BIOL368/F14:Isabel Gonzaga Week 3

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Annotated Bibliography for HIV Evolution Project

Questions

  1. Write three questions (or more) that you have about HIV that you would like answered.
    1. What is the current status of research on treatments on HIV today?
    2. With the high mutation rate of HIV sequences, has any success been found in targeting conserved sequence sections?
    3. What is the current understanding of the HIV replication pathway?
  2. Keyword Search
    1. What original keyword(s) did you use? How many results did you get?
      • Using Google Scholar: HIV+ENV: 103,000 Results
    2. Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
      • HIV+env+envelope: 6,220
      • HIV+ENV+Markham: 3,730
      • hiv+env+envelope+Markham: 2,990
      • The more search terms included, the more narrow the results
      • Including the author in the search terms greatly reduced the total number of results
  3. Advanced Search - For all three, which advanced search functions were most useful to narrow down the search? How many results did you get?
    1. Google Scholar
      • HIV Env OR Envelope: 390,000
      • allintitle: HIV Env OR Envelope: 42,600
      • allintitle: HIV Env OR Envelope author:Markham: 11
      • The more functions utilized greatly reduced the number of results. Searching by author and/or reducing the words to search titles only really narrowed the search.
      • The 'OR' function was important to use, so that more relevant articles are included in the initial search
    2. PubMed
      • (HIV[Title/Abstract]) AND ENV[Title/Abstract]: 5,251
      • (HIV-1[Title/Abstract]) AND (ENV[Title/Abstract] OR ENVELOPE[Title/Abstract]): 8,540
      • (HIV-1[Title/Abstract]) AND (ENV[Title/Abstract] OR ENVELOPE[Title/Abstract]) + 5 Years: 2,046
      • (HIV-1[Title/Abstract]) AND (Markham[Author]) AND (ENV[Title/Abstract] OR ENVELOPE[Title/Abstract]): 32
      • (HIV-1[Title/Abstract]) AND (Markham[Author]) AND (ENV[Title/Abstract] OR ENVELOPE[Title/Abstract]) + 5 Years: 1
      • Searching by Author greatly reduced the number of results. Using Title/Abstract and accounting for both 'env' and 'envelope' terms allowed for greater relevance in the results displayed, although it increased overall numbers. Additionally, applying a 'last 5 years' filter was also imperative to finding recent and relevant articles.
    3. Web of Science
      • TOPIC: (HIV-1) AND TOPIC: (ENV): 4,827
      • TOPIC: (HIV-1) AND TOPIC: (ENV) AND AUTHOR: (Markham): 26
      • TOPIC: (HIV-1) AND TOPIC: (ENV) Timespan: 2009-2014: 1,570
      • Searching by author and reducing the time span were, again, very useful in narrowing results. The feature 'Sort by Times Cited' was also very helpful, in terms of finding relevant articles.
      • The ability to go to an article and view articles which had cited the original makes it very easy to find more recent, related studies.
  4. Prospective Search on Markham et al. (1998)
    • Markham et al. (1998) cited 51 articles
    • Markham et al. (1998) has been cited 70 times

