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Unlabelled: Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system., Importance: HTLV-1 is a complex retrovirus that causes two distinct pathologies termed adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy in about 5% of infected individuals. Expression of the virus depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of virus expression. The findings reported in this study revealed a novel cis-acting regulatory element and indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. Our results add a layer of complexity to the mechanisms controlling the expression of alternatively spliced HTLV-1 mRNAs and suggest a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The present study employed mass sequencing of small RNA libraries to identify the repertoire of small noncoding RNAs expressed in normal CD4(+) T cells compared to cells transformed with human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma (ATLL). The results revealed distinct patterns of microRNA expression in HTLV-1-infected CD4(+) T-cell lines with respect to their normal counterparts. In addition, a search for virus-encoded microRNAs yielded 2 sequences that originated from the plus strand of the HTLV-1 genome. Several sequences derived from tRNAs were expressed at substantial levels in both uninfected and infected cells. One of the most abundant tRNA fragments (tRF-3019) was derived from the 3' end of tRNA-proline. tRF-3019 exhibited perfect sequence complementarity to the primer binding site of HTLV-1. The results of an in vitro reverse transcriptase assay verified that tRF-3019 was capable of priming HTLV-1 reverse transcriptase. Both tRNA-proline and tRF-3019 were detected in virus particles isolated from HTLV-1-infected cells. These findings suggest that tRF-3019 may play an important role in priming HTLV-1 reverse transcription and could thus represent a novel target to control HTLV-1 infection., Importance: Small noncoding RNAs, a growing family of regulatory RNAs that includes microRNAs and tRNA fragments, have recently emerged as key players in many biological processes, including viral infection and cancer. In the present study, we employed mass sequencing to identify the repertoire of small noncoding RNAs in normal T cells compared to T cells transformed with human T-cell leukemia virus type 1 (HTLV-1), a retrovirus that causes adult T-cell leukemia/lymphoma. The results revealed a distinct pattern of microRNA expression in HTLV-1-infected cells and a tRNA fragment (tRF-3019) that was packaged into virions and capable of priming HTLV-1 reverse transcription, a key event in the retroviral life cycle. These findings indicate tRF-3019 could represent a novel target for therapies aimed at controlling HTLV-1 infection.

The Rex protein of human T-cell leukemia virus (HTLV) acts posttranscriptionally to induce the cytoplasmic expression of the unspliced and incompletely spliced viral RNAs encoding the viral structural and enzymatic proteins and is therefore essential for efficient viral replication. Rex function requires nuclear import, RNA binding, multimerization, and nuclear export. In addition, it has been demonstrated that the phosphorylation status of HTLV-2 Rex (Rex-2) correlates with RNA binding and inhibition of splicing in vitro. Recent mutational analyses of Rex-2 revealed that the phosphorylation of serine residues 151 and 153 within a novel carboxy-terminal domain is critical for function in vivo. To further define the functional domain structure of Rex-2, we evaluated a panel of Rex-2 mutants for subcellular localization, RNA binding capacity, multimerization and trans-dominant properties, and the ability to shuttle between the nucleus and the cytoplasm. Rex-2 mutant S151A,S153A, which is defective in phosphorylation and function, showed diffuse cytoplasmic staining, whereas mutant S151D,S153D, previously shown to be functional and in a conformation corresponding to constitutive phosphorylation, displayed increased intense speckled staining in the nucleoli. In vivo RNA binding analyses indicated that mutant S151A,S153A failed to efficiently bind target RNA, while its phosphomimetic counterpart, S151D,S153D, bound twofold more RNA than wild-type Rex-2. Taken together, these findings provide direct evidence that the phosphorylation status of Rex-2 is linked to cellular trafficking and RNA binding capacity. Mutants with substitutions in either of the two putative multimerization domains or in the putative activation domain-nuclear export signal displayed a dominant negative phenotype as well as defects in multimerization and nucleocytoplasmic shuttling. Several carboxy-terminal mutants that displayed wild-type levels of phosphorylation and localized to the nucleolus were also partially impaired in shuttling. This is consistent with the hypothesis that the carboxy terminus of Rex-2 contains a novel domain that is required for efficient shuttling. This work thus provides a more detailed functional domain map of Rex-2 and further insight into its regulation of HTLV replication.

The activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the DeltaLoop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.

Three mRNA species encoding the x-III open reading frame are expressed in human T-cell leukemia virus type 2 (HTLV-2)-infected cells. An mRNA composed of exons 1, 2, and 3 produces the essential posttranscriptional regulator Rex; shorter 1-3 and 1-B mRNAs encode a family of x-III proteins of unknown function that represent truncated forms of Rex. This report presents an analysis of the functional interactions between Rex and the x-III proteins, results of which suggest a role for the x-III proteins as negative regulators of Rex function. Cotransfection assays demonstrated that the x-III proteins were able to inhibit the ability of Rex to activate the expression of a Rex-dependent mRNA. Analysis of intracellular compartmentalization in actinomycin D-treated cells showed that coexpression of the x-III proteins resulted in the sequestration of Rex into the nuclear compartment. Subcellular fractionation studies showed that Rex was preferentially localized in the cytoplasmic or nuclear fraction depending on its phosphorylation status and that coexpression of Rex with the x-III proteins changed the phosphorylation pattern of Rex and the intracellular distribution of the x-III proteins. In vitro protein binding assays demonstrated the formation of Rex-Rex homomultimeric complexes; however, mixed Rex/x-III multimers were not detected. These findings indicated a correlation between phosphorylation and intracellular trafficking of Rex and suggested that the mechanism underlying the inhibitory effects of the x-III proteins might result from an interference with these processes.

The X region of human T-cell lymphotropic virus type 1 (HTLV-1) encodes two nucleolar/nuclear proteins, the posttranscriptional regulator of mRNA expression Rex and a protein of unknown function named Tof. To gain insight into the possible biological role of Tof, we investigated the mechanism governing its intracellular trafficking and identified its nucleolar/nuclear localization signal (NLS). Mutational analysis of Tof revealed that its NLS was located between amino acids 71 and 98 and contained two arginine-rich domains that functioned in an interdependent manner. Studies of Tof-Rex hybrid proteins showed that the Tof NLS could functionally replace the NLS of Rex at the level of nuclear targeting. As the NLS of Rex is known to mediate its interaction with its RNA target, the Rex-responsive element (RXRE), we tested whether the NLS of Tof could replace that of Rex in mediating activation of a RXRE-containing mRNA. Results showed that the NLS of Tof was indeed able to mediate activation of RXRE-containing mRNAs, suggesting that Tof itself may function as a regulator of RNA expression and utilization. A comparison of their compartmentalization in response to actinomycin D treatment indicated that Tof did not share Rex's shuttling pathway. Expression of Tof from its natural multiply spliced mRNA required the presence of Rex, suggesting that Tof may regulate viral or cellular mRNA expression during the later stages of viral replication.