Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.

The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

Structural and cellular biology of adeno-associated virus attachment and entry.

Adeno-associated virus (AAV) is a nonenveloped, ssDNA virus in the parvovirus family, which has become one of the leading candidate vectors for human gene therapy. AAV has been studied extensively to identify host cellular factors involved in infection, as well as to identify capsid variants that confer clinically favorable transduction profiles ex vivo and in vivo. Recent advances in technology have allowed for direct genetic approaches to be used to more comprehensively characterize host factors required for AAV infection and allowed for identification of a critical multi-serotype receptor, adeno-associated virus receptor (AAVR). In this chapter, we will discuss the interactions of AAV with its glycan and proteinaceous receptors and describe the host and viral components involved in AAV entry, which requires cellular attachment, endocytosis, trafficking to the trans-Golgi network and nuclear import. AAV serves as a paradigm for entry of nonenveloped viruses. Furthermore, we will discuss the potential of utilizing our increased understanding of virus-host interactions during AAV entry to develop better AAV-based therapeutics, with a focus on host factors and capsid interactions involved in in vivo tropism.

Regulation of Mumps Virus Replication and Transcription by Kinase RPS6KB1.

Mumps virus (MuV) caused the most viral meningitis before mass immunization. Unfortunately, MuV has reemerged in the United States in the past several years. MuV is a member of the genus Rubulavirus, in the family Paramyxoviridae, and has a nonsegmented negative-strand RNA genome. The viral RNA-dependent RNA polymerase (vRdRp) of MuV consists of the large protein (L) and the phosphoprotein (P), while the nucleocapsid protein (NP) encapsulates the viral RNA genome. These proteins make up the replication and transcription machinery of MuV. The P protein is phosphorylated by host kinases, and its phosphorylation is important for its function. In this study, we performed a large-scale small interfering RNA (siRNA) screen targeting host kinases that regulated MuV replication. The human kinase ribosomal protein S6 kinase beta-1 (RPS6KB1) was shown to play a role in MuV replication and transcription. We have validated the role of RPS6KB1 in regulating MuV using siRNA knockdown, an inhibitor, and RPS6KB1 knockout cells. We found that MuV grows better in cells lacking RPS6KB1, indicating that it downregulates viral growth. Furthermore, we detected an interaction between the MuV P protein and RPS6KB1, suggesting that RPS6KB1 directly regulates MuV replication and transcription.IMPORTANCE Mumps virus is an important human pathogen. In recent years, MuV has reemerged in the United State, with outbreaks occurring in young adults who have been vaccinated. Our work provides insight into a previously unknown mumps virus-host interaction. RPS6KB1 negatively regulates MuV replication, likely through its interaction with the P protein. Understanding virus-host interactions can lead to novel antiviral drugs and enhanced vaccine production.

GPR108 Is a Highly Conserved AAV Entry Factor.

Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

Role of Small Hydrophobic Protein of J Paramyxovirus in Virulence.

J paramyxovirus (JPV) was first isolated from moribund mice with hemorrhagic lung lesions in Australia in 1972. It is a paramyxovirus classified under the newly proposed genus Jeilongvirus JPV has a genome of 18,954 nucleotides, consisting of eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. JPV causes little cytopathic effect (CPE) in tissue culture cells but severe disease in mice. The small hydrophobic (SH) protein is an integral membrane protein encoded by many paramyxoviruses, such as mumps virus (MuV) and respiratory syncytial virus (RSV). However, the function of SH has not been defined in a suitable animal model. In this work, the functions of SH of JPV, MuV, and RSV have been examined by generating recombinant JPV lacking the SH protein (rJPV-ΔSH) or replacing SH of JPV with MuV SH (rJPV-MuVSH) or RSV SH (rJPV-RSVSH). rJPV-ΔSH, rJPV-MuVSH, and rJPV-RSVSH were viable and had no growth defect in tissue culture cells. However, more tumor necrosis factor alpha (TNF-α) was produced during rJPV-ΔSH infection, confirming the role of SH in inhibiting TNF-α production. rJPV-ΔSH induced more apoptosis in tissue culture cells than rJPV, rJPV-MuVSH, and rJPV-RSVSH, suggesting that SH plays a role in blocking apoptosis. Furthermore, rJPV-ΔSH was attenuated in mice compared to rJPV, rJPV-MuVSH, and rJPV-RSVSH, indicating that the SH protein plays an essential role in virulence. The results indicate that the functions of MuV SH and RSV SH are similar to that of JPV SH even though they have no sequence homology.IMPORTANCE Paramyxoviruses are associated with many devastating diseases in animals and humans. J paramyxovirus (JPV) was isolated from moribund mice in Australia in 1972. Newly isolated viruses, such as Beilong virus (BeiPV) and Tailam virus (TlmPV), have genome structures similar to that of JPV. A new paramyxovirus genus, Jeilongvirus, which contains JPV, BeiPV, and TlmPV, has been proposed. Small hydrophobic (SH) protein is present in many paramyxoviruses. Our present study investigates the role of SH protein of JPV in pathogenesis in its natural host. Understanding the pathogenic mechanism of Jeilongvirus is important to control and prevent potential diseases that may emerge from this group of viruses.

