Polyomavirus BK-encoded microRNA suppresses autoregulation of viral replication

Polyomavirus BK (BKV) infection is an important cause of renal allograft failure. Viral microRNAs are known to play a crucial role in viral replication. This study investigated the expression of BKV-encoded microRNAs (miR-B1) in patients with polyomavirus-associated nephropathy (PVAN) and their role in viral replication. Following BKV infection in renal proximal tubular cells, the 3p and 5p miR-B1 levels were significantly increased. Cells transfected with the vector containing the miR-B1 precursor (the miR-B1 vector) showed a significant increase in expression of 3p and 5p miR-B1 and decrease in luciferase activity of a reporter containing the 3p and 5p miR-B1 binding sites, compared to cells transfected with the miR-B1-mutated vector. Transfection of the miR-B1 expression vector or the 3p and 5p miR-B1 oligonucleotides inhibited expression of TAg. TAg-enhanced promoter activity and BKV replication were inhibited by miR-B1. In contrast, inhibition of miR-B1 expression by addition of miR-B1 antagomirs or silencing of Dicer upregulated the expression of TAg and VP1 proteins in BKV-infected cells. Importantly, patients with PVAN had significantly higher levels of 3p and 5p miR-B1 compared to renal transplant patients without PVAN. In conclusion, we demonstrated that (1) miR-B1 expression was upregulated during BKV infection and (2) miR-B1 suppressed TAg-mediated autoregulation of BKV replication. Use of miR-B1 can be evaluated as a potential treatment strategy against BKV infection.     

疱疹病毒膜融合的分子机制

囊膜病毒与宿主细胞的膜融合是病毒入侵宿主细胞的重要过程,这一过程涉及到病毒囊膜表面糖蛋白与宿主细胞表面受体之间的相互作用和构象变化．疱疹病毒有多个糖蛋白及不同类型的细胞作用受体,相应的受体-糖蛋白复合体构成方式也有多种,其引致的膜融合机制被认为是目前病毒融合机制研究中最复杂的,近年来被广泛研究并取得突破性进展．从病毒糖蛋白与相应受体的结构与功能、受体-糖蛋白复合体的形成与入侵途径,以及膜融合模式几个方面,全面综述疱疹病毒膜融合的分子机制,并展望了未来研究趋势．     

Substrate modulation of enzyme activity in the herpesvirus protease family

The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.     

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan^® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-10⁶ copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.     

A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L^d by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L^d, “mini-H2-L^d,” consisting of only the α1α2 platform domain. Mini-H2-L^d refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β₂-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β₂-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L^d MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.     

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.     

Divergent target recognition by coexpressed 5′-isomiRs of miR-142-3p and selective viral mimicry

Sequence heterogeneity at the ends of mature microRNAs (miRNAs) is well documented, but its effects on miRNA function are largely unexplored. Here we studied the impact of miRNA 5′-heterogeneity, which affects the seed region critical for target recognition. Using the example of miR-142-3p, an emerging regulator of the hematopoietic lineage in vertebrates, we show that naturally coexpressed 5′-variants (5′-isomiRs) can recognize largely distinct sets of binding sites. Despite this, both miR-142-3p isomiRs regulate exclusive and shared targets involved in actin dynamics. Thus, 5′-heterogeneity can substantially broaden and enhance regulation of one pathway. Other 5′-isomiRs, in contrast, recognize largely overlapping sets of binding sites. This is exemplified by two herpesviral 5′-isomiRs that selectively mimic one of the miR-142-3p 5′-isomiRs. We hypothesize that other cellular and viral 5′-isomiRs can similarly be grouped into those with divergent or convergent target repertoires, based on 5′-sequence features. Taken together, our results provide a detailed characterization of target recognition by miR-142-3p and its 5′-isomiR-specific viral mimic. We furthermore demonstrate that miRNA 5′-end variation leads to differential targeting and can thus broaden the target range of miRNAs.     

Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.     

Molecular and in vitro Characterization of Field Isolates of Bovine Herpesvirus-1

Bovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of this study was to isolate and to characterize (molecular and biological characterization) BoHV-1 from 29 immunosuppressed animals. It was possible to obtain 18 isolates, each from a different animal, such as from the respiratory and reproductive tracts. In some cases the cytopathic effect was visible 12 hours post-inoculation, and became characteristic after 36-48 hours. Biological characteristics were evaluated and compared with Iowa and Colorado-1 reference strains, and differences were found in plaque size, virus titer measured by TCID50 and PFU/mL, and one step virus curves. These results showed that some isolates had a highly virulent-like behavior in vitro, compared to the reference strains, with shorter eclipse periods, faster release of virus into the supernatants, and higher burst size and viral titer. There were no differences in glycoprotein expression of BoHV-1 isolates, measured by Western blot on monolayers. Moreover, using restriction endonucleases analysis, most of the viruses were confirmed as BoHV-1.1 and just one of them was confirmed as BoHV-1.2a subtype. These findings suggest that some wild-type BoHV-1 isolates could be useful as seeds to develop new monovalent vaccines.     

Sorting and Transport of Alpha Herpesviruses in Axons

The alpha herpesviruses, a subfamily of the herpesviruses, are neurotropic pathogens found associated with most mammalian species. The prototypic member of this subfamily is herpes simplex virus type 1, the causative agent of recurrent cold sores in humans. The mild nature of this disease is a testament to the complex and highly regulated life cycle of the alpha herpesviruses. The cellular mechanisms used by these viruses to disseminate infection in the nervous system are beginning to be understood. Here, we overview the life cycle of alpha herpesviruses with an emphasis on assembly and transport of viral particles in neurons.