Distinct Roles for Nucleic Acid in In Vitro Assembly of Purified Mason-Pfizer Monkey Virus CANC Proteins

Abstract

In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (⌬ProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV ⌬ProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which ⌬ProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into ⌬ProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.

In Vitro Assembly of Virus-Like Particles of a Gammaretrovirus, the Murine Leukemia Virus XMRV

“…Particles of XMRV ⌬ProCANC and ⌬10CANC were not detected if the in vitro assembly was carried out in the absence of nucleic acid or at an NaCl concentration higher than 0.5 M (data not shown), suggesting that assembly must be initiated by the addition of nucleic acids under suitable conditions. Similar assembly requirements have been observed for the in vitro-assembled particles of RSV CANC (8), HIV-1 CANC (8,21,22), and M-PMV ⌬ProCANC (57).…”

“…It is known that the prevention of ␤-hairpin formation (by either the deletion or substitution of Pro1 or by N-terminal extension of CA) can shift the assembly mode to formation of immature-virus-like structures (21,46,50,57,61). We therefore decided to truncate the XMRV CANC construct by the deletion of the first proline residue (⌬ProCANC) with the aim of assembling immature-like structures.…”

“…1). To study the ability of XMRV CA and CANC to assemble into VLPs, we used both bacterial and in vitro assembly systems (50,57). TEM of thinsectioned E. coli BL21(DE3) expressing XMRV CA and CANC showed amorphous inclusion bodies instead of assembled mature-core-like structures ( Fig.…”

“…A number of studies have mimicked the assembly of both immature virus particles and mature cores in vitro using purified recombinant Gag-derived capsid-nucleocapsid (CANC) proteins (8,21,22,31,41,57,68). Although the in vitro assembly of retroviral genera, including the lentivirus HIV-1 (8,12,21,22), the betaretrovirus Mason-Pfizer monkey virus (M-PMV) (31,57), and the alpharetrovirus RSV (8,40,41,68), has been intensively studied, there is a lack of information on the in vitro assembly of gammaretroviruses. In vitro assembly was achieved by using His-tagged MLV CA molecules anchored to Ni 2ϩ -chelating lipid nanotubes as an assembly substrate (24).…”

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

“…The importance of RNA for assembly is well documented by in vitro assembly studies with various retroviruses, e.g., Rous sarcoma virus (RSV), HIV, murine leukemia virus (MLV), and M-PMV (5)(6)(7)(8)(9)(10)(11). Although cellular RNA is sufficient to promote Gag assembly (12), formation of an infectious retrovirus requires specific packaging of the viral genome (vRNA) into the assembling Gag particle.…”