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

Foamy Viruses, Bet, and APOBEC3 Restriction

Non-human primates (NHP) are an important source of viruses that can spillover to humans and, after adaptation, spread through the host population. Whereas HIV-1 and HTLV-1 emerged as retroviral pathogens in humans, a unique class of retroviruses called foamy viruses (FV) with zoonotic potential are occasionally detected in bushmeat hunters or zookeepers. Various FVs are endemic in numerous mammalian natural hosts, such as primates, felines, bovines, and equines, and other animals, but not in humans. They are apathogenic, and significant differences exist between the viral life cycles of FV and other retroviruses. Importantly, FVs replicate in the presence of many well-defined retroviral restriction factors such as TRIM5α, BST2 (Tetherin), MX2, and APOBEC3 (A3). While the interaction of A3s with HIV-1 is well studied, the escape mechanisms of FVs from restriction by A3 is much less explored. Here we review the current knowledge of FV biology, host restriction factors, and FV–host interactions with an emphasis on the consequences of FV regulatory protein Bet binding to A3s and outline crucial open questions for future studies

Encapsidation of Staufen-2 Enhances Infectivity of HIV-1

Staufen, the RNA-binding family of proteins, affects various steps in the Human Immuno-Deficiency Virus (HIV-1) replication cycle. While our previous study established Staufen-2–HIV-1 Rev interaction and its role in augmenting nucleocytoplasmic export of RRE-containing viral RNA, viral incorporation of Staufen-2 and its effect on viral propagation were unknown. Here, we report that Staufen-2 interacts with HIV-1 Gag and is incorporated into virions and that encapsidated Staufen-2 boosted viral infectivity. Further, Staufen-2 gets co-packaged into virions, possibly by interacting with host factors Staufen-1 or antiviral protein APOBEC3G, which resulted in different outcomes on the infectivity of Staufen-2-encapsidated virions. These observations suggest that encapsidated host factors influence viral population dynamics and infectivity. With the explicit identification of the incorporation of Staufen proteins into HIV-1 and other retroviruses, such as Simian Immunodeficiency Virus (SIV), we propose that packaging of RNA binding proteins, such as Staufen, in budding virions of retroviruses is probably a general phenomenon that can drive or impact the viral population dynamics, infectivity, and evolution

Enhancing the Catalytic Deamination Activity of APOBEC3C Is Insufficient to Inhibit Vif-Deficient HIV-1

The retroviral restriction factors of the APOBEC3 (A3) cytidine deaminase family catalyze the deamination of cytidines in single-stranded viral DNA. APOBEC3C (A3C) is a strong antiviral factor against viral infectivity factor (vif)-deficient simian immunodeficiency virus Δvif, which is, however, a weak inhibitor against human immunodeficiency virus (HIV)-1 for reasons unknown. The precise link between the antiretroviral effect of A3C and its catalytic activity is incompletely understood. Here, we show that the S61P mutation in human A3C (A3C.S61P) boosted hypermutation in the viral genomes of simian immunodeficiency virus Δvif and murine leukemia virus but not in human immunodeficiency virus HIV-1Δvif. The enhanced antiviral activity of A3C.S61P correlated with enhanced in vitro cytidine deamination. Furthermore, the S61P mutation did not change the substrate specificity of A3C, ribonucleoprotein complex formation, self-association, Zinc coordination, or viral incorporation features. We propose that local structural changes induced by the serine-to-proline substitution are responsible for the gain of catalytic activity of A3C.S61P. Our results are a first step toward an understanding of A3C's DNA binding capacity, deamination-dependent editing, and antiviral functions at the molecular level. We conclude that the enhanced enzymatic activity of A3C is insufficient to restrict HIV-1, indicating an unknown escape mechanism of HIV-1

