# Presentation: 030738

## HIV Database Workshop www.hiv.lanl.govseq-info@lanl.gov

_**Presenters**_**: Brian Gaschen, Bette Korber, Thomas Leitner, Brian Foley, Karina Yusim**
_**Database staff**_**: Werner Abfalterer, Chuck Calef, Robert Funkhouser, **
**Kristina Kommander, Dorothy Lang, John Mokili, Una Smith, Jim Szinger, Ming Zhang, ****Luciano Vieira de Araujo**
_**Regular Contributors**_**: John Mellors. Robert Shafer, and Christian Brander**
_**Project Officer**_**: James Bradac, NIAID, NIH**
**Carla Kuiken: www.hcv.lanl.gov**
*Theoretical Biology and Biophysics, T-10 *
*Los Alamos National Laboratory*

## Workshop Goals

- Understanding the database content, how information was obtained, and what is available
- Database searching
- Quality control tools
- Tools for analyses

## Workshop Topics

**Workshop Topics**

**Introduction **- 10 min
**Sequence Database**
- 	Brian Gaschen - 30 min
- 		*Basic sequence search interface and on-the-fly alignments*
*	**	**Geography search interface*
*	**	**GeneCutter - processing nucleotide sequences*
*	**	**N-glycosite - infer N-linked glycosylation (if time permits)*
- 	Thomas Leitner - 30 min
- 		*HIV database alignments and subtype reference sequences*
*	**	**Comparing “new” sequences with our reference sequences*
*	**	**Build a simple neighbor joining tree through the web*
*	**	**Using the new RIP tool for recombination analysis*
- 	Brian Foley - 10 min
- 		*3D views of HIV macromolecular structures*
**Break** - 15 min
**Immunology Database**
- 	Bette Korber - 30 min
- 		*HIV/SIV sequence locator tool*
*	**	**CTL search page*
*	**	**Ab search page*
*	**	**Epitope maps*
- 	Karina Yusim - 30 min
- 		*Peptgen - list peptides for reagent development*
*	**	**Motifscan - find HLA anchor residues in a protein sequence*
*	**	**ELF - epitope location finder*
**Vaccine Trial Database**
- 	Brian Foley - 25 min
- 		*SHIV maps*
*	**	**SIV alignments*
*	**	**Vaccine database searches*

## The HIV databases

- HIV Sequence database – founded 1986, G. Myers
- Relational database, data from GenBank with added fields from the literature
- Alignments – align indels and reduce multiple sequences per person
- Annual hard copy and reviews
- Web search interfaces: subtype, phenotype, geographic, sampling year...
- Analysis tools
- HIV Immunology database – founded 1995, B. Korber
- Comprehensive HIV epitope database , 300-400 papers a year
- Integrate HIV immunological and sequence data
- Annual hard copy and reviews
- Web search interfaces: epitope, protein, HLA type, immunogen, keywords
- Analysis tools for immunologists
- HIV Drug Resistance database, founded 1997, J. Mellors
- A searchable web listing of drug resistance mutations and literature links, updated annually by Dr. Mellors
- HIV Vaccine database, founded 2003, J. Mokili
- A searchable relational database of published primate vaccine trials

- Bette Korber

**Help**
- Tips at the top of the page are often overlooked
- Ranges, operators, wildcards, logical groupings
- Example: “Sampling country” gives two-letter ISO country codes
**Searches**
- Searches are case-insensitive
- Records are searchable through sequence, patient, genomic region, or publication information
- First seven fields will appear in search results page by default
- A “*” in a textbox will cause that field to be included in the results page
**Results**
- Can select not aligned, or aligned based on multiple pair wise alignments – alignments are good, but still need hand editing for an optimal alignment
- Select all or a subset of sequences for download

- Brian Gaschen

**Search Interface**

## Geography Tool 

- Another way to search/download sequences is by geographic region or country
- Results are biased as they show only the sampled individuals, not the true subtype distribution for a region’s population
- Results are selectable as in the search interface

- Brian Gaschen

## Gene Cutter 

- Useful for sequencing labs, particularly for rapid processing of new sets of full length genomes
- Cut out genes and proteins from aligned sets of DNA sequences
- Sequences do not need to be codon aligned – results can be codon-aligned on the fly with generally good results
- Currently, sequence alignments must contain HXB2 as a reference for the program to function