Parainfluenza Virus 5 Expressing Wild-Type or Prefusion Respiratory Syncytial Virus (RSV) Fusion Protein Protects Mice and Cotton Rats from RSV Challenge.

Human respiratory syncytial virus (RSV) is the leading cause of pediatric bronchiolitis and hospitalizations. RSV can also cause severe complications in elderly and immunocompromised individuals. There is no licensed vaccine. We previously generated a parainfluenza virus 5 (PIV5)-vectored vaccine candidate expressing the RSV fusion protein (F) that was immunogenic and protective in mice. In this work, our goal was to improve the original vaccine candidate by modifying the PIV5 vector or by modifying the RSV F antigen. We previously demonstrated that insertion of a foreign gene at the PIV5 small hydrophobic (SH)-hemagglutinin-neuraminidase (HN) junction or deletion of PIV5 SH increased vaccine efficacy. Additionally, other groups have demonstrated that antibodies against the prefusion conformation of RSV F have more potent neutralizing activity than antibodies against the postfusion conformation. Therefore, to improve on our previously developed vaccine candidate, we inserted RSV F at the PIV5 SH-HN gene junction or used RSV F to replace PIV5 SH. We also engineered PIV5 to express a prefusion-stabilized F mutant. The candidates were tested in BALB/c mice via the intranasal route and induced both humoral and cell-mediated immunity. They also protected against RSV infection in the mouse lung. When they were administered intranasally or subcutaneously in cotton rats, the candidates were highly immunogenic and reduced RSV loads in both the upper and lower respiratory tracts. PIV5-RSV F was equally protective when administered intranasally or subcutaneously. In all cases, the prefusion F mutant did not induce higher neutralizing antibody titers than wild-type F. These results show that antibodies against both pre- and postfusion F are important for neutralizing RSV and should be considered when designing a vectored RSV vaccine. The findings also that indicate PIV5-RSV F may be administered subcutaneously, which is the preferred route for vaccinating infants, who may develop nasal congestion as a result of intranasal vaccination.IMPORTANCE Despite decades of research, human respiratory syncytial virus (RSV) is still a major health concern for which there is no vaccine. A parainfluenza virus 5-vectored vaccine expressing the native RSV fusion protein (F) has previously been shown to confer robust immunity against RSV infection in mice, cotton rats, and nonhuman primates. To improve our previous vaccine candidate, we developed four new candidates that incorporate modifications to the PIV5 backbone, replace native RSV F with a prefusion-stabilized RSV F mutant, or combine both RSV F and PIV5 backbone modifications. In this work, we characterized the new vaccine candidates and tested their efficacies in both murine and cotton rat models of RSV infection. Most importantly, we found that PIV5-based RSV vaccine candidates were efficacious in preventing lower respiratory tract infection as well as in reducing the nasal viral load when administered via the subcutaneous route.

Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1.

UNLABELLED: The viral RNA-dependent RNA polymerases (vRdRps) of nonsegmented, negative-sense viruses (NNSVs) consist of the enzymatic large protein (L) and the phosphoprotein (P). P is heavily phosphorylated, and its phosphorylation plays a critical role in viral RNA synthesis. Since NNSVs do not encode kinases, P is phosphorylated by host kinases. In this study, we investigate the roles that viral proteins play in the phosphorylation of mumps virus (MuV) P. We found that nucleoprotein (NP) enhances the phosphorylation of P. We have identified the serine/threonine kinase Polo-like kinase 1 (PLK1) as a host kinase that phosphorylates P and have found that phosphorylation of P by PLK1 is enhanced by NP. The PLK1 binding site in MuV P was mapped to residues 146 to 148 within the S(pS/T)P motif, and the phosphorylation site was identified as residues S292 and S294. IMPORTANCE: It has previously been shown that P acts as a chaperone for NP, which encapsidates viral genomic RNA to form the NP-RNA complex, the functional template for viral RNA synthesis. Thus, it is assumed that phosphorylation of P may regulate NP's ability to form the NP-RNA complex, thereby regulating viral RNA synthesis. Our work demonstrates that MuV NP affects phosphorylation of P, suggesting that NP can regulate viral RNA synthesis by regulating phosphorylation of P.

Roles of Phosphorylation of the Nucleocapsid Protein of Mumps Virus in Regulating Viral RNA Transcription and Replication.

UNLABELLED: Mumps virus (MuV) is a paramyxovirus with a negative-sense nonsegmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV minigenome system were examined. The S439A mutant was found to have higher activity, four mutants had lower activity, and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had 90% reduced phosphorylation of NP and enhanced viral RNA synthesis and viral protein expression at early time points after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in downregulating viral RNA synthesis. IMPORTANCE: Mumps virus (MuV), a paramyxovirus, is an important human pathogen that is reemerging in human populations. Nucleocapsid protein (NP) of MuV is essential for viral RNA synthesis. We have identified the major phosphorylation site of NP. We have found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. The work will lead to a better understanding of viral RNA synthesis and possible novel targets for antiviral drug development.