Bibliography

Primary Research Articles

  1. Hoot S., McGuire A.T., Cohen K.W., Strong R.K., Hangartner L., Klein, F. Diskin, R., Sceid, J.F., Sather, D.N., Burton, D.R., & Stamatatos, L. (2013). Recombinant HIV envelope proteins fail to engage germline versions of anti-CD4bs bNAbs. PLoS Pathog 9(1): e1003106. doi:10.1371/journal.ppat.1003106
  2. Rolland, M., Edlefsen, P.T., Larsen, B.B., Tovanabutra, S., Sanders-Buell, E, Hertz, T. deCamp A.C., Carrico, C., Menis, S., Magaret, C.A., Ahmed, H., Juraska, M., Chen, L., Konopa, P., Nariya, S., Stoddard, J.N., Wong, K., Zhao, H., Deng, W., Maust, B.S., Bose, M., Howell, S., Bates, A., Lazzaro, M., O'Sullivan, A., Lei, E., Bradfield, A., Ibitamuno, G., Assawadarachai, V., O'Connell, R.J., deSouza, M.S., Nitayaphan, S., Rerks-Ngarm, S., Robb, M.L., McLellan, J.S., Georgiev, I., Kwong, P.D., Carlson, J.M., Michael, N.L., Schief, W.R., Gilbert, P.B., Mullins, J.I., & Kim, J.H. (2012). Increased HIV-1 vaccine efficacy against viruses with genetic signatures in Env V2. Nature. 490(7420), 417-20. doi: 10.1038/nature11519.
  3. White, T.A., Bartesaghi, A., Borgnia, M.J., Meyerson, J.R., de la Cruz, M.J., Bess, J.W., Nandwani, R., Hoxie, K.A., Lifson, K.D., Milne, J.L.S., & Subramaniam, S. (2010). Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure. PLoS Pathog 6(12): e1001249. doi:10.1371/journal.ppat.1001249.

Review Article

  1. Affranchino, J.L., & Gonzalez, S.A. (2014). Understanding the process of envelope glycoprotein incorporation into virions in simian and feline immunodeficiency viruses. Viruses. 6(1), 264-83. doi: 10.3390/v6010264.

Preparation for Week 4 Journal Club

Vocabulary

  1. Chemostat: (n.) a device for the continuous culture of bacterial (and other) cells. Growth occurs in an aerated fermenter vessel and its rate is controlled by the rate of addition of fresh nutrient from a reservoir (i.e. the dilution rate); this in turn controls the rate of removal of cells (and culture medium). In a chemostat, as opposed to an auxostat, the dilution rate is constant. At equilibrium, the rate of production of new cells by multiplication is equal to the rate of removal of grown cells. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed
  2. Epitope: (n.) any immunological determinant group of an antigen.Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed
  3. Monophyletic: (adj.) a group of organisms stemming from a most recent common ancestor. Source: Oxford Dictionary of Biology, 6ed
  4. Nonprogressor
  5. Sanger Chain Termination: (n.) a rapid technique for determining nucleotide sequences in DNA in which the 2′,3′‐dideoxy analogues of the normal deoxynucleoside triphosphates are used as specific chain‐terminating inhibitors of DNA polymerase Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed
  6. Seroconverting
  7. Seropositive: (adj.): denoting a connection within or origin in serum. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed
  8. Synonymous (adj.) a nucleotide substitution that does not result in an amino acid substitution in the translation product owing to the redundancy of the genetic code. ie. a silent mutation. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed
  9. Tamura-Nei
  10. Visit

Article Outline

Introduction

  • HIV-1 has high mutation and replication rates and are highly adaptable to dynamic host environments
  • Immune systems cause variable effects on virus DNA because they are unstable.
    • If immune systems indiscriminately target HIV coreceptors, the most abundant one is likely to be leftover (high load, low variability)
    • If immune systems target the most abundant variant, the total load will be decreased (low load, high variability)
      • Diverse strains mutate and continue to replicate and may develop resistance to immune system
      • Necessary to examine diversity patterns in HIV-1 evolution to view forces of evolution causing adaptation
  • Limitations of previous work: small samples, did not directly examine sequence patterns, or limited timeframe
  • The present research:
    • HIV evolution studied for 4 year span for 15 subjects
    • Shows that the number of CD4 T Cells within certain time frames lead to different selection patterns in evolution
    • Shows that rapid CD4 T Cell decline is linked to greater diversity for HIV-1 viral strains
      • Contradictory to previous studies

Materials and Methods

Study Population

  • 15 Participants selected from AIDS Linked to Intravenous Experiences (ALIVE) study in Baltimore
    • Followed infected or at-risk IV drug users and studied blood
  • Participants selected were followed from point of HIV-1 seroconversion and identified by CD4 T Cell levels
    • Rapid Progressors: less than 200 CD4 T Cells within 2 years of seroconversion
    • Moderate Progressors: 200-650 CD4 T Cells within 4 years of seroconversion
    • Nonprogressors: 650+ CD4 T Cells through observation period

