HIV-1 Dimer Initiation Complex

Ann Palcisco '01 and
Lauren Stancik '02

Chime Index

Contents:

I. Introduction

Retroviruses are a family of RNA viruses that replicate through a DNA intermediate.  They incorporate two copies of their single-stranded RNA genome into each viral particle (7, HIV Website).  Within the particle, these RNAs exist as a non-covalent homodimer in which two strands are aligned parallel and in register.  Dimerization might modulate several steps of the retroviral life cycle and thus infectivity, such as (1) translation of the genome into viral proteins; (2) preferential packaging (or encapsidation) of two genomic RNAs within the budding viral shell (or capsid) against an excess of cellular RNAs; (3) recombination between the two homologous genomic RNAs in order to increase the rate of viral evolution; (4) stabilization of the genome against degradation; and (5) reverse transcription1.  Within type 1 human immunodeficiency virus (HIV-1), the crucial first steps in dimer formation are mediated by a specific 35-nucleotide RNA stem-loop structure2, the dimer initiation complex.

Dimerization begins when the stem-loops (SL1) of two genomic strands associate by Watson-Crick base-pairing of their loops (a and b strands) <>, forming a transient kissing-loop complex.  This complex is then believed to isomerize into a linear duplex, sometimes referred to as the mature SL1 dimer initiation complex.  Within this complex the intrastrand base pairs of the stems have melted and reformed as interstrand pairs, creating a more stable linkage between the two strands2.

II. General Structure

The authentic HIV-1 dimer initiation complex consists of a highly-conserved hairpin, SL1,whose location coincides with that of the dimer linkage site (DLS).  SL1 is nine bases long, per strand, and includes the six-base palindrome GCGCGC (from the b strand) <>.  Another six-base palindrome GCGCGC is represented on the a strand, in conjunction with the palindrome of the b strand <>.  The palindrome is usually flanked by one 3'and two 5' adenines <>.

The SL1 stems form canonical A-type helices <>.  The loop palindromes, by contrast, form a right-handed helix <>, whose minor groove is fully exposed along the 'right side' of the complex.  However, this loop helix deviates significantly from A-type geometry: specifically, it is underwound and manifests high positive roll values between base pairs, as well as outward buckling of its lateral base pairs.  This has the effect of flattening and flexing the loop helix so that it undergoes a half turn with a significantly narrower helical radius and smaller pitch height than a conventional A-helix3.

The flanking adenines remain unbound, instead, through stacking interactions they help to partially stabilize the kissing-loop form.  To form the kissing-loop, each A9 / A9 base <> must rotate around the backbone to stack against bases of the opposite strand.  This angle allows for only partial stacking of A9, A8, A16 and G7.

III. Linear Conformation

The conformation of the mature linear duplex is that of a roughly linear double helix punctuated by two relatively flexible bulges that each encompass three unpaired adenines.  There is less distortion of the helices in the mature form than in the kissing-loop form.  The overall structure is that of a standard A-type helix, but the palindromic minihelix located between the two bulges is slightly distorted.   This slight distortion allows for A16 <> to be displaced away from its complement, C15 <>, and stack against A8 and A9    <>, stabilizing the adenine bulges.

Despite their divergent overall structures, the NMR spectum of the linear duplex and the kissing-loop are remarkably similar.  This is due to the fact that the local environment and bonding interactions are the same in both structures, except that all intramolecular base pairs of the kissing-loop are converted to intermolecular base pairs in the linear duplex.  One difference is the presence of the G7-C17 <> base pair, absent in the kissing-loop, in the mature dimer.  This reformed base pair normalizes the A-helices conformation in the linear form, thus increasing  stacking, relieving strain on the helix, and granting favorable thermodynamic effects.  These benefits likely account for the tendency of the kissing-loop complex to spontaneously convert to the linear form. 

