Cellular and Structural Characterization of VP1 and VP2 Knockout Mutants of AAV3B Serotype and Implications for AAV Manufacturing.

AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WT > VP2 > VP1 > VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing.

Development of reverse genetics systems and investigation of host response antagonism and reassortment potential for Cache Valley and Kairi viruses, two emerging orthobunyaviruses of the Americas.

Orthobunyaviruses such as Cache Valley virus (CVV) and Kairi virus (KRIV) are important animal pathogens. Periodic outbreaks of CVV have resulted in the significant loss of lambs on North American farms, whilst KRIV has mainly been detected in South and Central America with little overlap in geographical range. Vaccines or treatments for these viruses are unavailable. One approach to develop novel vaccine candidates is based on the use of reverse genetics to produce attenuated viruses that elicit immune responses but cannot revert to full virulence. The full genomes of both viruses were sequenced to obtain up to date genome sequence information. Following sequencing, minigenome systems and reverse genetics systems for both CVV and KRIV were developed. Both CVV and KRIV showed a wide in vitro cell host range, with BHK-21 cells a suitable host cell line for virus propagation and titration. To develop attenuated viruses, the open reading frames of the NSs proteins were disrupted. The recombinant viruses with no NSs protein expression induced the production of type I interferon (IFN), indicating that for both viruses NSs functions as an IFN antagonist and that such attenuated viruses could form the basis for attenuated viral vaccines. To assess the potential for reassortment between CVV and KRIV, which could be relevant during vaccination campaigns in areas of overlap, we attempted to produce M segment reassortants by reverse genetics. We were unable to obtain such viruses, suggesting that it is an unlikely event.

Infectious Bursal Disease Virus VP3 Upregulates VP1-Mediated RNA-Dependent RNA Replication.

Genome replication is a critical step in virus life cycles. Here, we analyzed the role of the infectious bursal disease virus (IBDV) VP3, a major component of IBDV ribonucleoprotein complexes, on the regulation of VP1, the virus-encoded RNA-dependent RNA polymerase (RdRp). Data show that VP3, as well as a peptide mimicking its C-terminal domain, efficiently stimulates the ability of VP1 to replicate synthetic single-stranded RNA templates containing the 3' untranslated regions (UTRs) from the IBDV genome segments.

Generation of genetically stable recombinant rotaviruses containing novel genome rearrangements and heterologous sequences by reverse genetics.

The rotavirus (RV) genome consists of 11 segments of double-stranded RNA (dsRNA). Typically, each segment contains 5' and 3' untranslated regions (UTRs) that flank an open reading frame (ORF) encoding a single protein. RV variants with segments of atypical size owing to sequence rearrangements have been described. In many cases, the rearrangement originates from a partial head-to-tail sequence duplication that initiates after the stop codon of the ORF, leaving the protein product of the segment unaffected. To probe the limits of the RV genome to accommodate additional genetic sequence, we used reverse genetics to insert duplications (analogous to synthetic rearrangements) and heterologous sequences into the 3' UTR of the segment encoding NSP2 (gene 8). The approach allowed the recovery of recombinant RVs that contained sequence duplications (up to 200 bp) and heterologous sequences, including those for FLAG, the hepatitis C virus E2 epitope, and the internal ribosome entry site of cricket paralysis virus. The recombinant RVs grew to high titer (>10(7) PFU/ml) and remained genetically stable during serial passage. Despite their longer 3' UTRs, rearranged RNAs of recombinant RVs expressed wild-type levels of protein in vivo. Competitive growth experiments indicated that, unlike RV segments with naturally occurring sequence duplications, genetically engineered segments were less efficiently packaged into progeny viruses. Thus, features of naturally occurring rearranged segments, other than their increased length, contribute to their enhanced packaging phenotype. Our results define strategies for developing recombinant RVs as expression vectors, potentially leading to next-generation RV vaccines that induce protection against other infectious agents.

The capsid protein of infectious bursal disease virus contains a functional alpha 4 beta 1 integrin ligand motif.

