Parvovirus Diversity and DNA Damage Responses

Parvoviruses have a linear single-stranded DNA genome, around 5 kb in length, with short imperfect terminal palindromes that fold back on themselves to form duplex hairpin telomeres. These contain most of the cis-acting information required for viral “rolling hairpin” DNA replication, an evolutionary adaptation of rolling-circle synthesis in which the hairpins create duplex replication origins, prime complementary strand synthesis, and act as hinges to reverse the direction of the unidirectional cellular fork. Genomes are packaged vectorially into small, rugged protein capsids ∼260 Å in diameter, which mediate their delivery directly into the cell nucleus, where they await their host cell’s entry into S phase under its own cell cycle control. Here we focus on genus-specific variations in genome structure and replication, and review host cell responses that modulate the nuclear environment.Viruses from the family Parvoviridae are unique in having a linear single-stranded DNA genome, ∼5 kb in length, which terminates in short (120–420 base) imperfect palindromes that fold into self-priming hairpin telomeres. These viruses replicate via a “rolling hairpin” mechanism, with strong evolutionary and mechanistic links to “rolling-circle” replication, as reviewed in detail in previous editions of this work (Cotmore and Tattersall 1996, 2006). Rolling hairpin synthesis relies on the ability of each hairpin to give rise to a duplex origin sequence, which can be nicked by a viral initiator nuclease to create a base-paired DNA primer, and to function as a hinge, allowing quasi-circular synthesis by alternately folding and unfolding to shuttle a continuous unidirectional replication fork back and forth along linear DNA. Together with a few adjacent nucleotides, these palindromes provide all of the cis-acting information required for viral DNA replication and packaging. However, the size, sequence, and predicted structures of the hairpins can vary substantially between genera, or even between the two ends of a single genome, and they appear to have adapted to fulfill multiple additional roles in the life cycle of specific viruses. Parvoviral DNA amplification proceeds via a unidirectional single-strand displacement mechanism through a series of monomeric and concatemeric duplex replicative-form (RF) intermediates, and while the viral initiator protein, variously called NS1 or Rep, serves both as a site- and strand-specific nickase and as a 3′-to-5′ helicase, all other replicative functions are co-opted from the host cell. This mechanism benefits from suppression of host DNA synthesis, and generates long stretches of single-stranded DNA with alien terminal structures that invoke host damage responses, which impact both positively and negatively on viral replication. Although details of the mechanisms that mediate parvoviral replication have received relatively little attention since our previous review (Cotmore and Tattersall 2006), recognition that infection is invariably associated with host DNA damage responses (DDRs), some of which are specifically required for efficient viral DNA amplification, has led to significant reappraisal of the nuclear environment and replicative machinery available to these viruses. In parallel, major advances have occurred in our knowledge of genome diversity and cell specificity in this ever-expanding family, which provide novel insight into mechanisms of replication control.The International Committee on Taxonomy of Viruses (ICTV, Tijssen et al. 2011), classifies viruses in the family Parvoviridae that infect vertebrates as the subfamily Parvovirinae, which currently contains just five genera: the Parvoviruses, Dependoviruses, Amdoviruses, Erythroviruses, and Bocaviruses, although there are at least two additional genera, tentatively called Partetraviruses and Avetalviruses, that await ICTV recognition. This reflects a major jump in known virus diversity, with many new species and genera first identified in clinical or veterinary samples using PCR-based virus discovery methods (Allander et al. 2005; Jones et al. 2005; Day and Zsak 2010). Potential human pathogens that are still pending recognition include genetic variants of Human Bocavirus (HBoV 1–4), which are particularly common in the respiratory and gastrointestinal tracts of children (Kapoor et al. 2010; Kantola et al. 2011), and two broadly distributed genotypes of a “PARV4”-based genus (the aforementioned Partetraviruses), parenterally transmitted among injecting drug users, hemophiliacs, and polytransfused individuals (Sharp et al. 2009; Lahtinen et al. 2011). Most recently, viruses from another potential genus, with sequences resembling both Parvoviruses and Amdoviruses, were detected in fecal samples from children in Burkina Faso, and tentatively named Bufaviruses (Phan et al. 2012). In vitro culture systems or high titer clinical samples are often not available for new members, which can thus only be studied by PCR amplification from patient tissue.Although the vast majority of parvoviruses replicate without the aid of a helper virus, the adeno-associated viruses (AAVs) from the genus Dependovirus establish latent infections that only become productive when cells are coinfected with a more complex virus, typically an adeno- or herpesvirus. To date the replication mechanisms of adeno-associated virus 2 (AAV2) and minute virus of mice (MVM), from the genus Parvovirus, have been studied in detail, as documented in previous editions of this work. Here we adopt a broader perspective, discussing inter-genera variations that shed light on replication control, and reviewing host cell responses to viral infection that modulate the nuclear environment.     

