BK Polyomavirus Activates HSF1 Stimulating Human Kidney Hek293 Cell Proliferation.

OBJECTIVES: Some DNA viruses, such as BKPyV, are capable of inducing neoplastic transformation in human tissues through still unclear mechanisms. The goal of this study is to investigate the carcinogenic potential of BK polyomavirus (BKPyV) in human embryonic kidney 293 (Hek293) cells, dissecting the molecular mechanism that determines the neoplastic transformation. MATERIALS AND METHODS: BKPyV, isolated from urine samples of infected patients, was used to infect monolayers of Hek293 cells. Subsequently, intracellular redox changes, GSH/GSSH concentration by HPLC, and reactive oxygen/nitrogen species (ROS/RNS) production were monitored. Moreover, to understand the signaling pathway underlying the neoplastic transformation, the redox-sensitive HFS1-Hsp27 molecular axis was examined using the flavonoid quercetin and polishort hairpin RNA technologies. RESULTS: The data obtained show that while BKPyV replication is closely linked to the transcription factor p53, the increase in Hek293 cell proliferation is due to the activation of the signaling pathway mediated by HSF1-Hsp27. In fact, its inhibition blocks viral replication and cell growth, respectively. CONCLUSIONS: The HSF1-Hsp27 signaling pathway is involved in BKPyV infection and cellular replication and its activation, which could be involved in cell transformation.

Severe hemorrhagic cystitis caused by the BK polyomavirus is associated with decreased survival post-allogeneic hematopoietic stem cell transplantation.

BACKGROUND: BK polyomavirus reactivation can occur following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may lead to hemorrhagic cystitis (BKPyV-HC). We hypothesized that development of BKPyV-HC is associated with increased mortality post allo-HSCT. METHODS: We retrospectively reviewed data on 133 adult patients (≥18 years old) who underwent allo-HSCT from 2007 until 2014 at Hospital Israelita Albert Einstein in São Paulo, Brazil. RESULTS: Thirty-six patients presented with BKPyV-HC after a median time of 42 days, with a 1-year cumulative incidence probability of 28.9% (95% CI 21.5%-36.7%). In a multivariate Cox model, risk factors for development of BKPyV-HC included younger age, male sex, development of grade 2-4 acute graft-versus-host disease and recipients of umbilical cord blood grafts. Development of grade 3-4 BKPyV-HC (but not grade 1-2) was associated with a decreased overall survival (OS) in a multivariate Cox model (hazard ratio [HR] 7.51, P < 0.0001) and an increased risk of TRM (HR 3.66, P < 0.0001). Grade 3-4 BKPyV-HC was also associated with an increased risk of relapse that did not reach statistical significance (HR 3.01, P = 0.07). Median overall survival (OS) post-BKPyV-HC was 4.7 months, and cidofovir had no impact on survival. CONCLUSION: Development of BKPyV-HC appears to be associated with decreased survival following allo-HSCT.

Role of BK polyomavirus (BKV) and Torque teno virus (TTV) in liver transplant recipients with renal impairment.

PURPOSE: Renal impairment is a common complication after liver transplantation (LT). While BK polyomavirus (BKV) has been linked to renal failure in kidney transplant recipients, Torque teno virus (TTV) is a surrogate marker for immunosuppression that does not have a clear association with any human disease. The impact of BKV and TTV on renal impairment after LT is unknown. METHODOLOGY: In this retrospective study, urine and serum samples from 136 liver transplant recipients were screened for BKV and TTV by quantitative PCR. In addition, serum was screened for BKV-specific antibodies and the VP1 typing region was sequenced for BKV genotyping. All parameters were correlated with clinical data.Results/Key findings. BK viruria was detected up to 21 years after transplantation in 16.9 % of cases. BK viraemia was detected in 8.7 % of patients with BK viruria up to 4 years after LT. BKV-specific antibodies were detected in 93.6 % of all LT recipients and correlated with BKV viral load in urine. There was no correlation between renal impairment and the detection of BK DNA in urine (OR 0.983). TTV DNA was detected in 84.6 % of serum samples and in 66.6 % of urine samples. The TTV viral load in serum correlated with the BKV viral load but had no impact on renal impairment. CONCLUSION: Our data indicate that the detection of BKV and TTV is not a risk factor for renal impairment after LT. A correlation of TTV and BKV viral load seems to be an indicator for the immune status of the host.

