Mechanism and consequences of herpes simplex virus 1-mediated regulation of host mRNA alternative polyadenylation.

Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3' UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.

Structural identification of conserved RNA binding sites in herpesvirus ORF57 homologs: implications for PAN RNA recognition.

Kaposi's sarcoma-associated herpesvirus (KSHV) transcribes a long noncoding polyadenylated nuclear (PAN) RNA, which promotes the latent to lytic transition by repressing host genes involved in antiviral responses as well as viral proteins that support the latent state. KSHV also expresses several early proteins including ORF57 (Mta), a member of the conserved multifunctional ICP27 protein family, which is essential for productive replication. ORF57/Mta interacts with PAN RNA via a region termed the Mta responsive element (MRE), stabilizing the transcript and supporting nuclear accumulation. Here, using a close homolog of KSHV ORF57 from herpesvirus saimiri (HVS), we determined the crystal structure of the globular domain in complex with a PAN RNA MRE, revealing a uracil specific binding site that is also conserved in KSHV. Using solution NMR, RNA binding was also mapped within the disordered N-terminal domain of KSHV ORF57, and showed specificity for an RNA fragment containing a GAAGRG motif previously known to bind a homologous region in HVS ORF57. Together these data located novel differential RNA recognition sites within neighboring domains of herpesvirus ORF57 homologs, and revealed high-resolution details of their interactions with PAN RNA, thus providing insight into interactions crucial to viral function.

HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression.

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

The herpes viral transcription factor ICP4 forms a novel DNA recognition complex.

The transcription factor ICP4 from herpes simplex virus has a central role in regulating the gene expression cascade which controls viral infection. Here we present the crystal structure of the functionally essential ICP4 DNA binding domain in complex with a segment from its own promoter, revealing a novel homo-dimeric fold. We also studied the complex in solution by small angle X-Ray scattering, nuclear magnetic resonance and surface-plasmon resonance which indicated that, in addition to the globular domain, a flanking intrinsically disordered region also recognizes DNA. Together the data provides a rationale for the bi-partite nature of the ICP4 DNA recognition consensus sequence as the globular and disordered regions bind synergistically to adjacent DNA motifs. Therefore in common with its eukaryotic host, the viral transcription factor ICP4 utilizes disordered regions to enhance the affinity and tune the specificity of DNA interactions in tandem with a globular domain.

The interaction of the cellular export adaptor protein Aly/REF with ICP27 contributes to the efficiency of herpes simplex virus 1 mRNA export.

Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.

Arginine methylation of the RGG box does not appear to regulate ICP27 import during herpes simplex virus infection.

Arginine methylation can regulate protein import and export and can modulate protein interactions. Herpes simplex virus 1 (HSV-1) ICP27 is a shuttling protein involved in viral mRNA export. We previously reported that ICP27 is methylated on three arginines within its RGG box and that arginine methylation regulates ICP27 export and its interaction with SRPK1 and Aly/REF. Here, we report that ICP27 was efficiently imported into the nucleus when hypomethylated as determined by Fluorescence Recovery After Photobleaching (FRAP). Furthermore, coimmunoprecipitation of ICP27 with ß-importin was not significantly affected by ICP27 hypomethylation. Thus, ICP27 import does not appear to be regulated by arginine methylation.

Bimolecular Fluorescence Complementation analysis to reveal protein interactions in herpes virus infected cells.

Protein interactions are at the basis of all processes in living organisms. In particular, regulatory proteins do not act alone but participate in multifaceted sets of interactions that are organized into complex networks. In herpes simplex virus (HSV-1) infected cells, viral proteins interact with cellular proteins and with other viral proteins to form the protein complexes required for virus production, including transcription complexes, replication complexes and virion assembly complexes. While a number of methods have been developed to investigate protein-protein interactions such as coimmunoprecipitation, GST-binding assays and yeast 2-hybrid analyses, these approaches require removal of the proteins from the cellular environment and do not provide information on the spatial localization of the protein-protein interaction in living cells. The fluorescence based approach Bimolecular Fluorescence Complementation (BiFC) allows direct visualization of the subcellular localization of the protein complex in living cells. In BiFC, two halves of a fluorescent protein are fused to each of two interacting proteins of interest, resulting in nonfluorescent fusion proteins. Interaction of the protein partners tethers the fused fluorescent fragments in close proximity, which facilitates their association and restoration of fluorescence. Two limitations of BiFC are that there is a delay between the time that the interacting proteins associate and fluorescence complex formation and thus complex formation cannot be measured in real-time, and fluorescence complex formation is irreversible in vivo. Despite these limitations, BiFC is a powerful and sensitive approach that can be performed using standard molecular biology and cell culture protocols and a fluorescence microscope.