the antibody formation.It is the target cell of AIDS virus, infecting and eventually killing the lymphocyte by the virus. biology-online.org dictionary
4. CD4- A 55-kD glycoprotein that serves as differentiation antigen found on the surface of T lymphocytes and macrophages. The CD in CD4 is an abbreviation for cluster of differentiation. It belongs to the immunoglobulin supergene family. The presence of CD4 characterizes the helper/inducer cell. It also serves as HIV receptors where the virus binds directly with its envelope protein gp120. biology-online.org dictionary
5. Nested PCR -Variety of polymerase chain reaction, in which specificity is improved by using two sets of primers sequentially. An initial PCR is performed with the ‘outer’ primer pairs, then a small aliquot is used as a template for a second round of PCR with the ‘inner’ primer pair. Dictionary of Cell and Molecular Biology
6. PBMC (peripheral blood mononuclear cells) - A mixture of monocytes and lymphocytes; blood leucocytes from which granulocytes have been separated and removed.Dictionary of Cell and Molecular Biology
7. epitope- That part of an antigenic molecule to which the t-cell receptor responds, a site on a large molecule against which an antibody will be produced and to which it will bind. biology-online.org dictionary
8. seroconversion- the development of detectable specific antibodies to microorganisms in the blood serum as a result of infection or immunization. Serology (the testing for antibodies) is used to determine antibody positivity. Prior to seroconversion, the blood test is seronegative for the antibody; after seroconversion, the blood test is seropositive for the antibody. The word is often used in reference to blood testing for anti-HIV antibodies. In particular, "seroconverted" has been used to mean "became HIV positive." Wikipedia
9. Frequency dependent selection - the term given to an evolutionary process where the fitness of a phenotype is dependent on its frequency relative to other phenotypes in a given population. In positive frequency dependent selection, the fitness of a phenotype increases as it becomes more common. In negative frequency dependent selection, the fitness of a phenotype increases as it becomes less common. Negative frequency dependent selection is a particular mechanism of balancing selection. Wikipedia
10. monophyletic -a group of organisms (usually species) that are more closely related to each other than any other group, implying a shared most recent common ancestor.biology-online.org dictionary

### Outline of the Article:

#### INTRODUCTION

1. stable host environment -breeds best adapted viruses to dominate,all mutations after are not represented in the gene pool as much.
2. Different viruses are genetically similar with few variants because mutations will be more neutral
3. Unstable host environment - caused by the host's immune response or specifically in HIV differential display of co-receptors.
4. If destabilizing force selected strongly against the variants,gene pool of the viruses that were most dominant initially will survive best.
5. (Frequency dependent selection)If destabilizing force selected strongly against the virus variant that is most abundant,viral load decreases overall. Genetic diversity maintains due to gene pool of viral variants in fewer numbers due to their different variants.
6. Remaining viral variants will propagate and lead to variants that can surpass the selective power (such as the specific immune system response )
7. This experiment analyzes the sequence patterns of the viral variants using a wider scope of time points than previous studies.
• ( Analysis has shown divergent patterns between nonprogressor patients and rapid progression patients. Higher genetic diversity patterns in viral variants point to faster CD4 T Cell decline.)

#### METHODS

1. 15 patients checked in 6 month intervals: Rapid progressors (<200 CD4T-cell count) Moderate (200-650) NonProgressors (>650)
2. Sequence HIV-1 env genes by amplifying a region of the env gene with Nested PCR from the PBMCs, cloned into pUC19, sequenced. Used only samples that could be detected at lowest dilution.
3. Significant because viral DNA is mostly acquired from these unactivated host (due to recent infection) PBMCs and can only last a few days in unactivated Tcells. (In other words they won't have time to change significantly) Therefore, the DNA in these cells should be very similar to the RNA of the viruses circulating in the plasma at the time.
4. Used the single round PCR samples that could be detected at the lowest dilution in the second round PCRs in order to specify clones that had unique viral genome template.
5. used reverse transcription-PCR to measure plasma viral loads
6. Correlation analysiswas used, comparing individuals with a different status (non, moderate, rapid) at similar time points over the course of a year in order to determine the correlation between genetic diversity or mutational divergence via CD4 T-cell count and sequencing results.
7. Phylogenetic trees have taxa color coded based on time point. red=V1,orange=V2, green=V3, light blue=V4, dark blue= V5, purple=V6, brown=V7, gray=V8
8. dS/dN Ratios- determined a baseline sequence for each individual and then compared it with observed strains over time. Differences between the base and the subsequent strain was labeled either synonymous (dS) or nonsynonymous(dN).
9. dS,dN and dS/dN ratios were averaged over all strains across the board for each visit.
10. Subjects 9 and 15 had a notably high genetic variation in the first visit. Three phylogenetic trees were made to compare them to clones from other individuals. -Result: Monophyletic
11. Comparison of the Rate of Change of Divergence and Diversity - Each individual has their own line measuring divergence over (/) diversity over time. Individuals' slopes from each group (non, mod, rapid) were averaged and then compared using random effects models.