Incidence, risk factors and the effect of polyomavirus infection in hematopoietic stem cell transplant recipients.

Objective The effect of polyomavirus infection in HSCT recipients is poorly understood. Methods We evaluated 38 HSCT recipients. Polyomavirus was detected by nested qualitative polymerase chain reaction (PCR) assays of urine. The risk factors for BK virus and JC virus were analysed. The kidney and liver functions of infected and uninfected patients were compared. Results BK virus, JC virus, and simian virus 40 were detected in 21%, 42%, and 0% of HSCT recipients respectively. HCMV infection was found to be an independent risk factor for JC virus infection (odds ratio (OR): 8.528), while transplants with mismatched HLA are more susceptible to BK virus infection (OR: 12.000). Liver function of JC virus-infected subjects was worse than that of uninfected subjects. Conclusion We must be vigilant for opportunistic polyomavirus infections in HSCT recipients, especially those with HCMV co-infection or a mismatched HLA transplant. When unexplained liver function deterioration is observed, JC virus infection should be considered.

BRAF inhibitor-associated cutaneous squamous cell carcinoma: new mechanistic insight, emerging evidence for viral involvement and perspectives on clinical management.

Mutations in the BRAF proto-oncogene occur in the majority of cutaneous melanomas. The commonly detected valine (V) to glutamate (E) mutation (V600E) is known to drive melanomagenesis and has thus been the target of two highly selective chemotherapeutic agents: vemurafenib and dabrafenib. While BRAF inhibitor therapy has revolutionized the treatment of metastatic melanoma, unanticipated cutaneous toxicities, including the development of cutaneous squamous cell carcinomas (cSCCs), are frequently reported and hinder therapeutic durability. However, the mechanisms by which BRAF inhibitors induce cutaneous neoplasms are poorly understood, thus posing a challenge for specific therapies. In this review, we summarize the clinical and molecular profiles of BRAF inhibitor-associated cSCCs, with a focus on factors that may contribute to disease pathogenesis. In particular, we discuss the emerging evidence pointing towards viral involvement in BRAF inhibitor-induced cutaneous neoplasms and offer new perspectives on future therapeutic interventions. Continued clinical and mechanistic studies along this line will not only allow for better understanding of the pathogenic progression of BRAF inhibitor-induced cSCCs, but will also lead to development of new therapeutic and preventative options for patients receiving targeted cancer therapy.

BRD4 is associated with raccoon polyomavirus genome and mediates viral gene transcription and maintenance of a stem cell state in neuroglial tumour cells.

Polyomavirus infection often results in persistence of the viral genome with little or no virion production. However, infection of certain cell types can result in high viral gene transcription and either cytolysis or neoplastic transformation. While infection by polyomavirus is common in humans and many animals, major questions regarding viral persistence of most polyomaviruses remain unanswered. Specifically, identification of target cells for viral infection and the mechanisms polyomaviruses employ to maintain viral genomes within cells are important not only in ascribing causality to polyomaviruses in disease, but in understanding specific mechanisms by which they cause disease. Here, we characterize the cell of origin in raccoon polyomavirus (RacPyV)-associated neuroglial brain tumours as a neural stem cell. Moreover, we identify an association between the viral genome and the host cell bromodomain protein, BRD4, which is involved in numerous cellular functions, including cell cycle progression, differentiation of stem cells, tethering of persistent DNA viruses, and regulation of viral and host-cell gene transcription. We demonstrate that inhibition of BRD4 by the small molecule inhibitors (+)-JQ1 and IBET-151 (GSK1210151A) results in reduced RacPyV genome within cells in vitro, as well as significant reduction of viral gene transcripts LT and VP1, highlighting its importance in both maintenance of the viral genome and in driving oncogenic transformation by RacPyV. This work implicates BRD4 as a central protein involved in RacPyV neuroglial tumour cell proliferation and in the maintenance of a stem cell state.

How Polyomaviruses Exploit the ERAD Machinery to Cause Infection.

To infect cells, polyomavirus (PyV) traffics from the cell surface to the endoplasmic reticulum (ER) where it hijacks elements of the ER-associated degradation (ERAD) machinery to penetrate the ER membrane and reach the cytosol. From the cytosol, the virus transports to the nucleus, enabling transcription and replication of the viral genome that leads to lytic infection or cellular transformation. How PyV exploits the ERAD machinery to cross the ER membrane and access the cytosol, a decisive infection step, remains enigmatic. However, recent studies have slowly unraveled many aspects of this process. These emerging insights should advance our efforts to develop more effective therapies against PyV-induced human diseases.

