Differential sensitivity of murine leukemia virus to APOBEC3-mediated inhibition is governed by virion exclusion

While members of the APOBEC3 family of human intrinsic resistance factors are able to restrict the replication of Vif-deflcient forms of human immunodeficiency virus type 1 (HIV-1), they are unable to block replication of wild-type HIV-1 due to the action of Vif, which induces their degradation. In contrast, HIV-1 Vif is unable to block inhibition mediated by APOBEC3 proteins expressed by several heterologous species, including mice. Here, we have asked whether the simple retrovirus murine leukemia virus (MLV) is sensitive to restriction by the cognate murine or heterologous, human APOBEC3 proteins. We demonstrate that MLV is highly sensitive to inhibition by human APOBEC3G and APOBEC3B but resistant to inhibition by murine APOBEC3 or by other human APOBEC3 proteins, including APOBEC3F. This sensitivity fully correlates with the ability of these proteins to be packaged into MLV virion particles: i.e., human APOBEC3G and APOBEC3B are packaged while murine APOBEC3 and human APOBEC3F are excluded. Moreover, this packaging in turn correlates with the differential ability of these APOBEC3 proteins to bind MLV Gag. Together, these data suggest that MLV Gag has evolved to avoid binding, and hence virion packaging, of the cognate murine APOBEC3 protein but that MLV infectivity is still restricted by certain heterologous APOBEC3 proteins that retain this ability. Moreover, these results suggest that APOBEC3 proteins may help prevent the zoonotic infection of humans by simple retroviruses and provide a mechanism for how simple retroviruses can avoid inhibition by APOBEC3 family members

Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m5C) and 2′O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m5C on HIV-1 RNAs. NSUN2 inactivation inhibits not only m5C addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of m5C dysregulates the alternative splicing of viral RNAs. These data identify m5C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression. Courtney et al. report that HIV-1 transcripts are modified by the addition of 5-methylcytosine (m5C) residues. The nuclear methyltransferase NSUN2 is the primary m5C writer and is required for appropriate HIV-1 translation. NSUN2 deficiency and concomitant loss of m5C residues inhibits ribosomal recruitment to and alternative splicing of HIV-1 mRNAs. © 2019 Elsevier Inc

CRM1/Ran-Mediated Nuclear Export of p27(Kip1) Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase

Nup98 is a mobile nucleoporin with transcription-dependent dynamics

Nucleoporin 98 (Nup98), a glycine-leucine-phenylalanine-glycine (GLFG) amino acid repeat-containing nucleoporin, plays a critical part in nuclear trafficking. Injection of antibodies to Nup98 into the nucleus blocks the export of most RNAs. Nup98 contains binding sites for several transport factors; however, the mechanism by which this nucleoporin functions has remained unclear. Multiple subcellular localizations have been suggested for Nup98. Here we show that Nup98 is indeed found both at the nuclear pore complex and within the nucleus. Inside the nucleus, Nup98 associates with a novel nuclear structure that we term the GLFG body because the GLFG domain of Nup98 is required for targeting to this structure. Photobleaching of green fluorescent protein-Nup98 in living cells reveals that Nup98 is mobile and moves between these different localizations. The rate of recovery after photobleaching indicates that Nup98 interacts with other, less mobile, components in the nucleoplasm. Strikingly, given the previous link to nuclear export, the mobility of Nup98 within the nucleus and at the pore is dependent on ongoing transcription by RNA polymerases I and II. These data give rise to a model in which Nup98 aids in direction of RNAs to the nuclear pore and provide the first potential mechanism for the role of a mobile nucleoporin