#### RESULTS

##### Table 1
1. CD4 decline among the individuals resulted in variable patterns.
2. Nonprogressors started with low viral loads at the earlier time points vs the progressors (mod,rap) but this data did not lead to a showing of statistically significant difference between the moderate and rapid progressors viral load numbers in the earlier time points.
3. Sequencing used viral env region because it is known to tolerate frequent mutations.
4. Subjects 9 and 15 may have had dual infection, recombinant viruses, or mistake in timing of the point of seroconversion used to generate the consensus point (implicated by heterogenous diversity and divergence lines at the beginning compared to the homogenous lines of all other individuals.
5. Median diversity over time = -2.94 to 5.10 nt per clone per year
6. Rate of change in divergence= 0.13% to 2.09% of the nts per clone per year
7. CD4 T-cells have significant negative correlation with both Diversity and Divergence
8. Mutation without selection (randomly); dS/dN= approx. 1
• Rapid and moderate progressors had selective advantage for NS changes (dS/dN=0.4)
• non progressors=no selection for viruses with changes,trend toward selection for NS (dS/dN=1.5)
• Median ratios sig. different between non and moderate or non and rapid due to differences in dN.
##### Figure 1
• Genetic Diversity and Divergence measured over time with CD4 T-Cell counts shows that Tcells decrease over time as Diversity and Divergence increase over time. Shows started at homogenous point of divergence/diversity for each individual (except 9,15)
##### Figure 2
1. Diversity and Divergence increased over time for all progressor groups.
2. Severity in increase of Divergence correlated with group ie. non progressors less severe, moderates more severe, rapids most severe.
3. These rates were statistically significant between non and rapid but not statistically significant between moderate and rapid progressors.
4. Differences in rates of Diversity were significant between non and moderate, non and rapid, however NOT significant between moderate and rapid progressors.
##### Figure 3
1. No predominance of a single virus strain over time period.
2. Subject 9 shows that mutated viruses may not branch from its direct ancestory; for example viruses at V8 emerge from V6 clones. This skips V7.
##### Figure 4
1. Shows the same pattern of virus mutations in 4 other randomly selected individuals. Viruses at later visits regress to become more similar to viruses observed in much earlier visits.
2. Also shows that the individual's immune system selects against the few clones that are most dominant in number at any given visit, but not strong enough to eradicate all variants of the virus present.

#### DISCUSSION

1. increase of diversity/divergence => decrease in CD4 T-Cell levels
2. with individuals that started out with similar CD4Tcell counts, genetic diversity affected CD4 TCell counts more over 12 month period.
non progressor viral strains may select AGAINST amino acid change
progressors viral strains showed slection FOR amino acid change

• This serves as evidence against hypothesis that progression is caused by the success of the viral strain that is most fit in the beginning.
##### Previous Studies
1. McDonald et al (conflicting)- divergence at 2 time points increase; diversity decreased in rapids compared to slow progressors
• diversity conclusions depended on:
• region of env
• fewer time points
• subjects not followed from point of seroconversion

2. Wolinsky et al (conflicting) - less diversity in rapid decreasing CD4 T-Cell vs slow decrease CD4 Tcell counts

This was found in Subject 11 in this study, which started with much higher viral load.
Individuals 11 and those in Wolinsky's may be exceptional with ineffective immune responses.


3. Nowak et al (results agree with conflicting reasoning)- increase in diversity as decrease in CD4Tcells. Progressive increase in Diversity and Divergence in those who progress to AIDS. Model: Viral clones develop epitopes(binding sites) that Tcells cannot bind to, causing immune response to fail to control virus.

• Immune system targets more frequent virus, leaving less frequent; NOT due to failure due to epitopes
• Viruses that replicate less are conserved because in lower numbers aren't targeted by immune system
• Selection against nonsynonymous in nonprogressors because those changes might produce numbers high enough to be recognized by the immune system and eradicated.