To probe the cellular phenotype and biochemical function associated with the G domains of Escherichia coli EngA (YfgK, Der), mutations were created in the phosphate binding loop of each. Neither an S16A nor an S217A variant of G domain 1 or 2, respectively, was able to support growth of an engA conditional null. Polysome profiles of EngA-depleted cells were significantly altered, and His(6)-EngA was found to cofractionate with the 50S ribosomal subunit. The variants were unable to complement the abnormal polysome profile and were furthermore significantly impacted with respect to in vitro GTPase activity. Together, these observations suggest that the G domains have a cooperative function in ribosome stability and/or biogenesis.
Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16 degrees C and 37 degrees C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.
The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5′-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.
Understanding the life cycle and pathogenesis of animal viruses requires that we have systems in which the viruses can replicate and cause disease. For the latter, we rely upon animal models or information that we can obtain from studying natural infections of humans and other animals. For the former, however, we are largely dependent on the availability of cell culture systems in which viruses can be propagated to investigate the molecular mechanisms of viral replication. For many years, it was assumed that replication in culture provided an accurate description of the life cycle of the organism. In this Gem, we will discuss two viruses, polyomavirus and cytomegalovirus, in which cell culture systems have accidentally provided unique potential insights into viral replication and persistence in their hosts.
Abstract Food turns out to be not only the nutrient supplier for our body but also a carrier of regulatory information. Interestingly, a recent study made the discovery that some plant/food‐derived microRNAs (miRNAs) accumulate in the serum of humans or plant‐feeding animals, and regulate mammalian gene expression in a sequence‐specific manner. The authors provided striking evidence that miRNAs could function as active signaling molecules to transport information across distinct species or even kingdoms. Although the mechanism of how miRNAs are shuttled between different organisms is still not well characterized, initial results point to the involvement of microvesicles and specific RNA‐transporter‐like proteins. These findings raise both speculation about the potential impact that plants may have on animal physiology at the molecular level, and an appealing possibility that food‐derived miRNAs may offer us another means to deliver necessary nutrients or therapeutics to our bodies.
ABSTRACT APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomaviruses and papillomaviruses use dominant oncoproteins to induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T-antigen. First, truncated T-antigen (truncT) is sufficient for APOBEC3B upregulation and the RB interacting motif (LXCXE), but not the TP53 inhibition domain, is required. Second, upregulated APOBEC3B is strongly nuclear and partially localized to virus replication centers. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B . Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B . Fifth, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB binding motif has in addition to RB inactivation at least one additional function for triggering APOBEC3B upregulation in virus-infected cells. IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpresssed in many different cancers and correlated with elevated frequencies of C-to-T and C-to-G mutations in 5’-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T-antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely enables one or more E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to non-viral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.
BK polyomavirus (PyV) infects the genitourinary tract of >90% of the adult population. Immunosuppression increases the risk of viral reactivation, making BKPyV a leading cause of graft failure in kidney transplant recipients. Polyomaviruses have a small double-stranded DNA (dsDNA) genome that requires host replication machinery to amplify the viral genome. Specifically, polyomaviruses promote S phase entry and delay S phase exit by activating the DNA damage response (DDR) pathway via an uncharacterized mechanism requiring viral replication. BKPyV infection elevates expression of MutSα, a mismatch repair (MMR) pathway protein complex that senses and repairs DNA mismatches and can activate the DDR. Thus, we investigated the role of the MMR pathway by silencing the MutSα component, Msh6, in BKPyV-infected primary cells. This resulted in severe DNA damage that correlated with weak DNA damage response activation and a failure to arrest the cell cycle to prevent mitotic entry during infection. Furthermore, silencing Msh6 expression resulted in significantly fewer infectious viral particles due to significantly lower levels of VP2, a minor capsid protein important for trafficking during subsequent infections. Since viral assembly occurs in the nucleus, our findings are consistent with a model in which entry into mitosis disrupts viral assembly due to nuclear envelope breakdown, which disperses VP2 throughout the cell, reducing its availability for encapsidation into viral particles. Thus, the MMR pathway may be required to activate the ATR (ATM-Rad3-related) pathway during infection to maintain a favorable environment for both viral replication and assembly.
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