- Brian Gaschen

## N-GLYCOSITE

- Tracks of patterns of N-linked glycosylation site (N-X-[ST]) change in sequences
- INPUT: A sequence alignment of interest
- OUTPUT:
- Tallies of numbers of Ngly sites in each sequence
-  Highlighted Ngly sites
-  Graphics illustrating frequency of Ngly patterns in the   alignment, and in sub-regions of the alignment
-  Frequencies of different patterns of X and Y in
-     N-X-[ST]-Y

- Brian Gaschen

## Sequence alignments

- Originally based on iterations of manual and HMM alignments
- Yearly updates using HMM and manual corrections
- Full length genomes updated throughout the year
- Alignments are in reading frame (codon aligned)
- Alignments non-redundant
- Compendia alignments show fewer sequences than web version
- Reference alignments contain up to four representatives
- Protein alignments may contain frameshift compensations
- Subtype consensus with ties resolved, as well as maximum likelihood ancestors, are available for reagent production

- Thomas Leitner

## Comparing ”new” and database sequences

- As an example, eight “new” *env* sequences have been aligned to subtype references (A-K) and sequences from previous search (Japanese *env*) using HMM, Genecutter & Se-Al
- TreeMaker produces a Neighbor Joining tree for a “quick-and-dirty” comparison
- TreeMaker is based on DNADIST & NEIGHBOR in the PHYLIP package
- HIV-BLAST is an option for looking for highly similar sequences or possible contamination

- Thomas Leitner

## Recombination Analysis

- Many methods and programs exist to investigate potential recombination
- http://bioinf.man.ac.uk/~robertson/recombination/
- Investigating recombination requires many steps
- A new version of RIP is available at HIV db
- Automatic alignment
- Selection of background sequences
- Different window/gap handling options
- Graphic & table output

- Thomas Leitner

## HIV and SIV Protein3D Structures

- Structures Determined by X-Ray Crystallographic and NMR Methods
- HIV and SIV Structural proteins (RT, Protease, Env Core)
- RNA Secondary Structures (TAR, PSI, RRE)
- Models of non-crystalized proteins
- Env Core with Variable Loops added
- Tutorials and Reviews
- Env, RT, Protease
- Links to Free 3D Viewer Software
- CHIME/Protein Explorer
- RasMol
- Visual Molecular Dynamics

- Brian Foley

## HIV/SIV Sequence Locator Tool

- Rapidly returns position numbers of an HIV or SIV DNA or protein sequence fragment relative to the HXB2r or SMM239 reference strains.
- Such numbers are often included in the literature, and are often incorrect
- Marks the location of the sequence on an HIV map
- For DNA sequences, a translation is provided
- Can be used for input into the search interface, to align a new sequence you have generated with the database set.

- Bette Korber

## Immunology Database Overview

- HIV T-Cell (CTL, T-helper) and Antibodies (Ab)
- Types of data recorded
- Epitope sequence and location: HXB2 numbering, subtype
- Immunogen
- Host HLA or MHC, and Ab isotype
- Notes summarize main findings
- Contents: data from 1985 through 2002
- 2300 CTL entries
- 600 T-helper entries
- 1100 Ab entries

- Bette Korber

## Immunology Database: Search

- Bette Korber

- T Cells
- Cytotoxic T Lymphocytes (CTL)
- Helper T Lymphocytes (T-helper)
- Biological distinction between CTL and T-helper is not always obvious
- Organization is identical for CTL and T-helper
- One reference per entry
- B Cells (Antibodies)
- One entry for each monoclonal antibodies
- Many references per entry (up to 100)

## Immunology Database: Additional Information

- Bette Korber

- All entries for a reference
- Medline links to papers
- Epitope Tables
- Epitope Maps
- Unique species/HLA for T cell epitopes
- MAb name, species code for Ab
- Epitope Alignments
- Extracted from HIV-sequence database, includes subtype, country and year of sampling

## PeptGen

- Creates maps of overlapping peptides on proteins to aid in peptide design for mapping epitopes
- Consensus sequences for all HIV subtypes for all proteins are available
- Use alignments to design comparable sets of peptides (for example, to compare clades)
- INPUT
- Query sequence or aligned sequences
- Desired length of peptides, peptide overlap, forbidden C- and N-terminal amino acids
- OUTPUT
- Maps of overlapping peptides (forbidden amino acids are taken into account)
- Highlighted forbidden amino acids
- Hydropathicity scores for the peptides are available

