Retroviruses package two copies of its unspliced RNA

genome into their viral particles. It was reported that

retroviral genomes in the virus particle are non-

covalently dimerized in the 5’ untranslated region (UTR).

Genome selection is mediated by RNA packaging

elements, called -sites, which are typically located

within the 5’UTR. -sites generally overlap with the

dimerization site, implicating that retroviral genome

packaging and genome dimerization are coupled.

Background

Conclusion

Materials and Methods

Introduction

IMPACT OF DYNAMICS OF HIGHLY CONSERVED RETROVIRAL RNA PACKAGING

ELEMENT ON GENOME DIMERIZATION AND NUCLEOCAPSID BINDING Gerardo Camarena Gomez, Sabrina Ngo, Trevor Mathias, Yasuyuki Miyazaki, Michael Summers

Figure 4| Evidence for two alternate conformations of SL-C.

On the left, portion of the 2D NOESY spectrum obtained for SL-C

hairpin RNA, showing chemical exchange cross peaks that

correlate protons of the three distinct SL-C conformations (shown

at top). Note that a kissing interaction forms when SL-C harbors

a GACG tetraloop. On the right, NOSEY spectrum of SL-C

construct containing U328A/A333U mutations.

1. D’Souza, V. & Summers, M. F. (2005). How

Retroviruses Select Their Genomes. Nature 3, 643-

655.

2. Miyazaki, Y., et al. Structure of a Conserved Retroviral

RNA Packaging Element by NMR Spectroscopy and

Cryo-Electron Tomography. (Unpublished).

Dr. Michael Summers, Dr. Yasuyuki Miyazaki. This work is

supported by NIH-ARRA 3R37HI030917-1951 (M.F.

SUMMERS) and Howard Hughes Medical Institute at

UMBC.

References

Acknowledgements

We have proposed that the conformational change of

the -site induced by dimerization exposes nucleocapsid

(NC)- binding sites. In the Moloney Murine Leukemia

Virus (MoMuLV), the core-encapsidation signal (CES)

was determined as the minimum region required for

genome packaging. CES is composed of stem loops

DIS-1, DIS-2, SL-C, and SL-D. DIS-1 and DIS-2 harbor

self-complementary sequences and can form extended

dimer conformations while SL-C and SL-D harbor a

GACG tetraloop. A stem loop harboring a GACG

tetraloop can interact by forming an intermolecular CG

base pairing at the loop (kissing interaction). GACG

sequences in SL-C and SL-D are highly conserved

among all -retroviruses, implicating its biological

significance. Although it was hypothesized that SL-C and

SL-D promote dimerization via kissing interactions, the

function of SL-C and SL-D remains unclear.

Figure 2|RNA Secondary Structure for Moloney Murine Leukemia Virus

Genome Packaging. a| Representation of the Moloney Murine Leukemia

Virus genome, showing the relative locations of the coding and non-coding

elements. b| Secondary structure predicted for the MoMuLV -site, showing

the location of the dimerization initiation site (DIS), SL-C, and SL-D.

c| Potential dimerization of DIS-2. Base pairing enables the AGCU segment

(green) to form a kissing interaction with another molecule. The GACG

tetraloop (blue) on SL-C and SL-D also interact to form a kissing complex.

Note that the NC-binding UAUCUG segment (red) is sequestered by base

pairing in the hairpin structure and exposed in the dimer.

• NMR data revealed that SL-C forms two

alternate conformations, where one contains a

GACG tetraloop and the other does not.

• CESC-KISS inhibited dimerization of DIS, while

CESC-NOKISS promoted dimerization.

• CESC-KISS showed tight NC-binding while

CESC-NOKISS has significantly weak NC-binding.

• These results collectively suggest that SL-C

and SL-D serve as a scaffold for NC-binding by

forming a tandem stem loop complex rather

than promoting genome dimerization.

H

H

hH

Figure 3| Structure of CD determined by NMR.

a,b| Orthogonal views of the 20 structures calculated with Cyana

showing the degree of convergence and overall asymmetrical

shape of the dimer. The position of the experimentally

unrestrained U319 residue is labeled. c,d| Stereoscopic view

showing the c| structure of the A-minor K-turn and its proximity to

the kissing interface and d| stacking of the A353 and A354

residues between the lower stems of SL-C and SL-C.

Figure 1|Retroviral Life Cycle. Illustration of the early and late phase of the

HIV Replication Cycle. The blue region on the Gag protein shows the

nucleocapsid domain that mediates packaging.

Figure 5| Effect of SL-C conformation on nucleocapsid

binding affinity. On the left, ITC results show that mutation of SL-

C to form the GACG tetraloop resulted in tight nucleocapsid (NC)-

binding. On the right, mutation of SL-C to form CGAGU showed

weak NC-binding.

Figure 6| Dimerization of SL-C mutants. On the left, gel

electrophoresis shows dimeric and monomeric RNA of

CESNATIVE, CES

C-NOKISS, and CESC-KISS. On the right, graphical

representation of the percentage of dimerization of each RNA.

Nuclear Magnetic Resonance (NMR) Study

2D NOESY was performed for 1.5 mM of SL-C native

RNA in 10 mM Tris-HCl (pH 7.0) and 140 mM KCl.

Isothermal Titration Calorimetry (ITC) Study

5 µM of RNA (CESC-KISS, CES

C-NOKISS and CESCD-

NOKISS) are titrated to 100 µM of NC protein.

RNA Dimerization by Gel Shift Mobility Assay

native, CESC-KISS, CES

C-NOKISS, and CESCD-NOKISS

RNA dimerization was performed in 10 mM Tris-HCl

(pH 7.0), 140 mM KCl and 1 mM MgCl2. RNA was

incubated for a series of time intervals at 37oC.

CESNATIVE

CESC-KISS

CESC-NOKISS

Dimer

Monomer

0 0.25 0.5 1.0 1.5 2.0 3.0 6.0 24 Hour

Dimer

Monomer

Dimer

Monomer