Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism.

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. Polyomavirus and papillomavirus oncoproteins 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, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. 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 Last, 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 at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells.IMPORTANCE 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.

The Diverse Roles of microRNAs at the Host⁻Virus Interface.

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. Through this activity, they are implicated in almost every cellular process investigated to date. Hence, it is not surprising that miRNAs play diverse roles in regulation of viral infections and antiviral responses. Diverse families of DNA and RNA viruses have been shown to take advantage of cellular miRNAs or produce virally encoded miRNAs that alter host or viral gene expression. MiRNA-mediated changes in gene expression have been demonstrated to modulate viral replication, antiviral immune responses, viral latency, and pathogenesis. Interestingly, viruses mediate both canonical and non-canonical interactions with miRNAs to downregulate specific targets or to promote viral genome stability, translation, and/or RNA accumulation. In this review, we focus on recent findings elucidating several key mechanisms employed by diverse virus families, with a focus on miRNAs at the host⁻virus interface during herpesvirus, polyomavirus, retroviruses, pestivirus, and hepacivirus infections.

Particulate matter exposure increases JC polyomavirus replication in the human host.

BACKGROUND: Human polyomaviruses (HPyVs) asymptomatically infect the human population during childhood and establish latency in the host. Viral reactivation and urinary excretion can occur when the immune system is impaired. Exposure to particulate air pollution, including the PM10/PM2.5 components, is a public health problem and has been linked to several disorders. Studies assessing the relationship between PM10/PM2.5 exposure and viral replication are lacking. OBJECTIVES: To investigate the relationship between HPyVs viruria and PM10/PM2.5 exposures. METHODS: Individual environmental exposure was assessed in 50 healthy adult volunteers using a chemical transport model (CTM) with a municipality resolution for daily PM10 and monitoring stations data for daily PM2.5 exposures. For each subject, a urine sample was collected, and HPyVs (JCPyV, BKPyV, MCPyV, HPyV6, HPyV7 and HPyV9) loads were determined. Zero-inflated negative binomial (ZINB) regression was used to model the count data, as it contained excessive zeros. Covariates were chosen by stepwise selection. RESULTS: HPyVs DNA was detected in 54% (median:87.6*105 copies/ml) of the urine samples. JCPyV was the prevalent (48%, (median viral load:126*105 copies/ml). Considering the load of the most frequently measured HPyVs, JCPyV, in the count-part of the ZINB model, every unitary in PM measured 2 days before urine collection (PM Day -2) was associated with an increase in JCPyV load (PM10: +4.0%, p-value = 0.002; PM2.5: +3.6%, p-value = 0.005). In the zero-part, the significant predictor was the PM10 measured 5 days before urine collection (+3%, p-value = 0.03). CONCLUSIONS: The environmental levels of PM10/PM2.5 increase the JCPyV viruria. Our findings emphasize the need for studies assessing the influence of air pollution exposure on the risk of viral reactivation.

Assessment of the Virological Quality of Marine and Running Surface Waters in NW Greece: A Case Study.

The virological quality of surface marine and running water samples collected from Igoumenitsa gulf and Kalamas river (NW Greece) was assessed from October 2012 to September 2013. Sampling sites were exposed to different land and/or anthropogenic effects. Seawater samples were collected monthly from five sampling stations (new harbor, old harbor, wastewater treatment plant outlet, protected Natura area, Drepano beach). Viral targets included human adenoviruses (hAdVs), as index human viruses, while noroviruses (NoVs) and hepatitis A virus (HAV) were also studied. Kalamas river samples were collected seasonally, from three sampling stations (Soulopoulo, Dam, Sagiada-estuaries), while viral targets included also porcine adenoviruses (pAdVs) and bovine polyoma viruses (bPyVs), as additional index viruses. All water samples were analyzed for standard bacterial indicators, as well. Physicochemical and meteorological data were also collected. Based on the standard bacterial indices, both sea and river water samples did not exceed the limits set according to Directive 2006/7/EU. However, positive samples for hAdVs were found occasionally in all sampling sites in Igoumenitsa gulf (23.3%, 14/60) showing fecal contamination of human origin. Moreover, HAV was detected once, in the sampling site of the old port (at 510 GC/L). Most of the Kalamas water samples were found positive for hAdVs (58.3%, 7/12), while human noroviruses GI (NoVGI) (8.3%, 1/12) and GII (NoVGII) (16.7%, 2/12) were also detected. HAV, pAdVs, and bovine polyomaviruses (bPyVs) were not detected in any of the analyzed samples. No statistically significant correlations were found between classic bacterial indicators and viral targets, nor between viruses and meteorological data. Overall, the present study contributed to the collection of useful data for the biomonitoring of the region, and the assessment of the overall impact of anthropogenic activities. It provided also valuable information for the evaluation of the risk of waterborne viral infections and the protection of public health. It was the first virological study in the area and one of the few in Greece.