Sequencing HIV-env Genes

  • Peripheral Blood Mononuclear Cells were used to extract viral DNA from, as supported in the literature
    • Cells were recently infected
  • 185bp region of env gene from PBMC amplified through nested PCR
    • external env and nested primers were identified, containing BamGU and EcoRI restriction sites
  • Cloned into pUC19 using 'standard methods'
  • Sequenced using Sagner chain termination method
  • Plasma Viral Load determined by reverse transcription-PCR

Generation of Phylogenetic Trees

  • Constructed using MEGA computer software, using neighbor gaining algorithms and correcting for base composition and transition/transversion bias (Tamura Nei distance measure)
  • Taxon labels identify when he strain was isolated, and number of replicates
  • Taxa color-coded chronically (based on visit number)
    • Red: V1, Orange: V2; Green: V3; Light Blue: V4; Dark Blue: V5; Purple: V6; Brown: V7; Gray: V8; Black: V9
    • Sequences between visit dates were assigned the color of the MRCA

Correlation Analysis

  • Units of analysis defined as pairs within individuals (ie. comparing sequence data and CD4 T Cell count 1 year later)

Determination of dS/dN Ratios

  • Initial consensus sequence and observed sequences classified as synonymous or nonsynonymous
    • Corrections for nonsynonymous bias was made using Jukes-Cantor correction
  • dS/dN ratio found and averaged for all strains within a visit to remove bias in varying sample sizes
  • Median value used to determine average

Examination of Source of Greater Initial Visit Diversity in Subjects 9 and 15

  • High genetic variation between subjects 9 and 15 in their first visit - Needed to confirm if they were two different viruses are not
  • Clones from subjects 9&15 were compared against randomly selected clones from the study to determine relationship
  • Viruses shown to be monophyletic
  • History was reexamined to confirm HIV-1 seronegative status prior to first visit
  • Analyses on Subject 15 showed that, even without recombinations, the subject's diversity value was still higher than the rest at the 6month visit

Comparison of Rate of Change of Divergence and Diversity

  • Subject data was graphed for divergence/diversity over time with a line of best fit; slope used to characterize relationship
  • Slope averages between the three groups (defined by progression) were linked to the decline of CD4 T Cell counts

Results

  • CD4 decline patterns variable between all subjects
  • Nonprogressors had significantly lower viral loads than progressors (ANOVA; p<.02)
  • A total of 872 clones of the env region were sequenced and analyzed; changes defined by diversity and divergence
    • Diversity and divergence increased over time in all three progressor groups
    • Rates of diversity and divergence increase in order from nonprogressor, to moderate progressor to rapid progressor
      • Difference between progressor and nonprogressor was significant, close but insignificant for rapid progressor to moderate
  • Diversity and divergence negatively correlated with CD4 T Cell count 1 year later at a significant level
    • ie. subjects with increased diversity or divergence showed greater CD4 t cell decline in the next year
  • Ratio of dS/dN (synonnyous mutation per potential site of synonymous mutation to nonsynonyous mutation per potential site of non synonymous mutation) trended toward selection to synonynous selection.
    • Suggests that there is no advantage in changing envelope protein structure for nonprogressors
    • Progressors favored nonynonymous mutation
  • Phylogeneitc trees showed no predominance of a strain for extended period of time; viruses sequenced from each visit are interspersed within each others' branches

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Figure 4 shows the phylogenetic trees generated for four subjects, using the MEGA computer software. Each tree shows the genetic evolution of cloned sequences

Discussion

Comparison to Previous Literature

McDonald et al.
  • Compared env gene variation in rapid and slow progressors (2.5 years, 10 subjects, 2 time points)
  • Similar findings
**greater divergence in rapid progress ors
Wolinsky et al.
Nowak et al.

Figure 3

Figure 4

Weekly Assignments

Class Journals

Electronic Lab Notebook

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