Loss of the Yeast SR Protein Npl3 Alters Gene Expression Due to Transcription Readthrough.

Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression.

The innate antiviral factor APOBEC3G targets replication of measles, mumps and respiratory syncytial viruses.

The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adeno-associated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1-2 logs (90-99 %) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50-70 %, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (C→U) or guanine to adenine (G→A) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, A→G and U→C transitions, with preference for next neighbour-nucleotides U = A>C>G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells.

APOBEC-mediated viral restriction: not simply editing?

The APOBEC family of cytidine deaminases inhibit the mobility of diverse retroviruses, retrotransposons and other viruses. Initial reports proposed that these effects were due to the DNA editing capabilities of these enzymes; however, many recent studies have provided evidence suggesting that APOBEC proteins can inhibit these elements by several mechanisms, including editing-dependent and editing-independent processes. Investigating these modes of action and the potential contribution that each one makes to the antiviral activities of various APOBEC proteins is vital if we are to understand how APOBEC proteins protect host genomes from invading nucleic acids.

Comparison of cellular ribonucleoprotein complexes associated with the APOBEC3F and APOBEC3G antiviral proteins.

The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3F (APOBEC3F [A3F]) and A3G proteins are effective inhibitors of infection by various retroelements and share approximately 50% amino acid sequence identity. We therefore undertook comparative analyses of the protein and RNA compositions of A3F- and A3G-associated ribonucleoprotein complexes (RNPs). Like A3G, A3F is found associated with a complex array of cytoplasmic RNPs and can accumulate in RNA-rich cytoplasmic microdomains known as mRNA processing bodies or stress granules. While A3F RNPs display greater resistance to disruption by RNase digestion, the major protein difference is the absence of the Ro60 and La autoantigens. Consistent with this, A3F RNPs also lack a number of small polymerase III RNAs, including the RoRNP-associated Y RNAs, as well as 7SL RNA. Alu RNA is, however, present in A3F and A3G RNPs, and both proteins suppress Alu element retrotransposition. Thus, we define a number of subtle differences between the RNPs associated with A3F and A3G and speculate that these contribute to functional differences that have been described for these proteins.

Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity.

The antiretroviral activity of the cellular enzyme APOBEC3G has been attributed to the excessive deamination of cytidine (C) to uridine (U) in minus strand reverse transcripts, a process resulting in guanosine (G) to adenosine (A) hypermutation of plus strand DNAs. The HIV-1 Vif protein counteracts APOBEC3G by inducing proteasomal degradation and exclusion from virions through recruitment of a cullin5 ECS E3 ubiquitin ligase complex. APOBEC3G belongs to the APOBEC protein family, members of which possess consensus (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C cytidine deaminase motifs. Earlier analyses of APOBEC-1 have defined specific residues that are important for zinc coordination, proton transfer, and, therefore, catalysis within this motif. Because APOBEC3G contains two such motifs, we used site-directed mutagenesis of conserved residues to assess each region's contribution to anti-HIV-1 activity. Surprisingly, whereas either the N- or C-terminal domain could confer antiviral function in tissue culture-based infectivity assays, only an intact C-terminal motif was essential for DNA mutator activity. These findings reveal the nonequivalency of APOBEC3G's N- and C-terminal domains and imply that APOBEC3G-mediated DNA editing may not always be necessary for antiviral activity. Accordingly, we propose that APOBEC3G can achieve an anti-HIV-1 effect through an undescribed mechanism that is distinct from cytidine deamination.

Cytidine deamination of retroviral DNA by diverse APOBEC proteins.