Identification and genetic characterization of polyomaviruses in estrildid and fringillid finches.

Polyomavirus infections were detected in 40 companion bird individuals belonging to a broad species range of estrildid and fringillid finches and originating from 21 different bird aviaries. Based on partial virus protein 1 (VP1) sequences, the viruses were identified as Serinus canaria polyomavirus 1 and Pyrrhula pyrrhula polyomavirus 1. Serinus canaria polyomavirus 1 was found in 18 birds belonging to one estrildid and four fringillid species. Pyrrhula pyrrhula polyomavirus 1 was detected in 22 birds of six estrildid and three fringillid species. There was a large overlap in host range. Increased mortality was frequently found in the affected bird aviaries while clinical signs were diverse. Co-infections with other viruses, bacteria or fungal pathogens were common and might have influenced the clinical signs. Sequence analyses, including partial VP1 sequences of the 40 virus strains, and full genome sequences of selected strains revealed a high genetic heterogeneity among virus subgroups of Serinus canaria polyomavirus 1 and Pyrrhula pyrrhula polyomavirus 1, indicating the existence of two virus variants for both virus species. For Pyrrhula pyrrhula polyomavirus 1, two genotypes were found that associated with the family of the finches, Estrildidae or Fringillidae.

Oncogenic papillomavirus and polyomavirus in urban sewage in Egypt.

Recently, the occurrence of oncogenic viruses in contaminated water and their potential for waterborne transmission has been reported. We addressed an environmental surveillance of both HPyVs (JCPyV and BKPyV) and HPVs in three wastewater treatment plants in Egypt. A high level of dissemination was found for both viruses. HPyVs (JCPyV and BKPyV) were found in ~73% of examined samples, while HPVs were detected in 30.5%. Sequence analysis of HPV positive samples revealed a wide variety of circulating genotypes representing both anogenital (HPV-6, HPV-16, HPV-53, HPV-44, HPV-31, HPV-43) and cutaneous (HPV-37, HPV-21, HPV-120, HPV-111, HPV-5) types. In addition, two unclassified sequences were identified, suggesting putative types. The median concentrations of HPyVs in inflow samples were 3.03×1005, 3.9×1005, and 1.44×1005GC/l in the three WWTPs, respectively. Whereas, the viral concentration in outflow reduced by one order of magnitude in WWTP-A and WWTP-C and two orders of magnitude in WWTP-B. On the other hand, the mean concentration of the quantified HPVs positive samples was 1.68×1003GC/l for inflow and a quite similar pattern in the outflow as well. These data provide an evidence about the actual circulation pattern of both viruses in the population. Also, the high abundance of HPyVs supports its potential as a possible fecal indicator. However, further investigations are required for both viruses to elucidate the potential health risk via contaminated water.

Polyomavirus Replication and Smoking Are Independent Risk Factors for Bladder Cancer After Renal Transplantation.

BACKGROUND: Solid organ transplant recipients are at increased risk for developing malignancies. Polyomaviruses (PV) have been historically associated with experimental tumor development and recently described in association with renourinary malignancies in transplant patients. The aim of this study was to investigate the relationship between PV replication and smoking, and the development of malignant neoplasms in kidney transplant recipients. METHODS: A retrospective case-control study was conducted for PV replication in all kidney biopsies and urine cytologies performed between 1998 and 2014 from kidney transplant recipients at the University of Maryland Medical Center. Polyomavirus-positive patients (n = 943) were defined as having any of the following: a kidney biopsy with PV associated nephropathy, any urine cytology demonstrating "decoy" cells, and/or significant polyomavirus BK viremia. Polyomavirus-negative matched patients (n = 943) were defined as lacking any evidence of PV replication. The incidence of malignancy (excluding nonmelanoma skin tumors) was determined in these 1886 patients and correlated with demographic data and history of smoking. RESULTS: There was a 7.9% incidence of malignant tumors after a mean posttransplant follow-up of 7.9 ± 5.4 years. Among all cancer subtypes, only bladder carcinoma was significantly associated with PV replication. By multivariate analysis, only PV replication and smoking independently increased the risk of bladder cancer, relative risk, 11.7 (P = 0.0013) and 5.6 (P = 0.0053), respectively. CONCLUSIONS: The findings in the current study indicate that kidney transplant recipients with PV replication and smoking are at particular risk to develop bladder carcinomas and support the need for long-term cancer surveillance in these patients.

Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen.

It is speculated that bats are important reservoir hosts for numerous viruses, with 27 viral families reportedly detected in bats. Majority of these viruses have not been isolated and there is little information regarding their biology in bats. Establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. Currently, few bat cell lines have been developed and only Tb1-Lu, derived from Tadarida brasiliensis is commercially available. Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). To our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. The cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture.

Mouse Polyomavirus: Propagation, Purification, Quantification, and Storage.

Mouse polyomavirus (MPyV) is a member of the Polyomaviridae family, which comprises non-enveloped tumorigenic viruses infecting various vertebrates including humans and causing different pathogenic responses in the infected organisms. Despite the variations in host tropism and pathogenicity, the structure of the virions of these viruses is similar. The capsid, with icosahedral symmetry (ø, 45 nm, T = 7d), is composed of a shell of 72 capsomeres of structural proteins, arranged around the nucleocore containing approximately 5-kbp-long circular dsDNA in complex with cellular histones. MPyV has been one of the most studied polyomaviruses and serves as a model virus for studies of the mechanisms of cell transformation and virus trafficking, and for use in nanotechnology. It can be propagated in primary mouse cells (e.g., in whole mouse embryo cells) or in mouse epithelial or fibroblast cell lines. In this unit, propagation, purification, quantification, and storage of MPyV virions are presented.

The role of Merkel cell polyomavirus and other human polyomaviruses in emerging hallmarks of cancer.

Polyomaviruses are non-enveloped, dsDNA viruses that are common in mammals, including humans. All polyomaviruses encode the large T-antigen and small t-antigen proteins that share conserved functional domains, comprising binding motifs for the tumor suppressors pRb and p53, and for protein phosphatase 2A, respectively. At present, 13 different human polyomaviruses are known, and for some of them their large T-antigen and small t-antigen have been shown to possess oncogenic properties in cell culture and animal models, while similar functions are assumed for the large T- and small t-antigen of other human polyomaviruses. However, so far the Merkel cell polyomavirus seems to be the only human polyomavirus associated with cancer. The large T- and small t-antigen exert their tumorigenic effects through classical hallmarks of cancer: inhibiting tumor suppressors, activating tumor promoters, preventing apoptosis, inducing angiogenesis and stimulating metastasis. This review elaborates on the putative roles of human polyomaviruses in some of the emerging hallmarks of cancer. The reciprocal interactions between human polyomaviruses and the immune system response are discussed, a plausible role of polyomavirus-encoded and polyomavirus-induced microRNA in cancer is described, and the effect of polyomaviruses on energy homeostasis and exosomes is explored. Therapeutic strategies against these emerging hallmarks of cancer are also suggested.

The Cre/loxP recombination system for production of infectious mouse polyomavirus.

Murine polyomavirus mutants are frequently produced for experimental as well as therapy purposes. Commonly used methods for preparation of mutant viral genomes from recombinant vectors are laborious and give variable yields and quality. We describe an efficient and reproducible Cre/loxP-mediated recombination system that generates polyomavirus genomes from recombinant plasmid in vivo. We designed and constructed two variants of recombinant vectors containing the wild-type polyomavirus genome flanked by loxP homologous sites. The loxP sites were introduced either into the intronic region of early genes or between the two poly(A) signal sites of convergent transcriptional units. After cotransfection of the recombinant plasmids with the Cre-expressing vector into mouse 3T6 cells, we obtained infectious virus from the genome variant containing loxP site in the intronic region, but we failed to isolate any infectious virus from the viral genome containing loxP site between poly(A) signals. We show that the Cre/loxP-based method of polyomavirus production is simple, expedient, and reproducible and works with satisfactory efficiency.

Host response to polyomavirus infection is modulated by RNA adenosine deaminase ADAR1 but not by ADAR2.

