HBV genotypes differ in pathogenicity. In addition, genotype-specific differences in the regulation of transcription and virus replication exist in HBV, but the underlying mechanisms are unknown. Here, we show the presence of a G-quadruplex motif in the promoter of the preS2/S gene; this G-quadruplex is highly conserved only in HBV genotype B but not in other HBV genotypes. We demonstrate that this G-quadruplex motif forms a hybrid intramolecular G-quadruplex structure. Interestingly, mutations disrupting the G-quadruplex in HBV genotype B reduced the preS2/S promoter activity, leading to reduced hepatitis B surface antigen (HBsAg) levels. G-quadruplex ligands stabilized the G-quadruplex in genotype B and enhanced the preS2/S promoter activity. Furthermore, mutations disrupting the G-quadruplex in the full-length HBV genotype B constructs were associated with impaired virion secretion. In contrast to typical G-quadruplexes within promoters which are negative regulators of transcription the G-quadruplex in the preS2/S promoter of HBV represents an unconventional positive regulatory element. Our findings highlight (a) G-quadruplex mediated enhancement of transcription and virion secretion in HBV and (b) a yet unknown role for DNA secondary structures in complex genotype-specific regulatory mechanisms in virus genomes.

Background: G-quadruplexes are increasingly recognized as regulatory elements in human, animal, bacterial and plant genomes. The presence and function of G-quadruplexes are not well studied among herpesviruses; in particular, there are no systematic genome-wide analysis of these important secondary structures in herpesvirus genomes.Results: We performed genome-wide analysis of putative quadruplex sequences (PQS) in human herpesviruses. We found unusually high PQS densities among human herpesviruses. PQS are enriched in the repeat regions and regulatory regions of human herpesviruses. Interestingly, PQS densities are higher in regulatory regions of immediate early genes compared to early and late genes in most herpesviruses. In addition, the majority of genes functionally conserved across human herpesviruses contain one or more PQS within the regulatory regions. We also describe the existence of unique intramolecular PQS repeats or repetitive G-quadruplex motifs in herpesviruses. Functional studies confirm a role for G-quadruplexes in regulating the gene expression of human herpesviruses. Conclusion: The pervasiveness of PQS, their enrichment and conservation at specific genomic locations suggest that these structural entities may represent a novel class of functional elements in herpesviruses. Our findings provide the necessary framework for studies on the biological role of G-quadruplexes in herpesviruses.

Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.T he relative abundance of dinucleotides, particularly the CpG dinucleotide, has received much attention recently. The CpG dinucleotide content is normally expressed as a ratio of the actual (observed) number of CpG dinucleotides and the expected number of CpG dinucleotides (O/E ratio). The relative abundance of CpG dinucleotides varies greatly across viruses (1, 2). Depletion of CpG dinucleotides has been reported to occur in all small DNA viruses (Ͻ30 kb) infecting humans (2), while most large DNA viruses (Ͼ30 kb) infecting humans show little or no CpG dinucleotide depletion. Several possible reasons have been suggested to play a role in the depletion of CpG dinucleotides, including (i) lower transcription rate for CpG-containing codons (3), (ii) stimulation of Toll-like receptor 9-mediated innate immune response by unmethylated CpGs (4), and (iii) spontaneous deamination of methylated cytosines in CpG dinucleotides (5). Deamination of unmethylated cytosines results in C-to-U transitions which are amenable to mismatch repair mechanisms, whereas the deamination of 5-methylcytosine leads to C-to-T transitions that are often irreversible, resulting in high mutation rates in methylated CpGs and a depletion of CpG dinucleotides (6, 7).In most vertebrate genomes, the O/E ratio for CpG dinucleotides is Ͻ0.35, suggesting that vertebrate genomes have lost about two-thirds of CpG dinucleotides (5, 8). In contrast, invertebrate genomes show minor or no depletion of CpG dinucleotides (5). Early investigations on insects showed that...

Packaging signals ( pac1 and pac2) of human herpesviruses (HHVs) that contain GC-rich elements are essential for cleavage and packaging of the virus. Here, we report the presence of putative G-quadruplex sequences (PQSs) in the packaging signal ( pac1) of all HHVs. Importantly, the residues critical for the formation of G-quadruplex structures were highly conserved as compared to those not critical for the formation of this DNA secondary structure, indicating that G-quadruplexes are positively selected within pac1 in the evolution of herpesviruses. CD spectroscopy, NMR spectroscopy, native/denaturing gel, and DMS footprinting confirmed the formation of G-quadruplex structures in all pac1 PQS oligonucleotides analyzed; the majority of the PQS had the propensity to form intermolecular structures. The presence of highly conserved G-quadruplex motifs at genomic locations critical for virus packaging has not been previously recognized. Our findings provide a new perspective on the putative functions of G-quadruplexes in virus genomes.

The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. While the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native mass spectrometry to investigate RNA binding of wild-type Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically promotes nucleation of Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.