Recent Advances in Molecular Biology of Human Bocavirus 1 and Its Applications

Abstract

Human bocavirus 1 (HBoV1) was discovered in human nasopharyngeal specimens in 2005. It is an autonomous human parvovirus and causes acute respiratory tract infections in young children. HBoV1 infects well differentiated or polarized human airway epithelial cells in vitro. Unique among all parvoviruses, HBoV1 expresses 6 non-structural proteins, NS1, NS1-70, NS2, NS3, NS4, and NP1, and a viral non-coding RNA (BocaSR), and three structural proteins VP1, VP2, and VP3. The BocaSR is the first identified RNA polymerase III (Pol III) transcribed viral non-coding RNA in small DNA viruses. It plays an important role in regulation of viral gene expression and a direct role in viral DNA replication in the nucleus. HBoV1 genome replication in the polarized/non-dividing airway epithelial cells depends on the DNA damage and DNA repair pathways and involves error-free Y-family DNA repair DNA polymerase (Pol) η and Pol κ. Importantly, HBoV1 is a helper virus for the replication of dependoparvovirus, adeno-associated virus (AAV), in polarized human airway epithelial cells, and HBoV1 gene products support wild-type AAV replication and recombinant AAV (rAAV) production in human embryonic kidney (HEK) 293 cells. More importantly, the HBoV1 capsid is able to pseudopackage an rAAV2 or rHBoV1 genome, producing the rAAV2/HBoV1 or rHBoV1 vector. The HBoV1 capsid based rAAV vector has a high tropism for human airway epithelia. A deeper understanding in HBoV1 replication and gene expression will help find a better way to produce the rAAV vector and to increase the efficacy of gene delivery using the rAAV2/HBoV1 or rHBoV1 vector, in particular, to human airways. This review summarizes the recent advances in gene expression and replication of HBoV1, as well as the use of HBoV1 as a parvoviral vector for gene delivery.

Keywords: gene expression; human bocavirus; parvovirus; replication; viral vector.

FIGURE 1

Sequence and structure of the HBoV1 left and right end hairpins. The structures of HBoV1 LEH and REH are shown with the 3′ end and 5′ end sequences, respectively, which were predicted using the DNAMAN program (Lynnon, Co., Quebec, Canada). The ear and bubble are indicated, as well as the start and end nucleotides of HBoV1 genome. The sequence refers the full-length HBoV1 genome of the isolate Salvador1 (GenBank accession no.: JQ923422).

FIGURE 2

Transcription map of HBoV1. The major transcription landmarks including the terminal repeats (LEH and REH), promoter (P), splice donors (D) and acceptors (A), and (pA)p and (pA)d sites, are depicted. All identified mRNA transcripts are listed below the map (designated R1 to R6), with their respective sizes shown on the left and the detected molecular weight of the expressed proteins shown to the right. Different ORFs are illustrated in blue, red or green colors. The expressed non-coding RNA (BocaSR) is diagrammed with the size. NCR, non-coding region.

FIGURE 3

Comparisons between BocaSR and VAI RNA. (A) RNA sequence alignment. The sequences of BocaSR and adenovirus 5 (Ad5) VAI RNA were aligned using the CLUSTALW algorithm. Identical nucleotides are colored. Consensus sequences of the A- and B-boxes of the intergenic RNA Pol III are indicated. R represents G/A; Y, C/T, and N any nucleotides. (B,C) The structure of BocaSR (B) was predicted using the KineFold algorithm, with Ad5 VAI RNA serving as a reference (C). Nucleotide numbers shown in blue and black are the positions of the BocaSR RNA (VAI RNA) sequence and the HBoV1 genome, respectively. Stem structures are indicated, and the central tetranucleotide pairs are highlighted in gray.

FIGURE 4

Domains of HBoV1 NS proteins. HBoV1 NS1 (GenBank: AFR53039) and AAV5 Rep78 (GenBank: AAD13755) are aligned. N-terminal origin DNA binding domain (OBD; in red) and helicase domain (in purple) are diagramed. The regions positioned between the OBD and helicase domains (shown in green) are predicted to be the oligomerization signal. The C-terminal region (shown in yellow) is predicted to serve potentially transcriptional activation function. Dashed lines in the OBD indicate residues that are structured as endonuclease core/DNA binding loop (Tewary et al., 2013), and dashed rectangles in the helicase domain indicate Walker boxes (Koonin, 1993). Oligo. indicates a putative oligomerization signal. NS2, NS3, NS4, and NS1-70 proteins are diagramed in colored blocks with thin lines indicating excised aa sequences due to ligation of the neighboring exons of their mRNAs.