Structural features of antiviral DNA cytidine deaminases

The APOBEC3 (A3) family of cytidine deaminases plays a vital role for innate defense against retroviruses. Lentiviruses such as HIV-1 evolved the Vif protein that triggers A3 protein degradation. There are seven A3 proteins, A3A-A3H, found in humans. All A3 proteins can deaminate cytidines to uridines in single-stranded DNA (ssDNA), generated during viral reverse transcription. A3 proteins have either one or two cytidine deaminase domains (CD). The CDs coordinate a zinc ion, and their amino acid specificity classifies the A3s into A3Z1, A3Z2, and A3Z3. A3 proteins occur as monomers, dimers, and large oligomeric complexes. Studies on the nature of A3 oligomerization, as well as the mode of interaction of A3s with RNA and ssDNA are partially controversial. High-resolution structures of the catalytic CD2 of A3G and A3F as well as of the single CD proteins A3A and A3C have been published recently. The NMR and X-ray crystal structures show globular proteins with six α-helices and five β sheets arranged in a characteristic motif (α1-β1-β2/2'-α2-β3-α3-β4-α4-β5-α5-α6). However, the detailed arrangement and extension of individual structure elements and their relevance for A3 complex formation and activity remains a matter of debate and will be highlighted in this review

Prototype Foamy Virus Bet Impairs the Dimerization and Cytosolic Solubility of Human APOBEC3G

Cellular cytidine deaminases from the APOBEC3 family are potent restriction factors able to block the replication of retroviruses. Consequently, retroviruses have evolved a variety of different mechanisms to counteract inhibition by APOBEC3 proteins. Lentiviruses such as Human immunodeficiency virus (HIV) express Vif that interferes with APOBEC3 proteins by targeting the restriction factors for proteasomal degradation, hence blocking their ability to access the reverse transcriptase complex in the virions. Other retroviruses use less well characterized mechanisms to escape APOBEC3s' mediated cellular defence. Here we show that Prototype foamy virus Bet can protect foamy viruses and an unrelated simian immunodeficiency virus against human APOBEC3G (A3G). In our system, Bet binds to A3G and prevents its encapsidation without inducing its degradation. Bet failed to co-immunoprecipitate with A3G mutants unable to form homodimers, and dramatically reduced the recovery of A3G proteins from soluble cytoplasmic cell fractions. The Bet - A3G interaction is probably a direct binding and seems to be independent of RNA. Together, this data suggest a novel model whereby Bet uses two possibly complementary mechanisms to counteract A3G: (1) Bet prevents encapsidation of A3G by blocking A3G dimerization, and (2) sequesters A3G in immobile complexes, impairing its ability to interact with nascent virions

Tough Way In, Tough Way Out: The Complex Interplay of Host and Viral Factors in Nucleocytoplasmic Trafficking during HIV-1 Infection

Human immunodeficiency virus-1 (HIV-1) is a retrovirus that integrates its reverse-transcribed genome as proviral DNA into the host genome to establish a successful infection. The viral genome integration requires safeguarding the subviral complexes, reverse transcription complex (RTC) and preintegration complex (PIC), in the cytosol from degradation, presumably effectively secured by the capsid surrounding these complexes. An intact capsid, however, is a large structure, which raises concerns about its translocation from cytoplasm to nucleus crossing the nuclear membrane, guarded by complex nuclear pore structures, which do not allow non-specific transport of large molecules. In addition, the generation of new virions requires the export of incompletely processed viral RNA from the nucleus to the cytoplasm, an event conventionally not permitted through mammalian nuclear membranes. HIV-1 has evolved multiple mechanisms involving redundant host pathways by liaison with the cell’s nucleocytoplasmic trafficking system, failure of which would lead to the collapse of the infection cycle. This review aims to assemble the current developments in temporal and spatial events governing nucleocytoplasmic transport of HIV-1 factors. Discoveries are anticipated to serve as the foundation for devising host-directed therapies involving selective abolishment of the critical interactomes between viral proteins and their host equivalents

APOBEC4 Enhances the Replication of HIV-1

APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral LTR. A4 did not show detectable cytidine deamination activity in vitro and weakly interacted with single-stranded DNA. The presence of A4 in virus producer cells enhanced HIV-1 replication by transiently transfected A4 or stably expressed A4 in HIV-susceptible cells. APOBEC4 was capable of similarly enhancing transcription from a broad spectrum of promoters, regardless of whether they were viral or mammalian. We hypothesize that A4 may have a natural role in modulating host promoters or endogenous LTR promoters