Genetic Diversity of the Noncoding Control Region of the Novel Human Polyomaviruses

The genomes of polyomaviruses are characterized by their tripartite organization with an early region, a late region and a noncoding control region (NCCR). The early region encodes proteins involved in replication and transcription of the viral genome, while expression of the late region generates the capsid proteins. Transcription regulatory sequences for expression of the early and late genes, as well as the origin of replication are encompassed in the NCCR. Cell tropism of polyomaviruses not only depends on the appropriate receptors on the host cell, but cell-specific expression of the viral genes is also governed by the NCCR. Thus far, 15 polyomaviruses have been isolated from humans, though it remains to be established whether all of them are genuine human polyomaviruses (HPyVs). The sequences of the NCCR of these HPyVs show high genetic variability and have been best studied in the human polyomaviruses BK and JC. Rearranged NCCRs in BKPyV and JCPyV, the first HPyVs to be discovered approximately 30 years ago, have been associated with the pathogenic properties of these viruses in nephropathy and progressive multifocal leukoencephalopathy, respectively. Since 2007, thirteen novel PyVs have been isolated from humans: KIPyV, WUPyV, MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, HPyV10, STLPyV, HPyV12, NJPyV, LIPyV and QPyV. This review describes all NCCR variants of the new HPyVs that have been reported in the literature and discusses the possible consequences of NCCR diversity in terms of promoter strength, putative transcription factor binding sites and possible association with diseases.

Biological and molecular characterization of six Shiga toxin-producing Escherichia coli (STEC) strains encoding the stx2 gene in Colombia

ABSTRACTShiga toxin-producing Escherichia coli (STEC) is a bacterial pathogen that cause diarrhea and severe human diseases. Its principal virulence factor are the Shiga toxins Stx1 and Stx2 which have been identified diverse subtypes considered to be responsible for severe complications of STEC infection. These toxins are encoded in temperate bacteriophages and their expression is linked to phage lithic cycle, which is regulated by late genes and the Q anti-terminator protein. The aim of this study was to characterize biologically and molecularly STEC strains encoding stx2 gene isolated from cattle feces in Colombia. We selected six STEC strains, which were evaluated its Stx production, the Stx2 subtypes, induction of the lithic cycle of bacteriophages and its late region. The results evidenced two highlighted strains with high levels of Stx production and induction of the lithic cycle, compared with the others. Likewise, the strains evaluated showed three Stx2 subtypes: Stx2a, Stx2c, and Stx2d. Regarding the late region, most of the strains carried the qO111 allele and only one strain showed differences in the ninG gene. Although the sample was limited, variability was observed in the Stx production assay, induction of the lithic cycle, Stx2 subtypes and late region of the phages, which could indicate the diversity of the phages carrying STEC strains in Colombia.

Agnoprotein is an Essential Egress Factor during BK Polyomavirus Infection

BK polyomavirus (BKPyV) causes a lifelong chronic infection and is associated with debilitating disease in kidney transplant recipients. Despite its importance, aspects of the virus life cycle remain poorly understood. In addition to the structural proteins, the late region of the BK genome encodes for an auxiliary protein called agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to virion infectivity. Here, we demonstrate an essential role for agnoprotein in BK virus release. Viruses lacking agnoprotein do not propagate to wild-type levels and fail to release from host cells. Despite this, loss of agnoprotein does not impair virion infectivity or morphogenesis. Instead, agnoprotein expression correlates with nuclear egress of BK virions. We demonstrate that the agnoprotein binding partner α-SNAP is necessary for BK virion release, and siRNA knockdown of α-SNAP prevents nuclear release of wild-type BK virions. These data highlight a novel role for agnoprotein and begin to reveal the mechanism by which polyomaviruses leave an infected cell.