FIGURE 5

A proposed model of NP1-facilitated definition of the VP-encoding exon. The viral pre-mRNA is shown in part with the A3 splice site, (pA)p1, (pA)p2, (pA)d1, and (pA)d_REH sites. Key signals for mRNA processing, i.e., AG at the 3′ acceptor, CFIm25-binding site UGUA, CPSF-binding site AAUAAA, are shown with the downstream element (DSE) indicated. The 3′ acceptor U2 interacting complex (U2AF/U2 snRNP) and the CPSF complex composed of CFIm, Fip1, CPSF, and CstF are diagrammed. CPSF6 interacts with Fip1 through their RS and RE/D domains. Nucleotide (nt) numbers show the location of A3 3′ splice site, and the cleavage site of each pA signals. (A) Without NP1. There are potential SR proteins (SF) binding to the A3 acceptor to enhance the binding of U2 snRNP to the A3 acceptor. CFIm binds to UGUA enhancers at 17 (too close) and 179 nts (too far) upstream of the (pA)p2 and (pA)p1, respectively. The distance between the A3 acceptor and (pA)p sites is short, which favors defining the exon between A3 and (pA)p sites. (B) With NP1. Potential interaction of NP1 with CPSF6 disrupts the interaction between CPSF6 and CFIm25 that binds UGUA sites 17 (too close) and 179 nts (too far) upstream of the (pA)p2 and (pA)p1, respectively. While the CFIm25 binds UGUA signals at –39 and –50 nt (optimal distance) upstream of the (pA)d1 and (pA)d_REH, respectively, the interaction between CFIm25 and CPSF6 is tight and, therefore, difficult to be interrupted by the NP1. The overall interaction between the U2 snRNP complex at the A3 acceptor and the CPSF complex at the (pA)d sites determines the large exon between the A3 acceptor and (pA)d sites.

FIGURE 6

The infection life cycle of HBoV1. A ciliated airway epithelial cell is depicted with diagrams of the cilia and junction molecules. HBoV1 enters the cells through binding to an unknown viral receptor, which is expressed on both the apical (ciliated) and the basal cells as indicated, and through receptor-mediated endocytosis, followed by intracellular trafficking (Steps 1–3). The virus escapes from the late endosome and enters the nucleus (Step 4). In the nucleus, the uncoated ssDNA viral genome is converted to replicative form dsDNA that expresses viral NS proteins and BocaSR (Steps 5–8). The viral DNA further replicates in the nucleus (Steps 12–16) and expresses both viral NS and capsid proteins (Steps 9–11), followed by genome packaging into empty capsid (Steps 16–18). Lastly, the matured virus egresses out of the infected cells (Steps 19, 20). The HBoV1 infection cycle in the ciliated epithelial cell is illustrated based on the studies on HBoV1 and references from other parvoviruses, which are explained in the text.

FIGURE 7

rAAV2/HBoV1 vector production systems. (A) NS-free rAAV2/HBoV1 vector production system in HEK293 cells. pAAV2 carries the genes of interest in rAAV2 genome. pCMVoptVP1-AAV2Rep is a two-in-one plasmid, in which the VP1 and Rep2 (Rep78/52) are expressed in two independent cassettes. pAd4.1-CMVoptVP2/3 has the optimized (opt)VP2/3 expression cassette in the Ad helper gene expression plasmid pAd4.1. (B) rAAV2/HBoV1 baculovirus expression vector (BEV) production system. Bac-AAV2ITR-GFP-Luc carries an rAAV2 genome. Bac-AAV2Rep-HBoV1Cap expresses AAV2 Rep proteins and HBoV1 capsid proteins. Bac-HBoV1NP1 expresses HBoV1 NP1. P10 and Ph are baculoviral promoters, and CMV and F5 tg83 are a human cytomegalovirus major immediate-early promoter and a synthetic promoter, respectively. Various polyadenylation signals are indicated as polyA. Luc: firefly luciferase gene; GFP, enhanced green fluorescent protein gene, and mCherry, a monomeric red fluorescent protein gene. They can be replaced by any gene of interest (GOI) for expression in the vector.