3404–3417 Nucleic Acids Research, 2011, Vol. 39, No. 8 doi:10.1093/nar/gkq1314 Published online 23 December 2010 The relationship between HIV-1 genome RNA dimerization, virion maturation and infectivity y Masahisa Ohishi1, Takashi Nakano2, Sayuri Sakuragi1, Tatsuo Shioda1, Kouichi Sano2 and Jun-ichi Sakuragi1,* 1Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University Suita, Osaka 565-0871 and 2Department of Microbiology and Infection Control, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan Received November 2, 2010; Revised and Accepted December 9, 2010 ABSTRACT The relationship between virion protein maturation and genomic RNA dimerization of human immuno- deficiency virus type 1 (HIV-1) remains incompletely understood. We have constructed HIV-1 Gag cleavage site mutants to enable the steady state observation of virion maturation steps, and pre- cisely study Gag processing, RNA dimerization, virion morphology and infectivity. Within the virion maturation process, the RNA dimer stabilization begins during the primary cleavage (p2-NC) of Pr55 Gag. However, the primary cleavage alone is not sufficient, and the ensuing cleavages are required for the completion of dimerization. From our obser- vations, the increase of cleavage products may not put a threshold on the transition from fragile to stable dimeric RNA. Most of the RNA dimerization process did not require viral core formation, and par- ticle morphology dynamics during viral maturation did not completely synchronize with the transition of dimeric RNA status. Although the endogenous virion RT activity was fully acquired at the initial step of maturation, the following process was necessary for viral DNA production in infected cell, suggesting the maturation of viral RNA/protein plays critical role for viral infectivity other than RT process. INTRODUCTION The genome of retrovirus such as human immunodeﬁ- ciency virus type 1 (HIV-1) is a single-stranded, positive- sense RNA. The viral genome always occurs as a dimer in virus particles, and the interaction is non-covalent since heating easily dissociates puriﬁed dimeric genomes into monomers. Genomic RNA dimerization is believed to be a crucial step for the life cycle of retroviruses. Template strand switching between two genomes during reverse transcription is often observed in the retroviral lifecycle (1). It is likely that the presence of two genomes in one virion helps the virus survive by providing genetic variety for their progeny (2). However, this may not fully explain why the virion is required to carry two identical RNAs in spite of severe space limitation, since retroviruses with little sequence variety such as HTLV-1 (3) are also dimeric. Identiﬁcation of cis-acting signals for retrovirus genome dimerization, called the dimer linkage structure (DLS), was initially attempted in an in vitro assay (4–8). The proposed DLS regions of HIV-1 is located within the untranslated region between LTR and the gag gene (4,9). Although the DLS on viral RNA is suggested to be involved in dimer formation and its close relationship to the packaging signal has been studied (2), there remains incompletely understood issues about the overall mechan- isms and the precise nature of retroviral genome dimerization. The retrovirus dynamically converts the morphology of its particle interior during particle release, termed ‘matur- ation’. Maturation changes virion morphology from the immature particle, called donut-shaped particle, to the mature virion; a particle lined with viral matrix proteins containing a condensed core composed of a viral capsid shell caging ribonucleoprotein (RNP) complex, comprised of viral RNA, nucleocapsid and enzymes (10). Maturation prepares the virus for infection of adjacent hosts and is inevitably essential for particle infectivity. Although many aspects about how the process of virion maturation contributes to achieving infectivity remain unclear, it is a well-accepted idea that viral RNA within the virion forms a stable and uniform dimer only after complete virion maturation. Obviously, viral protease (PR) activity to process Gag precursor protein (Pr55) is required for stable genomic RNA dimerization in the virion. It has *To whom correspondence should be addressed. Tel: +81 6 6879 8348; Fax: +81 6 6879 8347; Email: sakuragi@biken.osaka-u.ac.jp yThis article is dedicated to the memory of Dr Toshiyuki Goto. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.