Infectious bursal disease virus (IBDV), a member of the dsRNA Birnaviridae family, is an important immunosuppressive avian pathogen. We have identified a strictly conserved amino acid triplet matching the consensus sequence used by fibronectin to bind the alpha 4 beta 1 integrin within the protruding domain of the IBDV capsid polypeptide. We show that a single point mutation on this triplet abolishes the cell-binding activity of IBDV-derived subviral particles (SVP), and abrogates the recovering of infectious IBDV by reverse genetics without affecting the overall SVP architecture. Additionally, we demonstrate that the presence of the alpha 4 beta 1 heterodimer is a critical determinant for the susceptibility of murine BALB/c 3T3 cells to IBDV binding and infectivity. Our data suggests that the IBDV might also use the alpha 4 beta 1 integrin as a specific binding receptor in avian cells.

Autoproteolytic activity derived from the infectious bursal disease virus capsid protein.

Viral capsids are envisioned as vehicles to deliver the viral genome to the host cell. They are nonetheless dynamic protective shells, as they participate in numerous processes of the virus cycle such as assembly, genome packaging, binding to receptors, and uncoating among others. In so doing, they undergo large scale conformational changes. Capsid proteins with essential enzymatic activities are being described more frequently. Here we show that the precursor (pVP2) of the capsid protein VP2 of the infectious bursal disease virus (IBDV), an avian double-stranded RNA virus, has autoproteolytic activity. The pVP2 C-terminal region is first processed by the viral protease VP4. VP2 Asp-431, lying in a flexible loop preceding the C-terminal most alpha-helix, is responsible for the endopeptidase activity that cleaves the Ala-441-Phe-442 bond to generate the mature VP2 polypeptide. The D431N substitution abrogates the endopeptidase activity without introducing a significant conformational change, as deduced from the three-dimensional structure of the mutant protein at 3.1 A resolution. Combinations of VP2 polypeptides containing mutations affecting either the cleavage or the catalytic site revealed that pVP2 proteolytic processing is the result of a monomolecular cis-cleavage reaction. The D431N mutation does not affect the assembly of the VP2 trimers that constitute the capsid building block. Although VP2 D431N trimers are capable of assembling both pentamers and hexamers, expression of a polyprotein gene harboring the D431N mutation does not result in the assembly of IBDV virus-like particles. Reverse genetics analyses demonstrate that pVP2 self-processing is essential for the assembly of an infectious IBDV progeny.

Structural insights into the multifunctional protein VP3 of birnaviruses.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most harmful poultry diseases. The IBDV genome encodes five mature proteins; of these, the multifunctional protein VP3 plays an essential role in virus morphogenesis. This protein, which interacts with the structural protein VP2, with the double-stranded RNA genome, and with the virus-encoded, RNA-dependent RNA polymerase, VP1, is involved not only in the formation of the viral capsid, but also in the recruitment of VP1 into the capsid and in the encapsidation of the viral genome. Here, we report the X-ray structure of the central region of VP3, residues 92-220, consisting of two alpha-helical domains connected by a long and flexible hinge that are organized as a dimer. Unexpectedly, the overall fold of the second VP3 domain shows significant structural similarities with different transcription regulation factors.

Activation mechanism of a noncanonical RNA-dependent RNA polymerase.

Two lineages of viral RNA-dependent RNA polymerases (RDRPs) differing in the organization (canonical vs. noncanonical) of the palm subdomain have been identified. Phylogenetic analyses indicate that both lineages diverged at a very early stage of the evolution of the enzyme [Gorbalenya AE, Pringle FM, Zeddam JL, Luke BT, Cameron CE, Kalmakoff J, Hanzlik TN, Gordon KH, Ward VK (2002) J Mol Biol 324:47-62]. Here, we report the x-ray structure of a noncanonical birnaviral RDRP, named VP1, in its free form, bound to Mg(2+) ions, and bound to a peptide representing the polymerase-binding motif of the regulatory viral protein VP3. The structure of VP1 reveals that the noncanonical connectivity of the palm subdomain maintains the geometry of the catalytic residues found in canonical polymerases but results in a partial blocking of the active site cavity. The VP1-VP3 peptide complex shows a mode of polymerase activation in which VP3 binding promotes a conformational change that removes the steric blockade of the VP1 active site, facilitating the accommodation of the template and incoming nucleotides for catalysis. The striking structural similarities between birnavirus (dsRNA) and the positive-stranded RNA picornavirus and calicivirus RDRPs provide evidence supporting the existence of functional and evolutionary relationships between these two virus groups.