Analysis of Plant-Specific ANTH Domain-Containing Protein in Marchantia polymorpha.

Membrane trafficking is a fundamental mechanism for protein and lipid transport in eukaryotic cells and exhibits marked diversity among eukaryotic lineages with distinctive body plans and lifestyles. Diversification of the membrane trafficking system is associated with the expansion and secondary loss of key machinery components, including RAB GTPases, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and adaptor proteins, during plant evolution. The number of AP180 N-terminal homology (ANTH) proteins, an adaptor family that regulates vesicle formation and cargo sorting during clathrin-mediated endocytosis, increases during plant evolution. In the genome of Arabidopsis thaliana, 18 genes for ANTH proteins have been identified, a higher number than that in yeast and animals, suggesting a distinctive diversification of ANTH proteins. Conversely, the liverwort Marchantia polymorpha possesses a simpler repertoire; only two genes encoding canonical ANTH proteins have been identified in its genome. Intriguingly, a non-canonical ANTH protein is encoded in the genome of M. polymorpha, which also harbors a putative kinase domain. Similar proteins have been detected in sporadic lineages of plants, suggesting their ancient origin and multiple secondary losses during evolution. We named this unique ANTH group phosphatidylinositol-binding clathrin assembly protein-K (PICALM-K) and characterized it in M. polymorpha using genetic, cell biology-based and artificial intelligence (AI)-based approaches. Our results indicate a flagella-related function of MpPICALM-K in spermatozoids, which is distinct from that of canonical ANTH proteins. Therefore, ANTH proteins have undergone significant functional diversification during evolution, and PICALM-K represents a plant-unique ANTH protein that is delivered by neofunctionalization through exon shuffling.

Immune Prophylaxis and Therapy for Human Cytomegalovirus Infection.

Human Cytomegalovirus (HCMV) infection is widespread and can result in severe sequelae in susceptible populations. Primary HCMV infection of naïve individuals results in life-long latency characterized by frequent and sporadic reactivations. HCMV infection elicits a robust antibody response, including neutralizing antibodies that can block the infection of susceptible cells in vitro and in vivo. Thus, antibody products and vaccines hold great promise for the prevention and treatment of HCMV, but to date, most attempts to demonstrate their safety and efficacy in clinical trials have been unsuccessful. In this review we summarize publicly available data on these products and highlight new developments and approaches that could assist in successful translation of HCMV immunotherapies.

Responsiveness to basement membrane extract as a possible trait for tumorigenicity characterization.

Immortalized cell lines used to produce vaccines are expected to be described in terms of their tumorigenicity. However, current in vivo tumorigenicity assays can be time-consuming and results can be equivocal, especially for weakly tumorigenic cells. Basement membrane extract (BME) derived from the Engelbreth-Holm-Swarm mouse tumor, such as Matrigel and Cultrex, consists of laminin, collagen IV, entactin, heparan sulfate, and proteoglycans, as well as biologically active peptides and growth factors. For nearly three decades, BME has been used in cancer research to enhance tumorigenicity assays (both tumor "take" as well as tumor growth are substantially improved). We assessed the feasibility of using BME to facilitate the evaluation of vaccine cell substrate tumorigenicity. Vero cells (WHO 10-87) were serially passaged and banked at every ten passages beginning with p140; for the present study, low-passage Vero cells (Vero LP, originating from cells banked at p140) and high-passage Vero cells (Vero HP, originating from cells banked at p250) were used. In addition, Vero TPX2 and Vero NM1, cell lines established from tumors formed in nude mice by Vero HP cells, as well as other cell lines relevant to vaccine production (HeLa, MDCK, 293, and ARPE-19), were assessed. Female adult athymic nude mice were injected subcutaneously with cells in the absence or presence of BME. We observed that the tumorigenicity of ARPE-19 cells as well as Vero cells below passage 258 (Vero LP and Vero HP; previously characterized as non-tumorigenic or weakly tumorigenic, respectively) was not enhanced by BME. In contrast, BME shortened the latency and decreased the tumor-producing cell dose of HeLa, 293, and MDCK cells as well as the tumorigenic Vero derivatives TPX2 and NM1. Thus, responsiveness to BME may reflect the status of the neoplastic process and possibly serve as a useful trait for better defining the tumorigenic phenotype of cells.

Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response.

Human cytomegalovirus (HCMV) is a major cause of disability in congenitally infected infants and in the immunosuppressed. There is currently no licensed prophylactic HCMV vaccine. The HCMV envelope glycoprotein B (gB) is considered a major vaccine target antigen based on its critical role in mediating viral-host cell fusion and thus viral entry. The natural conformation of HCMV gB within the viral envelope is a trimer, but there has been no reported success in producing a recombinant trimeric gB suitable for vaccine use. Phase II clinical trials of a monomeric recombinant gB protein demonstrated 50% efficacy in preventing HCMV infection in seronegative women of reproductive age, and in reducing viremia in solid organ transplantation recipients. We now report the production of a uniformly trimeric recombinant HCMV gB protein in Chinese ovary cells, as demonstrated by Western blot analysis under modified non-reducing conditions and size exclusion chromatography with multi-angle scattering. Immunization of mice with trimeric HCMV gB induced up to 11-fold higher serum titers of total gB-specific IgG relative to monomeric HCMV gB using Alum + CpG as adjuvants. Further, trimeric HCMV gB elicited 50-fold higher complement-independent and 20-fold higher complement-dependent HCMV neutralizing titers compared to monomeric HCMV gB using the fibroblast cell line, MRC-5, and up to 6-fold higher complement-independent and -dependent HCMV neutralizing titers using the epithelial cell line, ARPE-19. The markedly enhanced HCMV neutralizing activity in response to trimeric HCMV gB was also observed using an additional four distinct clinical HCMV isolates. These data support a role for trimeric HCMV gB as an important component for clinical testing of a prophylactic HCMV vaccine.

Binding and neutralizing anti-cytomegalovirus activities in immune globulin products.

Congenital infection as well as infection of immunocompromised individuals by cytomegalovirus (CMV) can be associated with significant morbidity, mortality, and long-term adverse health outcomes. Assessment of anti-viral activity using appropriate assays is essential for ensuring safe and efficacious use of therapeutic CMV immune globulin (IG) products. In this study, we used commercial ELISA kits to compare anti-CMV antibody binding activity and avidity for lots of CMV-specific and normal IG products available in the US market. Additionally, neutralizing activity of IG products was measured against CMV strains (AD169wt131 or TB40E-GFP) in MRC-5 human fibroblasts and ARPE-19 human epithelial cells. Our data revealed that, regardless of the method, anti-CMV activity was higher in CMV IG lots we tested compared with normal IG lots; CMV binding activity was at least 4-fold higher, and neutralizing activity at least 2- and 3-fold higher for epithelial and fibroblast cells, respectively, in CMV IG lots compared with normal IG lots. Furthermore, anti-CMV activity values from all three methods (ELISA, neutralization in MRC-5 cells, and neutralization in ARPE-19 cells) were highly correlated, whereas avidity, although higher in CMV IG lots, did not correlate well with either binding or neutralizing activities.

Data on the inhibition of RNase inhibitor activity by a monoclonal antibody as assessed by microfluidics-based RNA electrophoresis.

Using purified reaction components, a commercial monoclonal antibody (Ab) specific to RNase inhibitor (RI) was found to interfere with the activity of RI. Total RNA was mixed with a monoclonal Ab specific to either RI (clone 3F11) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNase A, RI, or a combination of the above. Following incubation for 1 h at 22 °C or 37 °C, RNA integrity of the mixtures was assessed using microfluidics-based Bio-Rad Experion RNA electrophoresis. The addition of Ab 3F11 prevented RI from effectively inhibiting RNase A and therefore resulted in extensive RNA degradation. The data presented are associated with the research article entitled "Endogenous RNase Inhibitor Contributes to Stability of RNA in Crude Cell Lysates: Applicability to Reverse Transcription Quantitative PCR (RT-qPCR)" (Wang et al., 2016) [1].

