Studies on the Restriction of Murine Leukemia Viruses by Mouse APOBEC3

APOBEC3 proteins function to restrict the replication of retroviruses. One mechanism of this restriction is deamination of cytidines to uridines in (−) strand DNA, resulting in hypermutation of guanosines to adenosines in viral (+) strands. However, Moloney murine leukemia virus (MoMLV) is partially resistant to restriction by mouse APOBEC3 (mA3) and virtually completely resistant to mA3-induced hypermutation. In contrast, the sequences of MLV genomes that are in mouse DNA suggest that they were susceptible to mA3-induced deamination when they infected the mouse germline. We tested the possibility that sensitivity to mA3 restriction and to deamination resides in the viral gag gene. We generated a chimeric MLV in which the gag gene was from an endogenous MLV in the mouse germline, while the remainder of the viral genome was from MoMLV. This chimera was fully infectious but its response to mA3 was indistinguishable from that of MoMLV. Thus, the Gag protein does not seem to control the sensitivity of MLVs to mA3. We also found that MLVs inactivated by mA3 do not synthesize viral DNA upon infection; thus mA3 restriction of MLV occurs before or at reverse transcription. In contrast, HIV-1 restricted by mA3 and MLVs restricted by human APOBEC3G do synthesize DNA; these DNAs exhibit APOBEC3-induced hypermutation

Murine Leukemia Virus Nucleocapsid Mutant Particles Lacking Viral RNA Encapsidate Ribosomes

A single retroviral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. Gag normally selects the genomic RNA of the virus with high specificity; the nucleocapsid (NC) domain of Gag plays a crucial role in this selection process. However, encapsidation of the viral RNA is completely unnecessary for particle assembly. We previously showed that mutant murine leukemia virus (MuLV) particles that lack viral RNA because of a deletion in the cis-acting packaging signal (“Ψ”) in the genomic RNA compensate for the loss of the viral RNA by incorporating cellular mRNA. The RNA in wild-type and Ψ− particles was also found to be necessary for virion core structure. In the present work, we explored the role of RNA in MuLV particles that lack genomic RNA because of mutations in the NC domain of Gag. Using a fluorescent dye assay, we observed that NC mutant particles contain the same amount of RNA that wild-type virions do. Surprisingly enough, these particles contained large amounts of rRNAs. Furthermore, ribosomal proteins were detected by immunoblotting, and ribosomes were observed inside the particles by electron microscopy. The biological significance of the presence of ribosomes in NC mutant particles lacking genomic RNA is discussed

Effect of APOBEC3s on ΔVif HIV-1 DNA synthesis.

<p>293-mCAT1 cells were infected with ΔVif HIV-1. Infectivity and RT activity were assayed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone.0038190-Derse1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone.0038190-Gorelick1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone.0038190-Gorelick2" target="_blank">[30]</a>. Twenty-four hours after infection, the cells were lysed and assayed by real-time PCR for <i>Luciferase</i> DNA (black and green lines) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#s4" target="_blank">Materials and Methods </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone.0038190-Gherghe1" target="_blank">[32]</a>. Specific infectivity is represented with red and blue lines.</p

Immunoblotting of virus particles. A) Western blot on produced virus.

<p>Chimera (lanes 1 to 5) or MoMLV (lanes 6 to 10) were prepared by transient transfection of 293T cells, using 0, 3, or 10 μg APOBEC3 DNA as well as viral DNA; control cells were transfected with DNA of pGCcos3Neo (a derivative of pSV2Neo) (lane 11). The culture supernatants were fractionated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#s4" target="_blank">Materials and Methods</a> and equal volumes of culture fluid were loaded into the lanes and analyzed by immunoblotting against P30^CA. M, molecular weight markers. <b>B) Encapsidation of different APOBEC3s in MoMLV and chimera viruses.</b> Virus particles shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone-0038190-g003" target="_blank">figure 3A</a> were analyzed by immunoblotting against the HA tag. Equal volumes of culture fluid were loaded into the lanes.</p

Comparison of the infectivity of the viruses with the synthesis of the viral DNA.

<p>Viruses were prepared by co-transfection of 293T-hygro cells (which contain pLXSH) with viral clones, pBABE-Luc, and 0, 3, or 10 μg of APOBEC3 DNA. 293-mCAT1 cells were then infected with the resulting culture fluids. Relative specific infectivities (black lines) were determined as in Fig. 2. Parallel cultures were lysed and assayed for <i>hph</i> DNA (red lines) and strong stop DNA (green lines) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#s4" target="_blank">Materials and Methods</a>. Values in each assay were divided by the value for the virus produced with no APOBEC3 plasmid.</p

Effects of the different APOBEC3s on the infectivity of MoMLV and chimera viruses.

<p>Virus particles were produced by transient transfection of viral clones together with pBABE-Luc and 0, 3, or 10 μg of either mA3 or hA3G expression plasmids. Specific infectivities were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#s4" target="_blank">Materials and Methods</a>, dividing the luciferase activity values by the RT activity values; specific infectivity of samples produced without APOBEC3 is set to 100%. Black line, MoMLV with mA3; green, chimera with mA3; red, MoMLV with hA3G; blue, chimera with hA3G. Results are plotted <i>vs.</i> the quantity of APOBEC3 plasmid used in the transfections to generate the viruses.</p