Herpes simplex virus 1 (HSV-1) protein ICP27, an important regulator for viral gene expression, directly recognizes and exports viral RNA through an N-terminal RGG box RNA binding motif, which is necessary and sufficient for RNA binding. An ICP27 N-terminal peptide, including the RGG box RNA binding motif, was expressed and its binding specificity was analyzed using EMSA and SELEX. DNA oligonucleotides corresponding to HSV-1 glycoprotein C (gC) mRNA, identified in a yeast three-hybrid analysis, were screened for binding to the ICP27 N-terminal peptide in EMSA experiments. The ICP27 N-terminus was able to bind most gC substrates. Notably, the ICP27 RGG box was unable to bind G-quartet structures recognized by the RGG domains of other proteins. SELEX analysis identified GC-rich RNA sequences as a common feature of recognition. NMR analysis of SELEX and gC sequences revealed that sequences able to bind to ICP27 did not form secondary structures and conversely, sequences that were not able to bind to ICP27 gave spectra consistent with base-pairing. Therefore, the ICP27 RGG box is unique in its recognition of nucleic acid sequences compared to other RGG box proteins; it prefers flexible, GC-rich substrates that do not form stable secondary structures.
Background: Ebolaviruses cause a severe and often fatal haemorrhagic fever in humans, with some species such as Ebola virus having case fatality rates approaching 90%. Currently, the worst Ebola virus outbreak since the disease was discovered is occurring in West Africa. Although thought to be a zoonotic infection, a concern is that with increasing numbers of humans being infected, Ebola virus variants could be selected which are better adapted for human-to-human transmission. Results: To investigate whether genetic changes in Ebola virus become established in response to adaptation in a different host, a guinea pig model of infection was used. In this experimental system, guinea pigs were infected with Ebola virus (EBOV), which initially did not cause disease. To simulate transmission to uninfected individuals, the virus was serially passaged five times in naïve animals. As the virus was passaged, virulence increased and clinical effects were observed in the guinea pig. An RNAseq and consensus mapping approach was then used to evaluate potential nucleotide changes in the Ebola virus genome at each passage. Conclusions: Upon passage in the guinea pig model, EBOV become more virulent, RNA editing and also coding changes in key proteins become established. The data suggest that the initial evolutionary trajectory of EBOV in a new host can lead to a gain in virulence. Given the circumstances of the sustained transmission of EBOV in the current outbreak in West Africa, increases in virulence may be associated with prolonged and uncontrolled epidemics of EBOV.
Adenovirus late mRNA export is facilitated by viral early proteins of 55 and 34 kDa. The 34-kDa protein contains a leucine-rich nuclear export signal (NES) similar to that of the human immunodeficiency virus Rev protein. It was proposed that the 34-kDa protein might facilitate the export of adenovirus late mRNA through a Rev-like NES-mediated export pathway. We have tested the role of NES-mediated RNA export during adenovirus infection, and we find that it is not essential for the expression of adenovirus late genes.
Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is phosphorylated. Phosphorylation can affect protein localization, protein interactions, and protein function. The major sites of ICP27 that are phosphorylated are serine residues 16 and 18, within a CK2 site adjacent to a leucine-rich region required for ICP27 export, and serine 114, within a PKA site in the nuclear localization signal. Viral mutants bearing serine-to-alanine or glutamic acid substitutions at these sites are defective in viral replication and gene expression. To determine which interactions of ICP27 are impaired, we analyzed the subcellular localization of ICP27 and its colocalization with cellular RNA export factors Aly/REF and TAP/ NXF1. In cells infected with phosphorylation site mutants, ICP27 was confined to the nucleus even at very late times after infection. ICP27 did not colocalize with Aly/REF or TAP/NXF1, and overexpression of TAP/NXF1 did not promote the export of ICP27 to the cytoplasm. However, in vitro binding experiments showed that mutant ICP27 was able to bind to the same RNA substrates as the wild type. Nuclear magnetic resonance (NMR) analysis of the N terminus of ICP27 from amino acids 1 to 160, compared to mutants with triple substitutions to alanine or glutamic acid, showed that the mutations affected the overall conformation of the N terminus, such that mutant ICP27 was more flexible and unfolded. These results indicate that these changes in the structure of ICP27 altered in vivo protein interactions that occur in the N terminus but did not prevent RNA binding.ICP27 is a multifunctional protein that acts at both the transcriptional and posttranscriptional levels (47). At the transcriptional level, ICP27 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNAP II) and recruits RNAP II to sites of herpes simplex virus 1 (HSV-1) transcription/ replication (13, 59). The interaction of ICP27 with RNAP II requires the N-terminal leucine-rich region (LRR) of ICP27, and the viral mutant dLeu, in which this region is deleted, cannot interact with and recruit RNAP II (13). As a result, viral early and late transcript levels are reduced to 10 to 20% of the levels seen in wild-type HSV-1 infection (32). At the posttranscriptional level, ICP27 interacts with SR proteins, which are essential splicing factors, and with SRPK1, an SR protein-specific kinase, to mediate the aberrant phosphorylation of SR proteins (50). Inappropriately phosphorylated SR proteins are unable to participate in spliceosome assembly, and host cell pre-mRNA splicing is inhibited (19, 50), which contributes to the shutoff of host protein synthesis (18). Beginning about 5 h after infection, ICP27 leaves splicing speckles and recruits the cellular mRNA export factor Aly/REF to viral transcription/replication sites (8, 9). ICP27 then binds viral RNAs (46) and begins shuttling between the nucleus and cytoplasm in its role as an RNA export factor (8,9,28,39,46,52). ICP27 is exported to the cytoplasm through the TAP/ ...
Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is posttranslationally modified by phosphorylation during viral infection. ICP27 has been shown to be phosphorylated on three serine residues, specifically serine residues 16 and 18, which are within casein kinase 2 (CK2) sites, and serine residue 114, which is within a protein kinase A (PKA) site. Phosphorylation is an important regulatory mechanism that is reversible and controls many signaling pathways, protein-protein interactions, and protein subcellular localization. To determine the role of phosphorylation in modulating the activities of ICP27, we constructed phosphorylation site mutations at each of the three serine residues. Single, double, and triple viral mutants were created in which alanine or glutamic acid was substituted for serines 16, 18, and 114. ICP27 phosphorylation site mutants were defective in viral replication and viral gene expression. Notably, ICP4-containing replication compartment formation was severely compromised, with the appearance of small ring-like structures that persisted even at late times after infection. Neither the colocalization of ICP27 with RNA polymerase II nor the formation of Hsc70 nuclear foci was observed during infection with the phosphorylation site mutants, both of which occur during wild-type HSV-1 infection. These data indicate that several key events in which ICP27 plays a role are curtailed during infection with ICP27 phosphorylation site mutants.Herpes simplex virus 1 (HSV-1) ICP27 is a multifunctional regulatory protein that interacts with a number of proteins and that binds RNA (32). The different activities of ICP27 are regulated in a temporal manner during infection. At early times, ICP27 is localized to the nucleus, whereas beginning about 5 to 6 h after infection, ICP27 continuously shuttles between the nucleus and cytoplasm. ICP27 mediates its nuclear activities through a series of protein-protein interactions. At very early times during infection, ICP27 recruits the predominantly cytoplasmic splicing factor kinase SRPK1 into the nucleus (34). This results in the aberrant phosphorylation of a family of essential splicing factors termed SR proteins, which are specifically phosphorylated by SRPK1. The inappropriate phosphorylation of SR proteins prevents their participation in spliceosome assembly, which causes splicing complex formation to stall, and host cell pre-mRNA splicing is impaired (15,34). This contributes to the shutoff of host protein synthesis (14). Also at early times after infection, ICP27 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNAP II) and causes RNAP II relocalization to sites of HSV-1 transcription/replication. This recruitment of RNAP II is necessary for highly active and efficient viral transcription (5). Beginning about 5 h after infection, ICP27 disassociates from splicing speckles, taking the cellular mRNA export factor Aly/REF with it to viral transcription/replication compartments (3, 4). ICP27 then binds viral tr...
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