Endogenous RNase inhibitor contributes to stability of RNA in crude cell lysates: Applicability to RT-qPCR.

Crude cell lysates are increasingly used as input for direct analysis by reverse transcription quantitative PCR (RT-qPCR), particularly for high-throughput applications. We previously demonstrated that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercial cell-lysis reagents for the preparation of RT-qPCR-ready cell lysates; addition of an exogenous RNase inhibitor (RI) to the lysis buffer was found to be unnecessary to maintain RNA stability in cell lysates either freshly prepared or previously stored frozen at -80 °C. In the present study, we have demonstrated that the stability of RNA observed in our cell lysates is due to the presence of the endogenous RI. Furthermore, we have established the generalizability and applicability of this phenomenon by evaluating lysates prepared from cell lines commonly used in virology (A549, HeLa, MDCK, and Vero). Awareness of the mechanism underlying RNA stability may engender greater confidence in generating cell lysates for RT-qPCR without relying on addition of exogenous RI (a substantial cost-saving benefit) and encourage appropriate practices for handling and storage of samples.

RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.

Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination and natural infection.

A mouse strain defective in both T cells and NK cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated H-Ras and c-Myc.

As part of safety studies to evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cells, we have been developing in vivo assays to detect and quantify the oncogenic activity of DNA. We generated a plasmid expressing both an activated human H-ras gene and murine c-myc gene and showed that 1 µg of this plasmid, pMSV-T24-H-ras/MSV-c-myc, was capable of inducing tumors in newborn NIH Swiss mice. However, to be able to detect the oncogenicity of dominant activated oncogenes in cellular DNA, a more sensitive system was needed. In this paper, we demonstrate that the newborn CD3 epsilon transgenic mouse, which is defective in both T-cell and NK-cell functions, can detect the oncogenic activity of 25 ng of the circular form of pMSV-T24-H-ras/MSV-c-myc. When this plasmid was inoculated as linear DNA, amounts of DNA as low as 800 pg were capable of inducing tumors. Animals were found that had multiple tumors, and these tumors were independent and likely clonal. These results demonstrate that the newborn CD3 epsilon mouse is highly sensitive for the detection of oncogenic activity of DNA. To determine whether it can detect the oncogenic activity of cellular DNA derived from four human tumor-cell lines (HeLa, A549, HT-1080, and CEM), DNA (100 µg) was inoculated into newborn CD3 epsilon mice both in the presence of 1 µg of linear pMSV-T24-H-ras/MSV-c-myc as positive control and in its absence. While tumors were induced in 100% of mice with the positive-control plasmid, no tumors were induced in mice receiving any of the tumor DNAs alone. These results demonstrate that detection of oncogenes in cellular DNA derived from four human tumor-derived cell lines in this mouse system was not possible; the results also show the importance of including a positive-control plasmid to detect inhibitory effects of the cellular DNA.

MicroRNAs as potential biomarkers for VERO cell tumorigenicity.

MicroRNA expression appears to capture the process of neoplastic development in vitro in the VERO line of African green monkey kidney (AGMK) cells (Teferedegne et al. PLoS One 2010;5(12):e14416). In that study, specific miRNA signatures were correlated with the transition, during serial tissue-culture passage, of low-density passaged 10-87 VERO cells from a non-tumorigenic phenotype at passage (p) 148 to a tumorigenic phenotype at p256. In the present study, six miRNAs (miR-376a, miR-654-3p, miR-543, miR-299-3p, miR-134 and miR-369-3p) were chosen from the identified signature miRNAs for evaluation of their use as potential biomarkers to track the progression of neoplastic development in VERO cells. Cells from the 10-87 VERO cell line at passage levels from p148 to p256 were inoculated into newborn and adult athymic nude mice. No tumors were observed in animals inoculated with cells from p148 to p186. In contrast, tumor incidences of 20% developed only in newborn mice that received 10-87 VERO cells at p194, p234 and p256. By qPCR profiling of the signature miRNAs of 10-87 VERO cells from these cell banks, we identified p194 as the level at which signature miRNAs elevated concurrently with the acquisition of tumorigenic phenotype with similar levels expressed beyond this passage. In wound-healing assays at 10-passage intervals between p150 to p250, the cells displayed a progressive increase in migration from p165 to p186; beginning at p194 and higher passages thereafter, the cells exhibited the highest rates of migration. By qPCR analysis, the same signature miRNAs were overexpressed with concomitant acquisition of the tumorigenic phenotype in another lineage of 10-87 VERO cells passaged independently at high density. Correlation between the passages at which the cells expressed a tumorigenic phenotype and the passages representing peaks in expression levels of signature miRNAs indicates that these miRNAs are potential biomarkers for the expression of the VERO cell tumorigenic phenotype.