Resolution of parvovirus dimer junctions proceeds through a novel heterocruciform intermediate

The minute virus of mice initiator protein, NS1, excises new copies of the left viral telomere in a single sequence orientation, dubbed flip, during resolution of the junction between monomer genomes in palindromic dimer intermediate duplexes. We examined this reaction in vitro using both (32)P-end-labeled linear substrates and similar unlabeled templates labeled by incorporation of [alpha-(32)P]TTP during the synthesis. The observed products suggest a resolution model that explains conservation of the hairpin sequence and in which a novel heterocruciform intermediate plays a crucial role. In vitro, NS1 initiates two replication pathways from OriL(TC), the single active origin embedded in one arm of the dimer junction. NS1-mediated nicking liberates a base-paired 3' nucleotide to prime DNA synthesis and, in a reaction we call "read-through synthesis," forks established while the substrate is a linear duplex synthesize DNA in the flop orientation, leading to DNA amplification but not to junction resolution. Nicking leaves NS1 covalently attached to the 5' end of the DNA, where it can serve as a 3'-to-5' helicase, unwinding the NS1-associated strand. In the second pathway, resolution substrates are created when such unwinding induces the palindrome to reconfigure into a cruciform prior to fork assembly. New forks can then synthesize DNA in the flip orientation, copying one cruciform arm and creating a heterocruciform intermediate. Resolution proceeds via hairpin transfer in the extended arm of the heterocruciform, which releases one covalently closed duplex telomere and a partially single-stranded junction intermediate. We suggest that the latter intermediate is finally resolved via an NS1-induced single-strand nick at the otherwise inactive origin, OriL(GAA).     

Effects of inhaled corticosteroids on sputum cell counts in stable chronic obstructive pulmonary disease: a systematic review and a meta-analysis

Background

Whether inhaled corticosteroids suppress airway inflammation in chronic obstructive pulmonary disease (COPD) remains controversial. We sought to determine the effects of inhaled corticosteroids on sputum indices of inflammation in stable COPD.

Methods

We searched MEDLINE, EMBASE, CINAHL, and the Cochrane Databases for randomized, controlled clinical trials that used induced sputum to evaluate the effect of inhaled corticosteroids in stable COPD. For each chosen study, we calculated the mean differences in the concentrations of sputum cells before and after treatment in both intervention and control groups. These values were then converted into standardized mean differences to accommodate the differences in patient selection, clinical treatment, and biochemical procedures that were employed across original studies. If significant heterogeneity was present (p < 0.10), then a random effects model was used to pool the original data. In the absence of significant heterogeneity, a fixed effects model was used.

Results

We identified six original studies that met the inclusion criteria (N = 162 participants). In studies with higher cumulative dose (≥ 60 mg) or longer duration of therapy (≥ 6 weeks), inhaled corticosteroids were uniformly effective in reducing the total cell, neutrophil, and lymphocyte counts. In contrast, studies with lower cumulative dose (< 60 mg) or shorter duration of therapy (< 6 weeks) did not demonstrate a favorable effect of inhaled corticosteroids on these sputum indices.

Conclusions

Our study suggests that prolonged therapy with inhaled corticosteroids is effective in reducing airway inflammation in stable COPD.     

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens.     

Maintenance of the Flip Sequence Orientation of the Ears in the Parvoviral Left-End Hairpin Is a Nonessential Consequence of the Critical Asymmetry in the Hairpin Stem

Parvoviral terminal hairpins are essential for viral DNA amplification but are also implicated in multiple additional steps in the viral life cycle. The palindromes at the two ends of the minute virus of mice (MVM) genome are dissimilar and are processed by different resolution mechanisms that selectively direct encapsidation of predominantly negative-sense progeny genomes and conserve a single Flip sequence orientation at the 3′ (left) end of such progeny. The sequence and predicted structure of these 3′ hairpins are highly conserved within the genus Parvovirus, exemplified by the 121-nucleotide left-end sequence of MVM, which folds into a Y-shaped hairpin containing small internal palindromes that form the “ears” of the Y. To explore the potential role(s) of this hairpin in the viral life cycle, we constructed infectious clones with the ear sequences either inverted, to give the antiparallel Flop orientation, or with multiple transversions, conserving their base composition but changing their sequence. These were compared with a “bubble” mutant, designed to activate the normally silent origin in the inboard arm of the hairpin, thus potentially rendering symmetric the otherwise asymmetric junction resolution mechanism that drives maintenance of Flip. This mutant exhibited a major defect in viral duplex and single-strand DNA replication, characterized by the accumulation of covalently closed turnaround forms of the left end, and was rapidly supplanted by revertants that restored asymmetry. In contrast, both sequence and orientation changes in the hairpin ears were tolerated, suggesting that maintaining the Flip orientation of these structures is a consequence of, but not the reason for, asymmetric left-end processing.     

Mutations at the base of the icosahedral five-fold cylinders of minute virus of mice induce 3'-to-5' genome uncoating and critically impair entry functions

The linear single-stranded DNA genome of minute virus of mice can be ejected, in a 3'-to-5' direction, via a cation-linked uncoating reaction that leaves the 5' end of the DNA firmly complexed with its otherwise intact protein capsid. Here we compare the phenotypes of four mutants, L172T, V40A, N149A, and N170A, which perturb the base of cylinders surrounding the icosahedral 5-fold axes of the virus, and show that these structures are strongly implicated in 3'-to-5' release. Although noninfectious at 37°C, all mutants were viable at 32°C, showed a temperature-sensitive cell entry defect, and, after proteolysis of externalized VP2 N termini, were unable to protect the VP1 domain, which is essential for bilayer penetration. Mutant virus yields from multiple-round infections were low and were characterized by the accumulation of virions containing subgenomic DNAs of specific sizes. In V40A, these derived exclusively from the 5' end of the genome, indicative of 3'-to-5' uncoating, while L172T, the most impaired mutant, had long subgenomic DNAs originating from both termini, suggesting additional packaging portal defects. Compared to the wild type, genome release in vitro following cation depletion was enhanced for all mutants, while only L172T released DNA, in both directions, without cation depletion following proteolysis at 37°C. Analysis of progeny from single-round infections showed that uncoating did not occur during virion assembly, release, or extraction. However, unlike the wild type, the V40A mutant extensively uncoated during cell entry, indicating that the V40-L172 interaction restrains an uncoating trigger mechanism within the endosomal compartment.