IV. Gag Protein

The Gag gene of HIV-1, and related retroviruses, gives rise to the 55-kilodalton Gag precursor polyprotein, also called p55, that is expressed from the unspliced viral mRNA.  This gene possesses the information necessary for virion particle formation and release from the host cell membrane.  During virion release, the Gag polyprotein is cleaved by the pol-encoded protease.  This cleavage results in the production of several proteins, such as (1) the matrix protein (p17), which lines the virion membrane envelope; (2) the capsid protein (p24), which forms the core of the virion; (3) and the nucleocapsid protein (p9), which coats the genomic RNA.

The matrix polypeptide is derived from the N-terminal end of the Gag gene, and facilitates the nuclear transport of the viral genome, which in turn allows HIV-1 to infect nondividing cells.  The capsid protein forms the conical core of viral particles.  Cyclophilin A interacts with the p24 region of the Gag gene, resulting in its incorporation into HIV-1 particles.  The nucleocapsid recognizes the packaging signal of HIV-1, which consists of four stem loop structures at the 5' end of the viral RNA, and mediates the incorporation of a heterologous RNA into HIV-1 virions
(www.columbia.edu/cu/cie/techlists/ patents/5773225.htm, 12/7/00).

V. Implications

The dimer initiation complex is one of the most highly conserved structures within human HIV-1.  Because it plays a key role in in vitro dimerization and because dimers may be preferentially encapsidated into the HIV-1 virus, alterations of the kissing-loop might affect the in vivo dimerization and encapsidation process.

Studies have shown that deletion mutations introduced into the kissing-loop decreased the infectivity of the resulting viruses, reduced the amount of genomic RNA packaged per virus, and the proportion of dimeric genomic RNA was reduced4.  However, long-term culture of the mutated RNAs in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations5.

Shen et al.6 found that destroying the stem-loop <> reduced genome dimerization and proviral DNA synthesis but this has yet to be further studied for compensatory mutations.  Nonetheless, the kissing-loop remains one of the top research subjects in the search for HIV-1 gene therapy.

VI. References

1. Laughrea M, Jette L. 1996. Kissing-Loop Model of HIV-1 Genome Dimerization: HIV-1 RNAs Can Assume Alternative Dimeric Forms, and All Sequences Upstream or Downstream of Hairpin 248-271 are Dispensable for Dimer Formation. Biochemistry 35:1589-1598.

2. Mujeeb A, Parslow TG, Zarrinpar A, Das C, James TL. 1999. NMR structure of the mature dimer initiation complex of HIV-1 genomic RNA. Federation of European Biochemical Societies 458:387-392.

3. Mujeeb A, Clever JL, Billeci TM, James TL, Parslow TG. 1998. Structure
of the dimer initiation complex of HIV-1 genomic RNA. Nature Structural Biology 5:432-436.

4. Laughrea M, Jette L, Mak J, Kleinman L, Liang C, Wainberg MA. 1997.  Mutations in the kissing-loop hairpin of human inmmunodeficiency virus type-1 reduce viral infectivity as well as genomic RNA packaging and dimerization.  Journal of Virology 71: 3397-406.

5. Liang C, Rong L, Quan Y, Laughrea M, Kleiman L, Wainberg MA. 1999.  Mutations within four distinct gag proteins are required to restore replication of human immunodeficiency virus type-1 after deletion mutagenesis within the dimerization initiation site.  Journal of Virology 73: 7014-20.

6. Shen N, Jette L, Liang C, Wainberg MA, Laughrea M. 2000.  Impact of human immunodeficiency virus type-1 RNA dimerization on viral infectivity and of stem-loop B on RNA dimerization and reverse transcription and dissociation of dimerization form packaging. Journal of Virology 74: 5729-35.

7. Understanding Human Immunodeficiency Virus: An Interactive Exploration of Recent Literature.  Judith Kandel, Biology 302, Department of Biological Science, California State University at Fullerton.  http://biology.fullerton.edu/biol302/Browser/moreabout.html.