Brincidofovir Use after Foscarnet Crystal Nephropathy in a Kidney Transplant Recipient with Multiresistant Cytomegalovirus Infection

Background. Cytomegalovirus (CMV) antiviral drug resistance constitutes an increasing challenge in transplantation. Foscarnet is usually proposed when resistance for ganciclovir is suspected, but its use is limited by its nephrotoxicity. Case Presentation. We report a case of multiresistant CMV disease in a kidney transplant recipient. Foscarnet was prescribed after ganciclovir treatment failure in a patient with two mutations in the UL97 viral gene. Foscarnet induced biopsy-proven kidney crystal precipitation that resulted in severe acute transplant failure and nephrotic syndrome. Despite a large decrease in immunosuppression, CMV disease was not controlled and a salvage therapy with Brincidofovir (BCV), which is an oral lipid conjugate of cidofovir with limited nephrotoxicity, was attempted. Clinical and virological remission was observed after a 21-day course of BCV, despite mild and reversible liver toxicity. However, a new relapse could not be effectively cured by BCV due to a new mutation in the UL54 gene, which is known to confer resistance to cidofovir. A new course of foscarnet finally resulted in prolonged CMV remission. Herein, we present a review of foscarnet nephropathy cases in solid-organ transplanted patients. Conclusions. This unique case highlights the potential benefit of BCV use during resistant CMV infection, although mutations in the UL54 gene may limit its therapeutic efficacy. These findings need to be confirmed in clinical trials.

Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability

ABSTRACTA key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication.IMPORTANCEThe DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.

Does Cytomegalovirus Develop Resistance following Antiviral Prophylaxis and Treatment in Renal Transplant Patients in Kuwait?

The resistance of cytomegalovirus (CMV) to ganciclovir or valganciclovir is a factor in therapeutic failure and disease progression. CMV strains resistant to ganciclovir or valganciclovir have been associated with specific mutations in the UL97 and UL54 genes. Sequencing of both CMV UL97 and UL54 genes was performed to detect the presence of CMV antiviral resistance in six patients who received ganciclovir (and/or valganciclovir) and had prolonged detectable CMV DNA in their blood during antiviral treatment. Sequencing results showed no specific mutations in either UL97 or UL54 gene of CMV and therefore the CMV strains in kidney transplant patients who received ganciclovir either prophylactically or therapeutically were from the wild type. Our results suggest that CMV management and immunosuppression protocols for kidney transplant patients followed in the Organ Transplant Centre, Kuwait, is very effective in reducing the opportunity of developing CMV antiviral resistance.

In Vitro Construction of Effective M1GS Ribozymes Targeting HCMV UL54 RNA Segments

Seven sequence-specific ribozymes (M1GS RNAs) derived in vitro from the catalytic RNA subunit of Escherichia coli RNase P and targeting the mRNAs transcribed by the UL54 gene encoding the DNA polymerase of human cytomegalovirus were screened from 11 ribozymes that were designed based on four rules: (1) the NCCA-3′terminal must be unpaired with the substrate; (2) the guide sequence (GS) must be at least 12 nt in length; (3) the eighth nucleotide must be U, counting from the site –1; and (4) around the cleavage site, the sites –1/+1/+2 must be U/G/C or C/G/C. Further investigation of the factors affecting the cleavage effect and the optimal ratio for M1GS/substrate was carried out. It was determined that the optimal ratio for M1GS/substrate was 2:1 and too much M1GS led to substrate degrading. As indicated above, several M1GS that cleaved HCMV UL54 RNA segments in vitro were successfully designed and constructed. Our studies support the use of ribozyme M1GS as antisense molecules to silence HCMV mRNA in vitro, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.