JC polyomavirus in the aetiology and pathophysiology of glial tumours.

Glial brain tumours with their poor prognosis, limited treatment modalities and unclear detailed pathophysiology represent a significant health concern. The purpose of the current study was to investigate and describe the possible role of the human polyomavirus JC as an underlying cancerogenic or co-cancerogenic factor in the complex processes of glial tumour induction and development. Samples from 101 patients with glial tumours were obtained during neurosurgical tumour resection. Small tissue pieces were taken from several areas of the histologically verified solid tumour core. Biopsies were used for DNA extraction and subsequent amplification reactions of sequences from the JC viral genome. Real-time polymerase chain reaction was used for detection and quantification of its non-coding control region (NCCR) and gene encoding the regulatory protein Large T antigen (LT). An average of 37.6% of all patients was found to be LT positive, whereas only 6.9% tested positive for NCCR. The analysis of the results demonstrated significant variance between the determined LT prevalence and the rate for NCCR, with a low starting copy number in all positive samples and threshold cycles in the range of 36 to 42 representing viral load in the range from 10 to 1000 copies/µl. The results most probably indicate incomplete JC viral replication. Under such conditions, mutations in the host cell genome may be accumulated due to interference of the virus with the host cell machinery, and eventually malignant transformation may occur.

Novel syndromes associated with JC virus infection of neurons and meningeal cells: no longer a gray area.

PURPOSE OF REVIEW: The availability of a growing number of immunomodulatory medications over the past few years has been associated with various JC virus (JCV)-associated brain syndromes in patients with autoimmune diseases, including multiple sclerosis, Crohn's disease, and psoriasis that had not been previously recognized as predisposing factors for progressive multifocal leukoencephalopathy. This review covers the three novel syndromes discovered in the last decade that are caused by JCV infection of neurons and meningeal cells. RECENT FINDINGS: For more than 30 years, JCV was thought to exclusively infect oligodendrocytes and astrocytes in the white matter of the brain of immunosuppressed individuals. We now recognize that JCV-infected glial cells are frequently located at the gray-white matter junction or exclusively within the gray matter causing demyelination in the cortex. Mutations in JCV can trigger a change in tropism leading to involvement of other cell types, such as neurons and meningeal cells, causing clinically distinct entities. These new features of JCV infection provide challenges for clinicians taking care of affected patients and investigators studying the biology of this polyomavirus, its pathogenesis, and tropism. SUMMARY: We hope that increasing awareness of these syndromes will lead to early diagnosis, and pave the way for new avenues of research to better understand all aspects of JCV pathogenesis and develop efficient therapies for our patients. However, we need to remain vigilant and open to the possibility that additional JC variants or yet unknown polyomaviruses may also be associated with neurological diseases.

The prevalence and implications of BK virus replication in non-renal solid organ transplant recipients: A systematic review.

The significance of BK viruria and viremia in non-renal solid organ transplants is poorly understood. A systematic review was performed reviewing the incidence and implications of BK virus replication in non-renal solid organ transplants. Ninety-seven studies were identified, of which 18 including lung, heart, liver and pancreas transplants were included. The overall incidence of BK viruria and viremia was 20% and 3% respectively and 17 cases of BK nephropathy were identified. Heart transplant recipients had a higher overall incidence of BK viremia than other non-renal organ types, and the majority of cases of BK virus-associated nephropathy were in heart transplant recipients. The incidence of BK viremia was significantly lower in non-renal solid organ transplants than that of renal transplant recipients and BK virus-associated nephropathy was rare. BK virus-associated nephropathy may be considered in heart transplant recipients who have unexplained and persistent renal dysfunction not attributable to other causes.

A nucleotide exchange factor promotes endoplasmic reticulum-to-cytosol membrane penetration of the nonenveloped virus simian virus 40.

