RT- Reverse Transcriptase

HIV RT forms a heterodimer with RT/RNase H polyprotein and reverse transcribes viral RNA into DNA.



PDB-1RTD: HIV-1 (isolate UNKNOWN) RT mutant (UNKNOWN) and DNA complex - produced using SwissModel/SPDBV/POVray

Key web links:
ViralZone: HIV-1, HIV replication cycle, HIV resource
PDB: 1RTD (HIV-1 RT)
UniProt: P04585 (HIV-1 HXB2 POL)
Chime Tutorial:  Online Macromolecular Museum
HIV-1/Human Protein Interaction DB: HIV-1 Pol
Los Alamos HIV structure DB: Reverse Transcriptase
EMBL: K03455 [EMBL/GenBank/DDBJ]


Isoforms:

  • p51 (440 amino acids) - Reverse Transcriptase
  • p66 (560 amino acids) - RT + RNase H


Cleavage sites:


Function:

  • RT makes a double-stranded DNA copy of the dimer of single-stranded RNA.
  • RT has both RNA-dependant and DNA-dependant polymerase activity.
  • Shortly after virus entry into the cell, RT converts the viral ssRNA genome into dsDNA, which can then be integrated into the host genome.
  • RT is a heterodimer p66/p51 and displays multiple enzymatic activities: RNA-dependent and DNA-dependent DNA polymerase and RNase H activities .

Localization:

  • Virion
  • Host cell cytoplasm: reverse transcription probably takes place in the cytoplasm during capsid transport and uncoating
  • Host cell nucleus

Additional Information:

  • Late timing of expression.
  • RT has low fidelity (high frequency of mutation).
  • Viral DNA can be completely synthesized within 6 hours of viral entry.
  • Major functional species of polymerase is a heterodimer of p51 (Reverse Transcriptase) and p66 (Reverse Transcriptase - RNase H) .
  • Many cis-acting elements in viral RNA are required for the generation of viral DNA .
  • TAR is a small RNA stem-loop structure located at 5'-end of viral RNA and is required for initiation of Reverse Transcriptase .

RT Function & Host-Virus Protein Interactions:[TOP]




Interactions highlighted in the image:


Potential interactions (not in the image):


  • RT interacts with Actin.


Genomic Location & Protein Sequence: [TOP]

HIV-1 (HXB2):

          10         20         30         40         50         60         70         80         90        100
| | | | | | | | | |
PISPIETVPV KLKPGMDGPK VKQWPLTEEK IKALVEICTE MEKEGKISKI GPENPYNTPV FAIKKKDSTK WRKLVDFREL NKRTQDFWEV QLGIPHPAGL
110 120 130 140 150 160 170 180 190 200
| | | | | | | | | |
KKKKSVTVLD VGDAYFSVPL DEDFRKYTAF TIPSINNETP GIRYQYNVLP QGWKGSPAIF QSSMTKILEP FRKQNPDIVI YQYMDDLYVG SDLEIGQHRT
210 220 230 240 250 260 270 280 290 300
| | | | | | | | | |
KIEELRQHLL RWGLTTPDKK HQKEPPFLWM GYELHPDKWT VQPIVLPEKD SWTVNDIQKL VGKLNWASQI YPGIKVRQLC KLLRGTKALT EVIPLTEEAE
310 320 330 340 350 360 370 380 390 400
| | | | | | | | | |
LELAENREIL KEPVHGVYYD PSKDLIAEIQ KQGQGQWTYQ IYQEPFKNLK TGKYARMRGA HTNDVKQLTE AVQKITTESI VIWGKTPKFK LPIQKETWET
410 420 430 440
| | | |
WWTEYWQATW IPEWEFVNTP PLVKLWYQLE KEPIVGAETF
[download in fasta format]

Length: 440 amino acids (residues 155 to 585)
Molecular Weight: 51330 Da
Theoretical pI: 8.64
Position relative to Pol protein: 156 - 595


Protein Domains/Folds/Motifs: [TOP]

InterPro signature for RNA-directed DNA polymerase - IPR000477
InterPro signature for Reverse Transcriptase connection domain - IPR010659
InterPro signature for Reverse Transcriptase thumb domain - IPR010661


Secondary Structure predictions:

Low Complexity Regions - seg:


Antigenic Sites - EMBOSS:

Predicted Motifs: Printer-friendly version

N-glycosylation:
N-myristoylation:
Amidation:
Protein kinase C:
Casein kinase II:
Tyrosine kinase:
cAMP / cGMP kinase:
Cell attachment motif:
Asp Protease motif:
Asp Prot Retro motif:
Cysteine-rich Region:
Tryptophan-rich Region:
Zinc-finger CCHC motif:
Leucine Zipper motif:

HIV Antiretrovirals and Drug Resistance Mutations: [TOP]

