Merkel Cell Polyomavirus (MCPyV) in the Context of Immunosuppression: Genetic Analysis of Noncoding Control Region (NCCR) Variability among a HIV-1-Positive Population

Abstract

Background: Since limited data are available about the prevalence of Merkel cell polyomavirus (MCPyV) and the genetic variability of its noncoding control region (NCCR) in the context of immunosuppression, this study aimed to investigate the distribution of MCPyV in anatomical sites other than the skin and the behavior of NCCR among an HIV-1-positive population. Methods: Urine, plasma, and rectal swabs specimens from a cohort of 66 HIV-1-positive patients were collected and subjected to quantitative real-time polymerase chain reaction (qPCR) for MCPyV DNA detection. MCPyV-positive samples were amplified by nested PCR targeting the NCCR, and NCCRs alignment was carried out to evaluate the occurrence of mutations and to identify putative binding sites for cellular factors. Results: MCPyV DNA was detected in 10/66 urine, in 7/66 plasma, and in 23/66 rectal samples, with a median value of 5 × 102 copies/mL, 1.5 × 102 copies/mL, and 2.3 × 103 copies/mL, respectively. NCCR sequence analysis revealed a high degree of homology with the MCC350 reference strain in urine, whereas transitions, transversions, and single or double deletions were observed in plasma and rectal swabs. In these latter samples, representative GTT and GTTGA insertions were also observed. Search for putative binding sites of cellular transcription factors showed that in several strains, deletions, insertions, or single base substitutions altered the NCCR canonical configuration. Conclusions: Sequencing analysis revealed the presence of numerous mutations in the NCCR, including insertions and deletions. Whether these mutations may have an impact on the pathogenic features of the virus remains to be determined. qPCR measured on average a low viral load in the specimens analyzed, with the exception of those with the GTTGA insertion.

Structural Analysis of Merkel Cell Polyomavirus (MCPyV) Viral Capsid Protein 1 (VP1) in HIV-1 Infected Individuals

“…Although MCPyV is constantly shed from healthy skin and up to 60-80% of healthy people have antibodies against this virus, MCC is rare with an incidence of 0.2-0.45 cases/100,000 individuals per year in most countries [13]. The MCC incidence increases with aging [31][32][33][34][35] and among immunocompromised individuals such as HIV-1/AIDS patients [15,[35][36][37][38]. To date, the events connecting initial MCPyV infection and subsequent cell transformation remain elusive, although it has been established that viral DNA integration into the host genome and continued expression of the C-terminal truncated LT are required for MCC development [20].…”

“…Total DNA was extracted from urine and plasma using the DNeasy ® Blood & Tissue Kit (QIAGEN, Milan, Italy), according to the manufacturer's instructions. Specific qPCR assays were performed using TaqMan-based qPCR, employing primers and probes for MCPyV sT, as previously described [35]. All samples were tested in triplicate, and the number of viral copies was calculated from standard curves constructed using a ten-fold dilution series of plasmid pMCV-R17a containing the entire genome of MCPyV (Addgene, #24729) (dilution range: 10 8 -10 copies/mL).…”

“…SPNTs collected two times a week during transfection experiments, were subjected to 6 cycles of freezing and thawing, followed by centrifugation at 2000 rpm for 5 min [67]. The resulting clarified SPNTs were quantified using TaqMan-based qPCR, employing primers and probes for MCPyV sT, as previously described [35] and virions corresponding to 10 4 copies/mL were used to infect freshly seeded A549 cells. Cells and SPNTs infected with MCPyV prototype DNA were used as controls during the experiments with ex vivo plasma and urine samples.…”

“…SPNTs from A549 cells were used directly in molecular biology assays. Extracted DNA and SPNTs were quantified using qPCR for MCPyV sT, [35]. The amount of cellular DNA was quantified simultaneously using a SYBR GREEN PCR for the house-keeping β-globin gene [68] and used to normalize the MCPyV DNA.…”

Merkel Cell Polyomavirus and Merkel Cell Carcinoma

“…However, MCPyV NCCR variation affects early and late promoter activities in a VN-MCC cell line and in human dermal fibroblast and wild-type LT inhibited both early and late promoter activities in both cell lines, whereas tLT had the opposite effect [ 25 ]. A recent study demonstrated the onset of insertions and deletions in the NCCR among an HIV-1-positive population [ 26 ]. Whether NCCR variation has an influence on viral replication and pathogenic properties of the virus remains to be investigated.…”