UNLABELLED: The nonenveloped simian polyomavirus (PyV) simian virus 40 (SV40) hijacks the endoplasmic reticulum (ER) quality control machinery to penetrate the ER membrane and reach the cytosol, a critical infection step. During entry, SV40 traffics to the ER, where host-induced conformational changes render the virus hydrophobic. The hydrophobic virus binds and integrates into the ER lipid bilayer to initiate membrane penetration. However, prior to membrane transport, the hydrophobic SV40 recruits the ER-resident Hsp70 BiP, which holds the virus in a transport-competent state until it is ready to cross the ER membrane. Here we probed how BiP disengages from SV40 to enable the virus to penetrate the ER membrane. We found that nucleotide exchange factor (NEF) Grp170 induces nucleotide exchange of BiP and releases SV40 from BiP. Importantly, this reaction promotes SV40 ER-to-cytosol transport and infection. The human BK PyV also relies on Grp170 for successful infection. Interestingly, SV40 mobilizes a pool of Grp170 into discrete puncta in the ER called foci. These foci, postulated to represent the ER membrane penetration site, harbor ER components, including BiP, known to facilitate viral ER-to-cytosol transport. Our results thus identify a nucleotide exchange activity essential for catalyzing the most proximal event before ER membrane penetration of PyVs. IMPORTANCE: PyVs are known to cause debilitating human diseases. During entry, this virus family, including monkey SV40 and human BK PyV, hijacks ER protein quality control machinery to breach the ER membrane and access the cytosol, a decisive infection step. In this study, we pinpointed an ER-resident factor that executes a crucial role in promoting ER-to-cytosol membrane penetration of PyVs. Identifying a host factor that facilitates entry of the PyV family thus provides additional therapeutic targets to combat PyV-induced diseases.

The endoplasmic reticulum membrane J protein C18 executes a distinct role in promoting simian virus 40 membrane penetration.

UNLABELLED: The nonenveloped simian virus 40 (SV40) hijacks the three endoplasmic reticulum (ER) membrane-bound J proteins B12, B14, and C18 to escape from the ER into the cytosol en route to successful infection. How C18 controls SV40 ER-to-cytosol membrane penetration is the least understood of these processes. We previously found that SV40 triggers B12 and B14 to reorganize into discrete puncta in the ER membrane called foci, structures postulated to represent the cytosol entry site (C. P. Walczak, M. S. Ravindran, T. Inoue, and B. Tsai, PLoS Pathog 10: e1004007, 2014). We now find that SV40 also recruits C18 to the virus-induced B12/B14 foci. Importantly, the C18 foci harbor membrane penetration-competent SV40, further implicating this structure as the membrane penetration site. Consistent with this, a mutant SV40 that cannot penetrate the ER membrane and promote infection fails to induce C18 foci. C18 also regulates the recruitment of B12/B14 into the foci. In contrast to B14, C18's cytosolic Hsc70-binding J domain, but not the lumenal domain, is essential for its targeting to the foci; this J domain likewise is necessary to support SV40 infection. Knockdown-rescue experiments reveal that C18 executes a role that is not redundant with those of B12/B14 during SV40 infection. Collectively, our data illuminate C18's contribution to SV40 ER membrane penetration, strengthening the idea that SV40-triggered foci are critical for cytosol entry. IMPORTANCE: Polyomaviruses (PyVs) cause devastating human diseases, particularly in immunocompromised patients. As this virus family continues to be a significant human pathogen, clarifying the molecular basis of their cellular entry pathway remains a high priority. To infect cells, PyV traffics from the cell surface to the ER, where it penetrates the ER membrane to reach the cytosol. In the cytosol, the virus moves to the nucleus to cause infection. ER-to-cytosol membrane penetration is a critical yet mysterious infection step. In this study, we clarify the role of an ER membrane protein called C18 in mobilizing the simian PyV SV40, a PyV archetype, from the ER into the cytosol. Our findings also support the hypothesis that SV40 induces the formation of punctate structures in the ER membrane, called foci, that serve as the portal for cytosol entry of the virus.

Significant overexpression of the Merkel cell polyomavirus (MCPyV) large T antigen in Merkel cell carcinoma.