- Karina Yusim

## HLA Binding Motif Scanner: MotifScan

- Finds HLA anchor motifs within protein sequences for specified HLA genotypes, serotypes, or supertypes
- HLA anchor motif dictionaries are available on line
- Main motif and supermotif sources:
- SYPHEITHI Database, Rammensee *et al*. www.syfpeithi.de
- HLA Facts Book, Marsh *et al*. 2000
- Sette & Sidney, *Immunogenetics* **50**:201-212, 1999
- INPUT:
- User defined query sequence or aligned sequences, or reference set
- Selected HLA anchor motifs, or user defined motif
- The user defined motif function could be used to search for other patterns of interest in sequences
- OUTPUT:
- Anchor residue positions are highlighted in the query sequence
- Potential epitopes and positions are listed
- Output can be downloaded as text, convenient for further analysis

- Karina Yusim

## ELF: Epitope Location Finder 

- Helps identify potential CTL epitopes in reactive peptides
- INPUT:
- Reactive peptide sequence
- Full protein sequence that peptides were based on
- Patient’s HLA information – genotype or serotype
- OUTPUT:
- If HLA serotype is submitted, associated HLA genotypes are given
- Potential epitopes in reactive peptides, based on anchor motifs
- Maps of all HIV epitopes for all HIV proteins, highlighting epitopes that use the patient’s HLA presenting molecules
- Location of the query peptide according to the HXB2 reference strain
- Alignment of query peptide against reference database alignments
- All known CTL HIV epitopes contained in the query peptide
- Epitopes presented by the patient’s HLA presenting molecules, that are potentially experimentally missed because of amino acid differences between the previously defined epitopes and the query strain

- Karina Yusim

## SHIV Maps

- SHIVs are Simian (almost always SIV-SMM or SIV-MAC) / Human (usually HIV-1 subtype B) hybrid infectious clones.
- They are frequently used as vaccine reagents and/or challenge virus.
- Many are derived from clones, but often derived via complex in-vivo passage histories.
- Some have been sequenced, but many are only known via sequence of parental lineages.
- Many authors do not adequately describe the SHIVs they are working with, so we have created a review paper to help describe them.
-  _http://www.hiv.lanl.gov/content/hiv-db/REVIEWS/VACCINE2001/Vaccine2001.html_

- Brian Foley

## Primate Lentiviruses

- Brian Foley

_**Primate Lentiviruses**_

_**x**_

**Pol**** ****tree**

**Gao, *****Nature*****  1999**
**Santiago, *****Science***** 2002**
**Corbet, *****J. Virol***** 2000**
**Brian Foley, HIV database**

**Pol**** gene ****tree**

**x**

**Positive Chimps**
**HIV-1 M, N, O**

*P. t. troglodytes** *

*P. t. schweinfurthii*

## SIV/PIV Alignments

- Any non-human lentivirus is a SIV, not just the SIV-SMM/SIV-MAC group from Sooty mangabeys.
- HIV-1s (M, N and O groups) are related to the SIV-CPZs from the *Pan troglodytes troglodytes* chimps.  We describe these alignments as HIV-1/CPZ
- HIV-2s and SIV-MACs are related to SIV-SMMs from Sooty mangabeys.  We describe these alignments as HIV-2/SMM
- Dozens of other diverse non-human primates, such as African green monkeys, carry SIVs.
- Alignments of the diverse SIVs, plus HIVs, can help to identify highly conserved codons and other features.  We describe these alignments as “other SIV” or HIV-1/HIV-2/SIV.
-  _http://www.hiv.lanl.gov/content/hiv-db/ALIGN_CURRENT/ALIGN-INDEX.html_

- Brian Foley

## Vaccine database

- Under construction
- Non-human primate models
- _[http://hiv-web.](http://hiv-web.lanl.gov/cgi-bin/vaccine/public/index.cgi)__[lanl](http://hiv-web.lanl.gov/cgi-bin/vaccine/public/index.cgi)__[.gov/cgi-bin/vaccine/public/index.cgi](http://hiv-web.lanl.gov/cgi-bin/vaccine/public/index.cgi)_
- Aims: To introduce the new database and to interactively retrieve information related HIV/SIV vaccine studies in nonhuman primates
- Search criteria:
- Vaccine
- Challenge
- Adjuvant
- Objective: Immunogenicity, challenge, etc.
- Sources of Databases
- LANL
- EMMS Corporation (Dr Jon Warren)
- Output:
- Reference
- Summary
- Results
- Example of search
- Results tabulation

- Brian Foley