A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR.

Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37 °C incubation for 1-2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at -80 °C.

Development of a luciferase immunoprecipitation system assay to detect IgG antibodies against human respiratory syncytial virus nucleoprotein.

The nucleoprotein of respiratory syncytial virus (RSV-N) is immunogenic and elicits an IgG response following infection. The RSV-N gene was cloned into a mammalian expression vector, pREN2, and the expressed luciferase-tagged protein (Ruc-N) detected anti-RSV-N-specific IgG antibodies using a high-throughput immunoprecipitation method (the luciferase immunoprecipitation system [LIPS]-N(RSV) assay). The specificity of the assay was evaluated using monoclonal antibodies (MAbs) and monospecific pre- and postimmunization rabbit antisera. Blood serum samples from chimpanzees and humans with proven/probable RSV infection were also tested. The pre- and postimmunization serum samples from rabbits given human metapneumovirus (HMPV) or measles virus were negative when tested by the LIPS-N(RSV) assay, while antisera obtained after immunization with either the RSV-A or RSV-B strain gave positive signals in a dose-dependent manner. RSV-N MAb 858-3 gave a positive signal in the LIPS-N(RSV) assay, while MAbs against other paramyxovirus nucleoproteins or RSV-F or RSV-G did not. Serum samples from chimpanzees simultaneously immunized with vaccinia-RSV-F and vaccinia-RSV-G recombinant viruses were negative in the LIPS-N(RSV) assay; however, anti-RSV-N IgG responses were detected following subsequent RSV challenge. Seven of the 12 infants who were seronegative at 9 months of age had detectable anti-RSV-N antibodies when they were retested at 15 to 18 months of age. The LIPS-N(RSV) assay detects specific anti-RSV-N IgG responses that may be used as a biomarker of RSV infection.

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment.

BACKGROUND: Few studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting. METHODS: Virus/antibody mixtures were incubated for one hour at 37°C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies. RESULTS: We have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law). CONCLUSIONS: This qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.

Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50)). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.

Mutations in the GM1 binding site of simian virus 40 VP1 alter receptor usage and cell tropism.

Polyomaviruses are nonenveloped viruses with capsids composed primarily of 72 pentamers of the viral VP1 protein, which forms the outer shell of the capsid and binds to cell surface oligosaccharide receptors. Highly conserved VP1 proteins from closely related polyomaviruses recognize different oligosaccharides. To determine whether amino acid changes restricted to the oligosaccharide binding site are sufficient to determine receptor specificity and how changes in receptor usage affect tropism, we studied the primate polyomavirus simian virus 40 (SV40), which uses the ganglioside GM1 as a receptor that mediates cell binding and entry. Here, we used two sequential genetic screens to isolate and characterize viable SV40 mutants with mutations in the VP1 GM1 binding site. Two of these mutants were completely resistant to GM1 neutralization, were no longer stimulated by incorporation of GM1 into cell membranes, and were unable to bind to GM1 on the cell surface. In addition, these mutant viruses displayed an infection defect in monkey cells with high levels of cell surface GM1. Interestingly, one mutant infected cells with low cell surface GM1 more efficiently than wild-type virus, apparently by utilizing a different ganglioside receptor. Our results indicate that a small number of mutations in the GM1 binding site are sufficient to alter ganglioside usage and change tropism, and they suggest that VP1 divergence is driven primarily by a requirement to accommodate specific receptors. In addition, our results suggest that GM1 binding is required for vacuole formation in permissive monkey CV-1 cells. Further study of these mutants will provide new insight into polyomavirus entry, pathogenesis, and evolution.