See Stanford University HIV Drug Resistance Database, which contains information different HIV-1 subtypes and CRFs 01 and 02


See a recent review on HIV-1 drug resistance and subtypes, with special focus on HIV-1 subtype C, the most prevalent HIV-1 strain in the world


See ViralZone HIV Drug Pages


Mechanism of Action:


Reverse transcription describes the process by which the HIV reverse transcriptase (RT) enzyme reverse transcribes the virus RNA genome to DNA. This newly synthesized HIV DNA genome will then be transported into the host cell nucleus and integrated into the host genome for replication. Nucleoside reverse transcriptase inhibitors (NRTIs) are structural nucleoside analogues of DNA nucleotides. When incorporated into the reverse transcibed HIV DNA strand during reverse transcription, DNA polymerisation is blocked. This prevents the action of RT and viral genome replication . The action of NRTIs is based on their ability to prevent phosphodiesterase bonds with the following nucleotide in a growing DNA strand . Human cellular kinases phosphorylate these nucleoside analogues to their triphosphate form, afterwhich these nucleoside analogues are incorporated into newly synthesized HIV DNA strands, resulting in chain termination .


Mechanism of Action of NNRTI:


Like NRTIs, NNRTIs target the reverse transcription step of the HIV life cycle, however through a different mechanism. Unlike NRTIs, NNRTIs bind directly to the RT enzyme, inhibiting its action and preventing HIV DNA strand elongation. NNRTIs bind to the pocket residue of the RT enzyme which induces allosteric changes to the enzymes' active site, inhibiting DNA polymerization . This conformational change to the RT enzyme decreases its nucleoside binding affinity, preventing viral replication .
NNRTIs do not require phosphorylation activation like NRTIs. Furthermore, the difference in inhibition mechanisms and lack of overlapping resistance mutations, demonstrates NRTIs and NNRTIs compatibility to be used in a treatment cocktail.


Drug Resistance Mutations:


NRTI:
Position Mutation Mutation Type Drugs Affected Reference
M41 L Thymidine Analog Mutation (TAM) ABC, DDI, TDF, D4T, ZDV
M184 V, I 3TC, FTC, ABC, DDI
D67 N TAM ABC, DDI, TDF, D4T, ZDV
K70 R TAM D4T, ZDV
L210 W TAM ABC, DDI, TDF, D4T, ZDV
T215 F, Y TAM ABC, DDI, TDF, D4T, ZDV
K219 Q, E TAM D4T, ZDV
K65 R, N Non-Thymidine Analog Regimen Mutations (NTARM) 3TC, FTC, ABC, DDI, TDF, D4T
K70 E, G NTARM 3TC, FTC, ABC, DDI, TDF
L74 V, I NTARM ABC, DDI
V75 T NTARM ABC, DDI, D4T
V75 M NTARM ABC, DDI, TDF, D4T
Y115 F NTARM ABC, TDF
T69 Ins NTARM 3TC, FTC, ABC, DDI, TDF, D4T, ZDV
Q151 M Multi-NRTI Resistance Mutations (MNRM) 3TC, FTC, ABC, DDI, TDF, D4T, ZDV
A62 V MNRM 3TC, FTC, ABC, DDI, TDF, D4T, ZDV
V75 I MNRM ABC, DDI, D4T, ZDV
F77 L MNRM ABC, DDI, D4T, ZDV
F116 Y MNRM ABC, DDI, D4T, ZDV
NNRTI:
Position Mutation Additional Information Drugs Affected Reference
A98 G NVP, DLV, EFV, ETR
L100 I Mutation is associated with a higher level of
drug resistance and decreased virologic response.
NVP, DLV, EFV, ETR
K101 E, P K101E/P mutations are associated with a higher level of
NVP and DLV resistance and decreased virologic response.
K101P mutation is associated with a higher level of
EFV and ETR resistance and decreased virologic response.
NVP, DLV, EFV, ETR
K103 N, S Mutations are associated with a higher level of
drug resistance and decreased virologic response.
NVP, DLV, EFV
K103 R Mutation greatly reduces NVP, DLV and EFV
susceptibility when in combination with the V179D mutation (~15-fold reduction).
NVP, DLV, EFV
V106 A, M V106A/M mutations are associated with a higher
level of NVP and DLV resistance and decreased virologic response.
V106M mutation is associated with a higher level of
EFV resistance and decreased virologic response.
NVP, DLV, EFV
V108 I NVP, DLV, EFV
V179 D, E, F V179F mutation is associated with a higher level of
ETR resistance and decreased virologic response.
NVP, DLV, EFV, ETR
Y181 C, I, V Mutations are associated with a higher level of
NVP, DLV and ETR resistance and decreased virologic response.
NVP, DLV, EFV, ETR
G190 A, S Mutations are associated with a higher level of
NVP and EFV resistance and decreased virologic response.
NVP EFV, ETR
G190 E Mutation is associated with a higher level of
drug resistance and decreased virologic response.
NVP, DLV, EFV, ETR
P225 H EFV
F227 L NVP
F227 C Mutation is associated with a higher level of
ETR resistance and decreased virologic response.
NVP, DLV, EFV, ETR
M230 L Mutation is associated with a higher level of
drug resistance and decreased virologic response.
NVP, DLV, EFV, ETR
P236 L Mutation is associated with a higher level of
drug resistance and decreased virologic response.
DLV
K238 T Mutation is associated with a higher level of
NVP and DLV resistance and decreased virologic response.
NVP, DLV, EFV, ETR
Primary and Secondary Database Entries: [TOP]