Adenosine deaminases acting on RNA (ADARs) catalyze the C-6 deamination of adenosine (A) to produce inosine (I), which behaves as guanine (G), thereby altering base pairing in RNAs with double-stranded character. Two genes, adar1 and adar2, are known to encode enzymatically active ADARs in mammalian cells. Furthermore, two size forms of ADAR1 are expressed by alternative promoter usage, a short (p110) nuclear form that is constitutively made and a long (p150) form that is interferon inducible and present in both the cytoplasm and nucleus. ADAR2 is also a constitutively expressed nuclear protein. Extensive A-to-G substitution has been described in mouse polyomavirus (PyV) RNA isolated late times after infection, suggesting modification by ADAR. To test the role of ADAR in PyV infection, we used genetically null mouse embryo fibroblast cells deficient in either ADAR1 or ADAR2. The single-cycle yields and growth kinetics of PyV were comparable between adar1(-/-) and adar2(-/-) genetic null fibroblast cells. While large T antigen was expressed to higher levels in adar1(-/-) cells than adar2(-/-) cells, less difference was seen in VP1 protein expression levels between the two knockout MEFs. However, virus-induced cell killing was greatly enhanced in PyV-infected adar1(-/-) cells compared to that of adar2(-/-) cells. Complementation with p110 protected cells from PyV-induced cytotoxicity. UV-irradiated PyV did not display any enhanced cytopathic effect in adar1(-/-) cells. Reovirus and vesicular stomatitis virus single-cycle yields were comparable between adar1(-/-) and adar2(-/-) cells, and neither reovirus nor VSV showed enhanced cytotoxicity in adar1(-/-)-infected cells. These results suggest that ADAR1 plays a virus-selective role in the host response to infection.

Prevalence of WU and KI polyomaviruses in plasma, urine, and respiratory samples from renal transplant patients.

WU and KI polyomaviruses (WUPyV, KIPyV) have been detected in respiratory, blood, stool, and lymphoid tissue, but not in urine samples. PCR based detection revealed higher frequency in immunocompromised individuals. In this study the prevalence of WUPyV and KIPyV was analyzed in respiratory, urine, and blood samples from renal transplant patients compared with healthy individuals. WUPyV and KIPyV were detected by nested PCR. The PCR products were sequenced and viral DNA loads were determined by quantitative real-time PCR. WUPyV and KIPyV were found in plasma (3.6%; 7/195), urine (14%; 7/50), and respiratory samples (10%; 9/90) of renal transplant patients, but not in plasma (0/200) and urine (0/36) specimens from healthy blood donors. WUPyV and KIPyV were detected mainly early after renal transplantation and the viral loads were low. A higher prevalence of WUPyV was found in plasma and urine samples, KIPyV was found more frequently in respiratory samples from renal transplant patients. It is hypothesized that immunosuppression due to the transplantation may result in reactivation of these viruses or may establish greater susceptibility to infection with KIPyV and WUPyV.

Heterogeneity among viral antigen-specific CD4+ T cells and their de novo recruitment during persistent polyomavirus infection.

Virus-specific CD4(+) T cells optimize antiviral responses by providing help for antiviral humoral responses and CD8(+) T cell differentiation. Although CD4(+) T cell responses to viral infections that undergo complete clearance have been studied extensively, less is known about virus-specific CD4(+) T cell responses to viruses that persistently infect their hosts. Using a mouse polyomavirus (MPyV) infection model, we previously demonstrated that CD4(+) T cells are essential for recruiting naive MPyV-specific CD8(+) T cells in persistently infected mice. In this study, we defined two dominant MPyV-specific CD4(+) T cell populations, one directed toward an epitope derived from the nonstructural large T Ag and the other from the major viral capsid protein of MPyV. These MPyV-specific CD4(+) T cells vary in terms of their magnitude, functional profile, and phenotype during acute and persistent phases of infection. Using a minimally myeloablative-mixed bone marrow chimerism approach, we further show that naive virus-specific CD4(+) T cells, like anti-MPyV CD8(+) T cells, are primed de novo during persistent virus infection. In summary, these findings reveal quantitative and qualitative differences in the CD4(+) T cell response to a persistent virus infection and demonstrate that naive antiviral CD4(+) T cells are recruited during chronic polyomavirus infection.

Quantitative considerations to gather maximum information from viral growth efficiency studies: the example of polyomavirus type BK (BKV).

This short communication shows how the application of simple mathematical formulae allows researchers to extract maximum information from viral growth efficiency studies at virtually no additional costs (in terms of time or money), thus improving the comparability of results (growth rates, replicative capacities, efficacies of antivirals) between in vitro and in vivo growth efficiency studies. This could help in elucidating kinetic links between the molecular basis of virus function and clinical findings.

RNA processing in the polyoma virus life cycle.

