Internal polyadenylation of parvoviral precursor mRNA limits progeny virus production

Huang, Qing-Guo; Deng, Xuefeng; Best, Sonja M.; Bloom, Marshall E.; Li, Yang; Qiu, Jianming

doi:10.1016/j.virol.2012.01.031

Cited by 12 publications

(13 citation statements)

References 42 publications

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“…We next examined the role of the (pA)p site in blocking the read-through of VP mRNA and capsid protein expression. First, we made mutations of the PAS AAUAAA site at nt 3485 (16), as well as mutations of its upstream and downstream regions, which often regulate polyadenylation (24,25), in pCMVNS*Cap. We failed to decrease the level of (pA)p mRNA or increase the level of VP mRNA (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Nonstructural Protein NP1 of Human Bocavirus 1 Plays a Critical Role in the Expression of Viral Capsid Proteins

Zou

Cheng

Shen

et al. 2016

J Virol

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100

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A novel chimeric parvoviral vector, rAAV2/HBoV1, in which the recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged by the human bocavirus 1 (HBoV1) capsid, has been shown to be highly efficient in gene delivery to human airway epithelia (Z. Yan et al., Mol Ther 21:2181-2194, 2013, http://dx.doi.org/10.1038/mt.2013.92). In this vector production system, we used an HBoV1 packaging plasmid, pHBoV1NSCap, that harbors HBoV1 nonstructural protein (NS) and capsid protein (Cap) genes. In order to simplify this packaging plasmid, we investigated the involvement of the HBoV1 NS proteins in capsid protein expression. We found that NP1, a small NS protein encoded by the middle open reading frame, is required for the expression of the viral capsid proteins (VP1, VP2, and VP3). We also found that the other NS proteins (NS1, NS2, NS3, and NS4) are not required for the expression of VP proteins. We performed systematic analyses of the HBoV1 mRNAs transcribed from the pHBoV1NSCap packaging plasmid and its derivatives in HEK 293 cells. Mechanistically, we found that NP1 is required for both the splicing and the read-through of the proximal polyadenylation site of the HBoV1 precursor mRNA, essential functions for the maturation of capsid protein-encoding mRNA. Thus, our study provides a unique example of how a small viral nonstructural protein facilitates the multifaceted regulation of capsid gene expression. IMPORTANCEA novel chimeric parvoviral vector, rAAV2/HBoV1, expressing a full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene, is capable of correcting CFTR-dependent chloride transport in cystic fibrosis human airway epithelium. Previously, an HBoV1 nonstructural and capsid protein-expressing plasmid, pHBoV1NSCap, was used to package the rAAV2/HBoV1 vector, but yields remained low. In this study, we demonstrated that the nonstructural protein NP1 is required for the expression of capsid proteins. However, we found that the other four nonstructural proteins (NS1 to -4) are not required for expression of capsid proteins. By mutating the cis elements that function as internal polyadenylation signals in the capsid protein-expressing mRNA, we constructed a simple HBoV1 capsid protein-expressing gene that expresses capsid proteins as efficiently as pHBoV1NSCap does, and at similar ratios, but independently of NP1. Our study provides a foundation to develop a better packaging system for rAAV2/HBoV1 vector production. Human bocavirus 1 (HBoV1), first identified in 2005 (1), belongs to the genus Bocaparvovirus in the subfamily Parvovirinae of the family Parvoviridae (2). The genus Bocaparvovirus consists of three groups of viruses, namely, HBoV1 to -4, bovine parvovirus 1 (BPV1), and minute virus of canines (MVC/CnMV) (3). HBoV1 causes respiratory tract infections in young children worldwide (4-11). In vitro, the virus infects only polarized (welldifferentiated) human airway epithelium cultured at an air-liquid interface (HAE-ALI) (12-15). We have constructed an infectious clone of HBoV1...

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Section: Resultsmentioning

confidence: 99%

Nonstructural Protein NP1 of Human Bocavirus 1 Plays a Critical Role in the Expression of Viral Capsid Proteins

Zou

Cheng

Shen

et al. 2016

J Virol

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100

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“…Besides TBEV, internal poly(A) or poly(U) tracts have been described in a number of different viruses282930313233. The negative-sense, vesicular stomatitis virus (VSV) generates a poly(A) tail through an RNA dependent RNA polymerase (RdRp) slippage mechanisms, which depends on specific sequences and a U7 tract on the RNA template34.…”

Section: Discussionmentioning

confidence: 99%

The role of the poly(A) tract in the replication and virulence of tick-borne encephalitis virus

Asghar

Lee

Nilsson

et al. 2016

Sci Rep

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The tick-borne encephalitis virus (TBEV) is a flavivirus transmitted to humans, usually via tick bites. The virus causes tick-borne encephalitis (TBE) in humans, and symptoms range from mild flu-like symptoms to severe and long-lasting sequelae, including permanent brain damage. It has been suggested that within the population of viruses transmitted to the mammalian host, quasispecies with neurotropic properties might become dominant in the host resulting in neurological symptoms. We previously demonstrated the existence of TBEV variants with variable poly(A) tracts within a single blood-fed tick. To characterize the role of the poly(A) tract in TBEV replication and virulence, we generated infectious clones of Torö-2003 with the wild-type (A)3C(A)6 sequence (Torö-6A) or with a modified (A)3C(A)38 sequence (Torö-38A). Torö-38A replicated poorly compared to Torö-6A in cell culture, but Torö-38A was more virulent than Torö-6A in a mouse model of TBE. Next-generation sequencing of TBEV genomes after passaging in cell culture and/or mouse brain revealed mutations in specific genomic regions and the presence of quasispecies that might contribute to the observed differences in virulence. These data suggest a role for quasispecies development within the poly(A) tract as a virulence determinant for TBEV in mice.

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“…Splicing of this intron to remove the polyadenylation signal helps govern access to the capsid gene for these viruses (27). The amdoviruses (28,29) and the bocaviruses (3) have polyadenylation sites that lie within their capsid gene; however, these potent motifs are retained in the capsid-encoding mRNA and must be somehow suppressed to allow export and accumulation of mRNAs that encode capsid protein information. We have shown here that the bocavirus MVC has evolved yet another way to allow essential access to its capsid protein gene, namely, by the action of its novel NP1 protein, which is required to read through its internal (pA)p site.…”

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confidence: 99%

Characterization of the Nonstructural Proteins of the Bocavirus Minute Virus of Canines

Sukhu

Fasina

Burger

et al. 2013

J Virol

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bWe present a detailed characterization of a single-cycle infection of the bocavirus minute virus of canines (MVC) in canine WRD cells. This has allowed identification of an additional smaller NS protein that derives from an mRNA spliced within the NS gene that had not been previously reported. In addition, we have identified a role for the viral NP1 protein during infection. NP1 is required for read-through of the MVC internal polyadenylation site and, thus, access of the capsid gene by MVC mRNAs. Although the mechanism of NP1's action has not yet been fully elucidated, it represents the first parvovirus protein to be implicated directly in viral RNA processing.

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Internal polyadenylation of parvoviral precursor mRNA limits progeny virus production

Cited by 12 publications

References 42 publications

Nonstructural Protein NP1 of Human Bocavirus 1 Plays a Critical Role in the Expression of Viral Capsid Proteins

Nonstructural Protein NP1 of Human Bocavirus 1 Plays a Critical Role in the Expression of Viral Capsid Proteins

The role of the poly(A) tract in the replication and virulence of tick-borne encephalitis virus

Characterization of the Nonstructural Proteins of the Bocavirus Minute Virus of Canines

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