Functional Study of Proteins Encoded by Human Bocavirus

Background of Human Bocavirus

Human bocavirus (HBoV) is another newly discovered member of the human pathogenic Parvoviridae family after parvovirus B19. Since its discovery in 2005, it has been detected in human respiratory tract, feces, serum and other specimens, and has been widely prevalent worldwide. So far, 4 genotypes (HBoV 1~4) have been discovered. According to the new classification system, HBoV 1~4 and gorilla bocavirus belong to the genus Bocavirus, of which HBoV1 and HBoV3 belong to the species Primate bocaparvovirus 1, while HBoV2 and HBoV4 belong to the species Primate bocaparvovirus 2. HBoV1 was mainly detected in respiratory samples of children with respiratory symptoms, with a detection rate ranging from 1.5% to 19%, and there have been reports of single infection causing death; HBoV2 was mainly detected in stool samples of patients with acute diarrhea, with a detection rate of 0.8% to 26.0%, indicating that HBoV2 may be the causative pathogen of childhood diarrhea. However, to date, the function of its encoded protein has not been fully revealed, and its replication mechanism and pathogenic mechanism are still unclear. Early studies mainly focused on HBoV epidemiological data analysis, gene sequence analysis, protein structure deduction and function prediction; with the establishment of HBoV1 infectious clones and reverse genetics systems, more progress has been made in the functional research of proteins encoded by various HBoV genes.

Figure 1. Immune response to HBoV infection: from viral persistence to acute and long consequences. (Sources: Trapani S, et al. 2023)

Structure and Function of Nonstructural Proteins

• NS Protein

Nonstructural protein NS includes NS1, NS1-70, NS2, NS3 and NS4. NS1 protein is translated from sheared R1 mRNA, with a length of 781aa and a molecular weight of 100 kD; unsheared R1mRNA translates into NS1-70 protein, with a length of 639aa and a molecular weight of 86kD; R2, R3 and R4 mRNA encode NS2 (472aa, 66kD), NS3 (507aa, 69kD), and NS4 (199aa, 34kD) proteins respectively by shearing off D1-A1', D1'-A1, or both.

NS1 protein has a DNA binding domain (DBD) at the amino terminus, a helicase activity domain in the middle region, and a transcription activation domain (TAD) at the carboxyl terminus, which is essential for viral DNA replication. The promoter transcription factor binding region is located at 96-145 nucleotides (nt) of the NS1 gene. After mutation of this region in 293T and HeLa cells, the promoter activity is 2.5 times lower than that of the wild type, suggesting that NS1 plays an important role in the viral transcription process. The results of HBoV1 NS1 structure prediction show that NS1 is first transcribed and translated at the early stage of HBoV1 infection. 397-552nt contains the helicase domain superfamily 3 necessary for DNA replication, especially the conserved motifs of Walker A and Walker B. The virus initiates viral DNA replication by recognizing the helicase replication origin and bypassing the host cell's regulatory pathway. From the structural analysis, it can be seen that NS2 contains the complete DNA binding domain (DBD) and transcription activation domain (TAD) of NS1, while NS3 and NS4 only contain TAD, suggesting that NS2 has an important function in HBoV1 replication. So far, NS3 and NS4 have not been found to have any role in the HBoV1 replication process.

Under the stimulation of tumor necrosis factor-α (TNF-α), both NS1 and NS1-70 can bind to the p65DNA domain, thereby preventing the interaction between p65 and HBoV1 DNA. Since NS1-70 lacks 142nt of important domains including TAD compared to NS1 protein, NS1 can inhibit the nuclear factor (NF-κB) signaling pathway more strongly than NS1-70. This result suggests that the NS1 carboxyl-terminal TAD domain plays an important role in regulating the function of the host's innate immune response. NS1 can also affect the activity of other transcription factors in cells, such as increasing the activity of transcription factors AP-1 and STAT3 by 3 to 6 times.

• NP1 Protein

The open reading frame of the NP1 gene is 657 nt, encoding a 219aa, 25kD NP1 protein. The structural prediction of the functional protein related to viral replication shows that NP1 has no homologous sequences in the species that have been sequenced so far. It is a protein unique to HBoVs. It contains multiple phosphorylation modification sites and plays an important regulatory role in the splicing, polyadenylation, and translation of viral mRNA precursors. Studies have shown that knocking out NP1 in recombinant infectious clones with mutations at the carboxyl terminus of NS1 can lead to varying degrees of reduction in viral replication, so it is inferred that NP1 can promote the replication of the viral genome. Systematic analysis of the mRNA of infectious clone derivatives showed that NP1 is necessary for the splicing and reading of the proximal polyadenylation site of the HBoV1 precursor RNA. NP1 regulates RNA processing by inhibiting polyadenylation of internal sites and promoting upstream splicing, which suggests that NP1 plays an important role in promoting capsid protein expression. The dual luciferase reporter gene system reported that NP1 can regulate the activity of transcription factor activator protein 1 (AP-1) and signal transducer and activator of transcription 1 (STAT1), but has no significant effect on the extracellular secretion level of interleukin type 6 (IL-6) and TNF-a, and has no effect on NF-κB activation. HBoV1 interacts with interferon regulatory factor 3 (IRF-3) through the non-structural protein NP1, blocking the transcription initiation process of IRF-3 and IFN-β promoter, thereby inhibiting the expression activity of IFN-β and blocking its antiviral effect, suggesting that HBoV1 can regulate the host's natural immune response through NP1.

• Structural Proteins VP1, VP2, and VP3

The coding genes of structural proteins VP1 (671aa, 74kD) share the 3' end sequence with VP2 (581aa, 64kD) and VP3 (542aa, 60kD), so the encoded proteins have a common carboxyl terminus. VP1 contains an amino terminus of 129aa called VP1 unique region (VP1u). Like other members of the Parvoviridae family, VP1u of HBoV1 has phospholipase A2 (PLA2) activity (a type of phospholipase that specifically hydrolyzes glycerophospholipid bonds and can dissolve cell membranes). Studies have found that any amino acid mutation in 21Pro, 41His, 42Asp, or 63Asp of VP1u may destroy the sPLA2 activity of HBoV VP1u, suggesting that these residues are essential for its PLA2 activity. According to the research on the PLA2 function, it can be inferred that the PLA2 activity of HBOV1 is related to its entry of host cells.

The recombinantly expressed HBoV VP3 protein can self-assemble into T=1 icosahedral virus-like particles (VLPs), which have similar morphology and spatial structure to natural viruses such as B19. It does not contain viral genetic material but has good safety and immunogenicity. It can effectively stimulate the body to produce a good immune response and is a potential target for HBoV antiviral drugs. The VLP capsid formed by VP3 contains receptor binding epitopes, which can be responsible for binding to receptors and mediating cell endocytosis, and can also induce cytokine responses in peripheral blood mononuclear cells. HBoV1-VP3VLPs can activate IL-13, IL-10 and IFN-γ of CD4+ T cells. VP3 protein regulates the IFN pathway by targeting RNF125 protein and promotes the synthesis of IFN at the protein level. For example, HBoV1 VP3 protein interacts with negative feedback regulators of the IFNs signaling pathway, inhibits the degradation of intracellular retinoic acid-induced gene protein RIG-I, and thus promotes the expression of IFNs.

HBoV Antibody