BACKGROUND: The purpose of this study was to determine the expression pattern of the Merkel cell polyomavirus (MCPyV) large T-protein antigen in patients with Merkel cell carcinoma. METHODS: A tissue microarray (TMA) containing 30 specimens was constructed and stained for the MCPyV large T protein. Immunohistochemical expression was determined semiquantitively and was compared to patients' outcome. RESULTS: Nuclear expression of MCPyV large T protein was detected in 29 of 30 specimens (97%). In particular, 60% to 100%, 30% to 60%, and 10% to 30% of tumor cells were positive in 27 specimens (90%), 1 (3%), and 1 (3%), respectively. There was no difference in positivity between primary and metastatic lesions. Clinical data could not be correlated to MCPyV large T-protein expression. CONCLUSION: MCPyV large T protein was significantly overexpressed in 97% of all specimens. Although we could not demonstrate a predictive effect, MCPyV large T protein may represent a molecular marker with utility in pathological diagnosis as well as a potential new therapeutic target in patients with Merkel cell carcinoma.

WU and KI polyomaviruses in respiratory samples from allogeneic hematopoietic cell transplant recipients.

Data are limited regarding 2 new human polyomaviruses, KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV), in immunocompromised patients. We used real-time PCR to test for these and 12 respiratory viruses in 2,732 nasal wash samples collected during the first year after allogeneic hematopoietic cell transplantation from 222 patients. Specimens were collected weekly until day 100; then at least every 3 months. One year after hematopoietic cell transplantation, the cumulative incidence estimate was 26% for KIPyV and 8% for WUPyV. Age <20 years predicted detection of KIPyV (hazard ratio [HR] 4.6) and WUPyV (HR 4.4), and detection of a respiratory virus in the previous 2 weeks predicted KIPyV detection (HR 3.4). Sputum production and wheezing were associated with detection of KIPyV in the past week and WUPyV in the past month. There were no associations with polyomavirus detection and acute graft versus host disease, cytomegalovirus reactivation, neutropenia, lymphopenia, hospitalization, or death.

Factors influencing viral clearing and renal function during polyomavirus BK-associated nephropathy after renal transplantation.

BACKGROUND: The course of BK virus nephropathy (BKVN) is difficult to predict. METHODS: Between 2008 and 2010, we diagnosed BKVN in 46 (5.5%) of 859 patients with transplant biopsies by simian virus 40 (SV40) staining and routine serum polymerase chain reaction. We measured the influence of different variables on glomerular filtration rate (ΔGFR increasing or decreasing) and the time for viral polymerase chain reaction reduction by 1 log (≤13 or >13 weeks). At diagnosis, we either reduced calcineurin inhibitor (CNI) and mycophenolate mofetil by 30% to 50% (n=23), or we switched from CNI to mammalian target of rapamycin (mTOR) inhibitor (n=7) or from CNI to mTOR inhibitor as a second step in patients with protracted viral reduction (n=16). Results are the following: GFR stabilized or increased in 61% of patients and decreased in 39% (graft failure, 15%). Viral reduction by 1 log was rapid in 54% (≤13 weeks) and slow in 46% (>13 weeks). Rapid viral reduction was associated with stable or increasing GFR (84%), compared with slow viral reduction (33%; P=0.0004). High peak viral load, tacrolimus treatment, and late diagnosis (biopsy for cause vs. protocol biopsy) had a negative influence on GFR and viral reduction time. Defining 1-log viral load reduction as an event, tacrolimus compared with cyclosporine was associated with slow viral reduction (P=0.0043). In 88% of patients with slow viral reduction, the secondary switch from CNI to mTOR inhibitor favored viral load decrease. CONCLUSIONS: We conclude that peak viral load, tacrolimus treatment, delayed diagnosis, and viral reduction time influence outcomes in patients with BKVN.

Polyomavirus BK replication in liver transplant candidates with normal renal function.

Polyomavirus-associated nephropathy (PVAN) has a predilection for kidney rather than for other solid organ transplants such as the liver. Immunosuppression is widely recognized to be a major risk factor for PVAN development. Since end-stage liver disease (ESLD) patients are immunocompromised and immunosuppression is a major cause of BK virus reactivation, we sought to evaluate BK virus replication in patients listed for liver transplantation. From April to October 2010, we enrolled 20 patients listed for liver transplantation. BK virus load was measured by quantitative real-time polymerase chain reaction on plasma and urine samples. Viremia occurred in only 1 among 20 patients. We hypothesized that in ESLD patients, the low prevalence of BK virus infection may be related to the prevalent impairment of antibacterial immunity rather than to the viral-specific one. In BK virus reactivation, not only the immunodepressive state itself, but also the specific immunologic mechanisms involved may have a role.