Not only is gene regulation in polyoma interesting, but it has also proven to be highly informative and illustrative of a number of novel concepts in gene regulation. Of special interest and importance are the mechanisms by which this virus switches from the expression of early gene products to late gene products after the onset of viral DNA replication. This switch is mediated at least in part by changes in transcription elongation and polyadenylation in the late region, and by the formation and editing of dsRNA in the nucleus. In this review we will summarize the regulation of RNA synthesis and processing during polyoma infection, and will point out in particular those aspects that have been most novel.

Alterations of the plasma membrane caused by murine polyomavirus proliferation: an electrorotation study.

In this report we investigate the alterations of the dielectric properties of the plasma membrane caused by the infection of cultured fibroblasts with murine polyomavirus. The approach consists in a well-established dielectric spectroscopy technique, electrorotation, which has been successfully used in our laboratory to study the alterations of the plasma membrane of cells exposed to various forms of stress. The response to viral proliferation was time dependent as shown by evaluation of the de novo synthesis of viral DNA. This response was paralleled by gradual damage of the membrane evidenced by alteration of the dielectric parameters, specific capacitance and conductance. The electrorotation results show a reduced effect on the dielectric properties of the membrane when infection is carried out in the presence of a natural oil (MEX). In this case a drastic reduction in viral DNA synthesis was also monitored, thus indicating an antiviral action of this product.

[Correlation between load of polyomavirus and hemorrhagic cystitis].

OBJECTIVE: To study the correlation between polyoma virus load and hemorrhagic cystitis after allogeneic stem cells transplantation for prevention of hemorrhagic cystitis. METHODS: Blood and urine specimens were collected from 40 healthy persons, 40 patient with stem cells transplantation and 20 cases complicated with hemorrhagic cystitis for determination of VP1 gene of polyomaviruses BK virus (BKV)/Jamestown Canyon virus (JCV) and simian virus 40 (SV40) by polymerase chain reaction (PCR) and EvaGreen stain fluorescence quantitative assay. RESULTS: In the peripheral blood, all genes of BKV/JCV and SV40 were negative, while BKV gene in urine and blood from healthy persons and patient with stem cells transplantation was 15% (6/40) and 100% (40/40), respectively. The gene of JCV was positive in 10% (4/40) and 12% (5/40), the gene of SV40 was negative. CONCLUSION: Genes of BKV and JCV was detectable in urine specimens of healthy persons and there was a correlation between the load of polyomavirus and incidence of hemorrhagic cystitis.

Polyomavirus small T antigen controls viral chromatin modifications through effects on kinetics of virus growth and cell cycle progression.

Minichromosomes of wild-type polyomavirus were previously shown to be highly acetylated on histones H3 and H4 compared either to bulk cell chromatin or to viral chromatin of nontransforming hr-t mutants, which are defective in both the small T and middle T antigens. A series of site-directed virus mutants have been used along with antibodies to sites of histone modifications to further investigate the state of viral chromatin and its dependence on the T antigens. Small T but not middle T was important in hyperacetylation at major sites in H3 and H4. Mutants blocked in middle T signaling pathways but encoding normal small T showed a hyperacetylated pattern similar to that of wild-type virus. The hyperacetylation defect of hr-t mutant NG59 was partially complemented by growth of the mutant in cells expressing wild-type small T. In contrast to the hypoacetylated state of NG59, NG59 minichromosomes were hypermethylated at specific lysines in H3 and also showed a higher level of phosphorylation at H3ser10, a modification associated with the late G(2) and M phases of the cell cycle. Comparisons of virus growth kinetics and cell cycle progression in wild-type- and NG59-infected cells showed a correlation between the phase of the cell cycle at which virus assembly occurred and histone modifications in the progeny virus. Replication and assembly of wild-type virus were completed largely during S phase. Growth of NG59 was delayed by about 12 h with assembly occurring predominantly in G(2). These results suggest that small T affects modifications of viral chromatin by altering the temporal coordination of virus growth and the cell cycle.

How a small DNA virus uses dsRNA but not RNAi to regulate its life cycle.

Mouse polyomavirus contains a circular DNA genome, with early and late genes transcribed from opposite strands. At early times after infection, genes encoded from the early transcription unit are predominantly expressed. After the onset of viral DNA replication, expression of genes encoded from the late transcription unit increases dramatically. At late times, late primary transcripts are inefficiently polyadenylated, leading to the generation of multigenomic RNAs that are precursors to mature mRNAs. These transcripts contain sequences complementary to the early RNAs and downregulate early-strand gene expression by inducing RNA editing. Our recent work leads to a model where the production of the multigenomic late RNAs is also controlled by the editing of poly(A) signals, directed by overlapping primary transcripts.