Identifiers:



ViralZone: HIV-1, HIV replication cycle, HIV resource
PDB/MMDB: Search for HIV-1 & RT

UniProt: P04585 (HIV-1 HXB2 POL)
EC: 2.7.7.49
EMBL: K03455; AAB50259.1 [EMBL/GenBank/DDBJ]

InterPro: IPR000477 - RNA-directed DNA polymerase (RT) family
IPR010659 - Reverse Transcriptase connection domain
IPR010661 - InterPro signature for Reverse Transcriptase thumb domain
Pfam: PF00078
Prints: none
SCOP: SSF56672 DNA/RNA polymerase
BLOCKS: P04585
Prosite: P04585
ProtoNet: P04585
Database of Interacting Proteins: P04585
ModBase: P04585
HIV-1/Human Protein Interaction DB: HIV-1 Pol
HIV-1 Sequence Database Los Alamos HIV Sequence Database

PDB:




Reviews and References: [TOP]

Cite the resource by citing the following paper:
Doherty R et al. BioAfrica's HIV-1 Proteomics Resource: Combining protein data with bioinformatics tools. Retrovirology (2005), 9;2(1):18.

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2 - Retroviruses
Coffin JM, Hughes SH, Varmus HE.
CD-ROM ed. (2002) Cold Spring Harbor Laboratory Press [Read it online: NCBI Bookshelf]
3 - Molecular Characteristics of HIV-1 Subtype C Viruses from KwaZulu-Natal, South Africa:
Implications for Vaccine and Antiretroviral Control Strategies.
Gordon M, De Oliveira T, Bishop K, Coovadia HM, Madurai L, Engelbrecht S, Janse van Rensburg E, Mosam A, Smith A, Cassol S.
Journal of Virology 77(4): 2587-2599 (2003) [pubmed: 12551997]
4 - Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.
Kohlstaedt LA, Wang J, Friedman JM.
Science 256: 1783-1790 (1992) [pubmed: 1377403]
5 - A critical role for the TAR element in promoting efficient human immunodeficiency virus type 1
reverse transcription.
Harrich D, Ulich C, Gaynor RB.
J Virol 70: 4017-4127 (1996) [pubmed: 8648739]
6 - [HIV RT Review] Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase:
implications for drug resistance.
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Science 282(5394): 1669-1675 (1998) [pubmed: 9831551]
7 - [Website] An accurate look into HIV
Teresa Larsen
The Scripp Research Institute - Website: http://www.sdsc.edu/GatherScatter/GSsummer96/larsen.html
8 - [Website] HIV-1 RT
Kaylan Das
Rutgers University - Website: http://www.cabm.rutgers.edu/~kalyan/RT_imgs/index.html
9 - [Website] HIV Drug Resistance Database
Stanford
Website: http://hivdb.stanford.edu
10 - [Website] HIV Drug Resistance Database
Los Alamos National Labs
Website: http://www.hiv.lanl.gov/content/sequence/RESDB/
11 - Molecular biology: an HIV secret uncovered.
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Viruses 1(3):873-94 (2009) [pubmed: 21994574]
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PLoS One 4(3):e4724 (2009) [pubmed: 19266092]
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Anderson KS.
Biochim Biophys Acta. 1587(2-3):296-9 (2002) [pubmed: 12084471]
17 - HIV-1 reverse transcriptase and antiviral drug resistance. Part 1.
Das K, Arnold E.
Curr Opin Virol. 3(2):111-8 (2013) [pubmed: 23602471]
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Clin Pharmacokinet. 40(12):893-905 (2001) [pubmed: 11735608]
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Virology 454-455:371-9 (2014) [pubmed: 24559861]
20 - The K101P and K103R/V179D mutations in human immunodeficiency virus type 1 reverse transcriptase confer resistance to nonnucleoside reverse transcriptase inhibitors.
Parkin NT, Gupta S, Chappey C, Petropoulos CJ.
Antimicrob Agents Chemother. 50(1):351-4 (2006) [pubmed: 16377709]



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