Molecular characterization of the first polyomavirus from a New World primate: squirrel monkey polyomavirus

DNA samples from a variety of New World monkeys were screened by using a broad-spectrum PCR targeting the VP1 gene of polyomaviruses. This resulted in the characterization of the first polyomavirus from a New World primate. This virus naturally infects squirrel monkeys (Saimiri sp.) and is provisionally named squirrel monkey polyomavirus (SquiPyV). The complete genome of SquiPyV is 5075 bp in length, and encodes the small T and large T antigens and the three structural proteins VP1, VP2 and VP3. Interestingly, the late region also encodes a putative agnoprotein, a feature that it shares with other polyomaviruses from humans, baboons and African green monkeys. Comparison with other polyomaviruses revealed limited sequence similarity to any other polyomavirus, and phylogenetic analysis of the VP1 gene confirmed its uniqueness.

High quality DNA from human papillomavirus (HPV) for PCR/RFLPs

The analysis of DNA in clinical samples for a secure diagnostic has become indispensable nowadays. Techniques approaching isolation of high molecular weigth DNA of HPV could lead to efficient amplification and early clinical diagnosis of the virus DNA by PCR (polymerase chain reaction). We describe a fast, non-toxical, efficient and cheap method for DNA isolation of human papilloma virus (HPV) from cervical smears using guanidine (DNAzol solution). A 450 bp DNA band correponding to the late region (L1) of the virus genome was detected by PCR, showing that the DNAzol extraction soluction generated a good viral DNA yield. The electrophoretic pattern after digestion with restriction endonucleases (RFLPs/PCR) revealed the predominance of HPV-16 and HPV-33 in the samples from the State of Alagoas, Brazil.

Expression of aquaporins in the renal connecting tubule

The renal connecting tubule (CNT) is a distinct segment that occurs between the distal convoluted tubule (DCT) and the cortical collecting duct. On the basis of its characterization in rabbit it is widely believed that connecting tubule cells have a low permeability to water and do not respond to vasopressin. Here we utilize segment-specific markers and specific aquaporin antibodies to characterize expression of water channels in CNT of the rat by immunocytochemistry. Colocalization of aquaporin 2 (AQP2), AQP3, and AQP4 with Na+, Ca2+ exchanger (NCX), a transporter characteristic of the connecting tubule, gave heterogeneous labeling. There was aquaporin labeling in many but not all regions labeled by NCX. Colocalization of AQP2 with AQP3 and with AQP4 showed that AQP3 and AQP4 labeling were always accompanied by AQP2. Immunogold labeling and electron microscopy showed that NCX-labeled cells with AQP2 labeling had the morphology of CNT cells, whereas NCX-labeled cells without AQP2 labeling were DCT cells. The latter regions were identified as the late region of the DCT known as DCT2. Additionally, regions of CNT lacking AQP2 labeling could be identified in Brattleboro rats not treated with vasopressin but not in such animals chronically treated with deamino-Cys1,d-Arg8-vasopressin (dDAVP). Quantitative analysis of labeling was consistent with expression of AQP2 over a longer region of CNT after dDAVP exposure.

Nucleotide Sequence, Genome Organization, and Transcription Map of Bovine Adenovirus Type 3

ABSTRACT The complete DNA sequence of bovine adenovirus type 3 is reported here. The size of the genome is 34,446 bp in length with a G+C content of 54%. All the genes of the early and late regions are present in the expected locations of the genome. However, the late-region genes are organized into seven families, instead of five as they are in human adenovirus type 2. The deduced amino acid sequences of open reading frames (ORFs) in the late regions and early region 2 (E2) and for IVa2 show higher degrees of homology, whereas the predicted amino acid sequences of ORFs in the E1, E3, and E4 regions and the pIX, fiber, and 33,000-molecular-weight nonstructural proteins show little or no homology with the corresponding proteins of other adenoviruses. In addition, the penton base protein lacks the integrin binding motif, RGD, but has an LDV motif instead of an MDV motif. Interestingly, as in other animal adenoviruses, the virus-associated RNA genes appear to be absent from their usual location. Sequence analysis of cDNA clones representing the early- and late-region genes identified splice acceptor and splice donor sites, polyadenylation signals and polyadenylation sites, and tripartite leader sequences.