Plaque purification as a method to mitigate the risk of adventitious-agent contamination in influenza vaccine virus seeds.

At present, the seed viruses for the manufacture of licensed seasonal inactivated influenza vaccines in the United States are derived from primary egg isolates as a result of concerns associated with adventitious agents. According to the prevailing view, the passage of influenza viruses through eggs serves as a filtering step to remove potential contaminating viruses. We have investigated the feasibility of addressing adventitious-agent risk by subjecting influenza virus to a plaque-purification procedure using MDCK cells. SV40 and canine adenovirus-1 (representing viruses for which MDCK cells are non-permissive and permissive, respectively) were used as challenge viruses to model agents of concern that might be co-isolated along with the influenza virus. By mixing influenza virus strain A/PR/8/34 with varying amounts of each challenge virus and then performing a plaque assay for influenza virus using MDCK cells, we have attempted to determine the efficiency by which the challenge virus is removed. Our data suggest that substantial removal can be achieved even after a single round of plaque purification. If cell-derived isolates were deemed to be acceptable following a plaque-purification procedure, the manufacture of seasonal influenza vaccine would be facilitated by: (1) the expansion of the repertoire of viruses from which seed virus candidates could be generated for licensed egg-derived vaccines as well as for vaccines manufactured in mammalian cells; and (2) the mitigation of adventitious-agent risk associated with the seed virus, and hence the elimination of the need to passage seed viruses in eggs for vaccines manufactured in mammalian cells.

Patterns of microRNA expression in non-human primate cells correlate with neoplastic development in vitro.

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines), progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

A quantitative PCR assay for SV40 neutralization adaptable for high-throughput applications.

A neutralization assay incorporating a quantitative SYBR Green PCR endpoint has been developed for SV40. The present study demonstrates that crude virus samples can serve as suitable amplification templates for quantitative PCR without the need for nucleic acid extraction. The denaturation temperature of thermocycling appears to be sufficient to release the encapsidated viral genome and allow its availability as a PCR template. Issues arising from inhibitors of PCR present in crude virus samples can be circumvented easily by a 100-fold dilution step. Using a streamlined procedure that eliminates sample nucleic acid extraction (a hitherto rate-limiting step that diminishes throughput substantially), quantitative PCR was applied in order to assess: (1) the replication kinetics of SV40 and (2) the inhibition of SV40 productive infection by neutralizing antibodies. A similar high-throughput approach might be feasible for related polyomaviruses (e.g., BKV and JCV) as well as for other families of viruses.

Identification of a neutralization epitope in the VP1 capsid protein of SV40.

Three SV40 escape mutants were identified by selection in the presence of monoclonal antibodies with neutralizing activity. The VP1 amino acid alterations in these mutants were: (1) K73-->E (in loop BC); (2) D77-->E (in loop BC); (3) K171-->R (in loop EF); and (4) Q175-->H (in loop EF). These residues are clustered in close proximity to each other on the surface of the native capsid protein, strongly suggesting that they form a conformational epitope directly recognized by the neutralizing antibody. To our knowledge, the present study represents the first experimental mapping of a neutralization epitope of a polyomavirus family member. Structural information regarding the neutralization epitope should be useful for clarifying the extent of cross-reactivity exhibited by the humoral immune response towards related primate polyomaviruses (e.g., SV40, BKV, and JCV).

Identification of a mutation in the SV40 capsid protein VP1 that influences plaque morphology, vacuolization, and receptor usage.

A plaque variant of SV40 that was first isolated in the 1960s, designated SV40-LP(KT), was molecularly cloned and subjected to sequence analysis. The genome of SV40-LP(KT) was found to be nearly identical to the previously described isolate known as 777. However, SV40-LP(KT) contained a mutation in the VP1 coding region resulting in a change of histidine 136 to tyrosine. This VP1 mutation was identified as a genetic determinant influencing a number of phenotypes associated with SV40-LP(KT) such as plaque morphology, intracellular vacuole formation, and ganglioside receptor usage.