An intronic G run within HIV-1 intron 2 is critical for splicing regulation of vif mRNA.

Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1.

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.

Murine leukemia virus resists producer cell APOBEC3A by its Glycosylated Gag but not target cell APOBEC3A.

The human APOBEC3A (A3A) polynucleotide cytidine deaminase has been shown to have antiviral activity against HTLV-1 but not HIV-1, when expressed in the virus producer cell. In viral target cells, high levels of endogenous A3A activity have been associated with the restriction of HIV-1 during infection. Here we demonstrate that A3A derived from both target cells and producer cells can block the infection of Moloney-MLV (MLV) and related AKV-derived strains of MLV in a deaminase-dependent mode. Furthermore, glycosylated Gag (glycoGag) of MLV inhibits the encapsidation of human A3A, but target cell A3A was not affected by glycoGag and exerted deamination of viral DNA. Importantly, our results clearly indicate that poor glycoGag expression in MLV gag-pol packaging constructs as compared to abundant levels in full-length amphotropic MLV makes these viral vectors sensitive to A3A-mediated restriction. This raises the possibility of acquiring A3A-induced mutations in retroviral gene therapy applications.

APOBEC3B Activity Is Prevalent in Urothelial Carcinoma Cells and Only Slightly Affected by LINE-1 Expression.

The most common mutational signature in urothelial carcinoma (UC), the most common type of urinary bladder cancer is assumed to be caused by the misdirected activity of APOBEC3 (A3) cytidine deaminases, especially A3A or A3B, which are known to normally restrict the propagation of exogenous viruses and endogenous retroelements such as LINE-1 (L1). The involvement of A3 proteins in urothelial carcinogenesis is unexpected because, to date, UC is thought to be caused by chemical carcinogens rather than viral activity. Therefore, we explored the relationship between A3 expression and L1 activity, which is generally upregulated in UC. We found that UC cell lines highly express A3B and in some cases A3G, but not A3A, and exhibit corresponding cytidine deamination activity in vitro. While we observed evidence suggesting that L1 expression has a weak positive effect on A3B and A3G expression and A3B promoter activity, neither efficient siRNA-mediated knockdown nor overexpression of functional L1 elements affected catalytic activity of A3 proteins consistently. However, L1 knockdown diminished proliferation of a UC cell line exhibiting robust endogenous L1 expression, but had little impact on a cell line with low L1 expression levels. Our results indicate that UC cells express A3B at levels exceeding A3A levels by far, making A3B the prime candidate for causing genomic mutations. Our data provide evidence that L1 activation constitutes only a minor and negligible factor involved in induction or upregulation of endogenous A3 expression in UC.