Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency

Mouse apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 (mA3), an intracellular antiviral factor, has 2 allelic variations that are linked with different susceptibilities to beta- and gammaretrovirus infections among various mouse strains. In virus-resistant C57BL/6 (B6) mice, mA3 transcripts are more abundant than those in susceptible BALB/c mice both in the spleen and bone marrow. These strains of mice also express mA3 transcripts with different splicing patterns: B6 mice preferentially express exon 5-deficient (Δ5) mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the Δ5 mRNA exerts stronger antiretroviral activities than the full-length protein, how exon 5 affects mA3 antiviral activity, as well as the genetic mechanisms regulating exon 5 inclusion into the mA3 transcripts, remains largely uncharacterized. Here we show that mA3 exon 5 is indeed a functional element that influences protein synthesis at a post-transcriptional level. We further employed in vitro splicing assays using genomic DNA clones to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and the single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary. Distribution of the above polymorphisms among different Mus species indicates that the inclusion of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of this exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function

Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses

Deaminase-Independent Mode of Antiretroviral Action in Human and Mouse APOBEC3 Proteins

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) proteins (APOBEC3s) are deaminases that convert cytosines to uracils predominantly on a single-stranded DNA, and function as intrinsic restriction factors in the innate immune system to suppress replication of viruses (including retroviruses) and movement of retrotransposons. Enzymatic activity is supposed to be essential for the APOBEC3 antiviral function. However, it is not the only way that APOBEC3s exert their biological function. Since the discovery of human APOBEC3G as a restriction factor for HIV-1, the deaminase-independent mode of action has been observed. At present, it is apparent that both the deaminase-dependent and -independent pathways are tightly involved not only in combating viruses but also in human tumorigenesis. Although the deaminase-dependent pathway has been extensively characterized so far, understanding of the deaminase-independent pathway remains immature. Here, we review existing knowledge regarding the deaminase-independent antiretroviral functions of APOBEC3s and their molecular mechanisms. We also discuss the possible unidentified molecular mechanism for the deaminase-independent antiretroviral function mediated by mouse APOBEC3

Nuclear and cytoplasmic effects of human CRM1 on HIV-1 production in rat cells

The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced gag and singly spliced env mRNAs by bridging viral RNA and the export receptor, CRM1. Recently, rat CRM1 was found to be less efficient than human CRM1 in supporting Rev function in rats. In this study, to understand the role of CRM1 in HIV propagation, the mechanism underlying the function of human and rat CRM1 in HIV-1 replication was investigated in rat cells. The production of viral particles, represented by the p24 Gag protein, was greatly enhanced by hCRM1 expression in rat cells; however, this effect was not simply due to the enhanced export of gag mRNA. The translation initiation rate of gag mRNA was not increased, nor was the Gag protein stabilized in the presence of hCRM1. However, the processing of the p55 Gag precursor and the release of viral particles were facilitated. These results indicated that hCRM1 exports gag mRNA to the cytoplasm, not only more efficiently than rCRM1 but also correctly, leading to efficient processing of Gag proteins and particle formation

The Carboxy-Terminal Region of the Human Immunodeficiency Virus Type 1 Protein Rev Has Multiple Roles in Mediating CRM1-Related Rev Functions

The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced and singly spliced viral mRNAs by bridging viral RNA and export receptor human CRM1 (hCRM1). Ribonucleoprotein complex formation, including the oligomerization of Rev proteins on viral RNA, must occur to allow export. We show here that Rev-Rev interactions, which are a basis of complex formation, can be initiated without cellular factors and are subsequently enhanced by hCRM1-Ran-GTP. Furthermore, we reveal functions for the Rev carboxy-terminal (C-terminal) region, which is well conserved among many HIV-1 strains, and for which no function has been reported. This region is required for the efficient binding of Rev to hCRM1 and consequently for nuclear export, Rev-Rev dimerization, and full Rev transactivator activity. Consistent with these results, a HIV-1 proviral plasmid that expresses a C-terminally truncated Rev mutant protein produces smaller amounts of the p24 antigen than does a plasmid that possesses an intact rev gene. These results indicate the functional importance of the C-terminal region for full Rev activity, which leads to efficient HIV-1 replication

Mouse APOBEC3 interferes with autocatalytic cleavage of murine leukemia virus Pr180gag-pol precursor and inhibits Pr65gag processing.

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3

Adjusting Heterodimeric Coiled-Coils (K/E Zipper) to Connect Autophagy-Inducing Peptide with Cell-Penetrating Peptide

A connection of a functional peptide with a cell-penetrating peptide (CPP) used a heterodimeric coiled-coil as a molecular zipper can improve the intracellular delivery and activity of the functional peptide. However, the chain length of the coiled coil required for functioning as the molecular zipper is unknown at present. To solve the problem, we prepared an autophagy-inducing peptide (AIP) that conjugates with the CPP via heterodimeric coiled-coils consisting of 1 to 4 repeating units (K/E zipper; AIP-Kn and En-CPP), and we investigated the optimum length of the K/E zipper for effective intracellular delivery and autophagy induction. Fluorescence spectroscopy showed that K/E zippers with n = 3 and 4 formed a stable 1:1 hybrid (AIP-K3/E3-CPP and AIP-K4/E4-CPP, respectively). Both AIP-K3 and AIP-K4 were successfully delivered into cells by the corresponding hybrid formation with K3-CPP and K4-CPP, respectively. Interestingly, autophagy was also induced by the K/E zippers with n = 3 and 4, more intensively by the former than by the latter. The peptides and K/E zippers used in this study did not show significant cytotoxicity. These results indicate that the effective induction of autophagy occurs via an exquisite balance of the association and dissociation of the K/E zipper in this system