The human cytidine deaminase APOBEC3G edits both nascent human immunodeficiency virus (HIV) and murine leukemia virus (MLV) reverse transcripts, resulting in loss of infectivity. The HIV Vif protein is able to protect both viruses from this innate restriction to infection. Here, we demonstrate that a number of other APOBEC family members from both humans and rodents can mediate anti-HIV effects, through cytidine deamination. Three of these, rat APOBEC1, mouse APOBEC3, and human APOBEC3B, are able to inhibit HIV infectivity even in the presence of Vif. Like APOBEC3G, human APOBEC3F preferentially restricts vif-deficient virus. Indeed, the mutation spectra and expression profile found for APOBEC3F indicate that this enzyme, together with APOBEC3G, accounts for the G to A hypermutation of proviruses described in HIV-infected individuals. Surprisingly, although MLV infectivity is acutely reduced by APOBEC3G, no other family member tested here had this effect. It is especially interesting that although both rodent APOBECs markedly diminish wild-type HIV infectivity, MLV is resistant to these proteins. This implies that MLV may have evolved to avoid deamination by mouse APOBEC3. Overall, our findings show that although APOBEC family members are highly related, they exhibit significantly distinct antiviral characteristics that may provide new insights into host-pathogen interactions.

APOBEC3F can inhibit the accumulation of HIV-1 reverse transcription products in the absence of hypermutation. Comparisons with APOBEC3G.

APOBEC3F (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like protein 3F) is a cytidine deaminase that, like APOBEC3G, is able to restrict the replication of HIV-1/delta vif. Initial studies revealed high numbers of mutations in the cDNA of viruses produced in the presence of these proteins, suggesting that cytidine deamination underpinned the inhibition of infection. However, we have recently shown that catalytically inactive APOBEC3G proteins, derived through mutation of the C-terminal cytidine deaminase motif, still exert a substantial antiviral effect. Here, we have generated a panel of APOBEC3F mutant proteins and show that the C-terminal cytidine deaminase motif is essential for catalytic activity and that catalytic activity is not necessary for the antiviral effect of APOBEC3F. Furthermore, we demonstrate that the antiviral activities of wild-type and catalytically inactive APOBEC3F and APOBEC3G proteins correspond well with reductions in the accumulation of viral reverse transcription products. Additional comparisons between APOBEC3F and APOBEC3G suggest that the loss of deaminase activity is more detrimental to APOBEC3G function than to APOBEC3F function, as reflected by perturbations to the suppression of reverse transcript accumulation as well as antiviral activity. Taken together, these data suggest that both APOBEC3F and APOBEC3G are able to function as antiviral factors in the absence of cytidine deamination, that this editing-independent activity is an important aspect of APOBEC protein-mediated antiviral phenotypes, but that APOBEC3F may be a better model in which to study it.

Antiviral potency of APOBEC proteins does not correlate with cytidine deamination.

The human cytidine deaminases APOBEC3G (hA3G) and APOBEC3F (hA3F) are intracellular antiretroviral factors that can hypermutate nascent reverse transcripts and inhibit the replication of human immunodeficiency virus type 1 (HIV-1). Both enzymes have two cytidine deaminase motifs, although only the C-terminal motif is catalytic. Current models of APOBEC protein function imply editing is the principal mechanism of antiviral activity. In particular, hA3G is a more potent inhibitor of HIV-1 infectivity than hA3F and also induces a greater frequency of mutations in HIV-1 cDNA. We used hA3G/hA3F chimeric proteins to investigate whether cytidine deaminase potential reflects antiviral potency. We show here that the origin of the C-terminal deaminase motif is sufficient to determine the degree of mutation induced in a bacterial assay that measures mutations in chromosomal DNA. In contrast, this was not the case in the context of HIV-1 infection where the N-terminal deaminase motif also modulated the editing capabilities of the chimeras. Surprisingly, although three of the chimeric proteins induced levels of mutation that approximated those of parental hA3F, they displayed lower levels of antiviral activity. Most importantly, real-time PCR experiments revealed that the quantity of reverse transcripts detected in target cells, rather than the mutational burden carried by such DNAs, corresponded closely with viral infectivity. In other words, the antiviral phenotype of APOBEC proteins correlates with their ability to prevent the accumulation of reverse transcripts and not with the induction of hypermutation.

APOBEC-mediated editing of viral RNA.

Retroviral DNA can be subjected to cytosine-to-uracil editing through the action of members of the APOBEC family of cytidine deaminases. Here we demonstrate that APOBEC-mediated cytidine deamination of human immunodeficiency virus (HIV) virion RNA can also occur. We speculate that the natural substrates of the APOBEC enzymes may extend to RNA viruses that do not replicate through DNA intermediates. Thus, cytosine-to-uracil editing may contribute to the sequence diversification of many viruses.