It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.
The herpes simplex virus (HSV) UL31 gene encodes a conserved member of the herpesvirus nuclear egress complex that not only functions in the egress of DNA containing capsids from the nucleus, but is also required for optimal replication of viral DNA and its packaging into capsids. Here we report that the UL31 protein from HSV-2 can be recruited to sites of DNA damage by sequences found in its N-terminus. The N-terminus of UL31 contains sequences resembling a poly (ADP-ribose) (PAR) binding motif suggesting that PAR interactions might mediate UL31 recruitment to damaged DNA. Whereas PAR polymerase inhibition prevented UL31 recruitment to damaged DNA, inhibition of signaling through the ataxia telangiectasia mutated DNA damage response pathway had no effect. These findings were further supported by experiments demonstrating direct and specific interaction between HSV-2 UL31 and PAR using purified components. This study reveals a previously unrecognized function for UL31 and may suggest that the recognition of PAR by UL31 is coupled to the nuclear egress of herpesvirus capsids, influences viral DNA replication and packaging, or possibly modulates the DNA damage response mounted by virally infected cells.
It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.
BACKGROUND AND PURPOSE: For patients with high-grade gliomas, the appearance of a new, enhancing lesion after surgery and chemoradiation represents a diagnostic dilemma. We hypothesized that MR perfusion without and with contrast can differentiate tumor recurrence from radiation necrosis. MATERIALS AND METHODS:In this prospective study, we performed 3 MR perfusion methods: arterial spin-labeling, DSC, and dynamic contrast enhancement. For each lesion, we measured CBF from arterial spin-labeling, uncorrected relative CBV, and leakage-corrected relative CBV from DSC imaging. The volume transfer constant and plasma volume were obtained from dynamic contrast-enhanced imaging without and with T1 mapping using modified Look-Locker inversion recovery (MOLLI). The diagnosis of tumor recurrence or radiation necrosis was determined by either histopathology for patients who underwent re-resection or radiologic follow-up for patients who did not have re-resection.RESULTS: There were 26 patients with 32 lesions, 19 lesions with tumor recurrence and 13 lesions with radiation necrosis. Compared with radiation necrosis, lesions with tumor recurrence had higher CBF (P ¼ .033), leakage-corrected relative CBV (P ¼ .048), and plasma volume using MOLLI T1 mapping (P ¼ .012). For differentiating tumor recurrence from radiation necrosis, the areas under the curve were 0.81 for CBF, 0.80 for plasma volume using MOLLI T1 mapping, and 0.71 for leakage-corrected relative CBV. A correlation was found between CBF and leakage-corrected relative CBV (r s ¼ 0.54), volume transfer constant, and plasma volume (0.50 , r s , 0.77) but not with uncorrected relative CBV (r s ¼ 0.20, P ¼ .29). CONCLUSIONS:In the differentiation of tumor recurrence from radiation necrosis in a newly enhancing lesion, the diagnostic value of arterial spin-labeling-derived CBF is similar to that of DSC and dynamic contrast-enhancement-derived blood volume.ABBREVIATIONS: ASL ¼ arterial spin-labeling; AUC ¼ area under the curve; DCE ¼ dynamic contrast-enhanced; K trans ¼ volume transfer constant; MOLLI ¼ modified Look-Locker inversion recovery; pCASL ¼ pseudocontinuous pulse ASL; rCBV ¼ relative CBV (CBV lesion/CBV normal contralateral white matter); ROC ¼ receiver operating characteristic; SI ¼ signal intensity; SMART 1 Map ¼ saturation method using adaptive recovery times for cardiac T1 mapping; Vp ¼ plasma volume
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