Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotypespecific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotypedependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.IMPORTANCE Assembly-activating protein (AAP) is a recently discovered adenoassociated virus (AAV) protein that promotes capsid assembly and provides new opportunities for research in assembly. Previous studies on AAV serotype 2 (AAV2) showed that assembly takes place in the nucleolus and is dependent on AAP and that capsids colocalize with AAP in the nucleolus during the assembly process. However, through the investigation of 12 different AAV serotypes (AAV1 to -12), we find that AAP is not an essential requirement for capsid assembly of AAV4, -5, and -11, and AAP, assembled capsids, and the nucleolus do not colocalize for all the serotypes. In addition, we find that there are both serotype-restricted and serotypepromiscuous AAPs in their assembly roles. These findings challenge widely held beliefs about the importance of the nucleolus and AAP in AAV assembly and show the heterogeneous nature of the assembly process within the AAV family.

Chikungunya virus (CHIKV) infections can cause severe and debilitating joint and muscular pain that can be long lasting. Current CHIKV vaccines under development rely on the generation of neutralizing antibodies for protection; however, the role of T cells in controlling CHIKV infection and disease is still unclear. Using an overlapping peptide library, we identified the CHIKV-specific T cell receptor epitopes recognized in C57BL/6 infected mice at 7 and 14 days post-infection. A fusion protein containing peptides 451, 416, a small region of nsP4, peptide 47, and an HA tag (CHKVf5) was expressed using adenovirus and cytomegalovirus-vectored vaccines. Mice vaccinated with CHKVf5 elicited robust T cell responses to higher levels than normally observed following CHIKV infection, but the vaccine vectors did not elicit neutralizing antibodies. CHKVf5-vaccinated mice had significantly reduced infectious viral load when challenged by intramuscular CHIKV injection. Depletion of both CD4+ and CD8+ T cells in vaccinated mice rendered them fully susceptible to intramuscular CHIKV challenge. Depletion of CD8+ T cells alone reduced vaccine efficacy, albeit to a lesser extent, but depletion of only CD4+ T cells did not reverse the protective phenotype. These data demonstrated a protective role for CD8+ T cells in CHIKV infection. However, CHKVf5-vaccinated mice that were challenged by footpad inoculation demonstrated equal viral loads and increased footpad swelling at 3 dpi, which we attributed to the presence of CD4 T cell receptor epitopes present in the vaccine. Indeed, vaccination of mice with vectors expressing only CHIKV-specific CD8+ T cell epitopes followed by CHIKV challenge in the footpad prevented footpad swelling and reduced proinflammatory cytokine and chemokines associated with disease, indicating that CHIKV-specific CD8+ T cells prevent CHIKV disease. These results also indicate that a T cell-biased prophylactic vaccination approach is effective against CHIKV challenge and reduces CHIKV-induced disease in mice.

The genetic manipulation of skeletal muscle cells in vitro is notoriously difficult, especially when using undifferentiated muscle cell lines (myoblasts) or primary muscle stem cells (myosatellites). We therefore optimized methods of gene transfer by overexpressing green fluorescent protein (GFP) in mouse C2C12 cells and in a novel system, primary rainbow trout myosatellite cells. A common lipid-based transfection reagent was used (Lipofectamine 2000) along with three different viral vectors: adeno-associated virus serotype 2 (AAV2), baculovirus (BAC) and lentivirus. Maximal transfection efficiencies of 49% were obtained in C2C12 cells after optimizing cell density and reagent:DNA ratio, although GFP signal rapidly dissipated with proliferation and was not maintained with differentiation. The transduction efficiency of AAV2 was optimized to 65% by extending incubation time and decreasing cell density, although only 30% of cells retained expression after passing. A viral comparison revealed that lentivirus was most efficient at transducing C2C12 myoblasts as 97% of cells were transduced with only 106 viral genomes (vg) compared to 54% with 108 vg AAV2 and 23% with 109 vg BAC. Lentivirus also transduced 90% of primary trout myosatellites compared to 1–10% with AAV2 and BAC. The phosphoglycerate kinase 1 promoter was 10-fold more active than the cytomegalovirus immediate-early promoter in C2C12 cells and both were effective in trout myosatellites. Maximal transduction of C2C12 myotubes was achieved by differentiating myoblasts previously transduced with lentivirus and the pgk promoter. Thus, our optimized protocol proved highly effective in diverse muscle cell systems and could therefore help overcome a common technological barrier.