Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus.

Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.

Herpes simplex virus co-infection facilitates rolling circle replication of the adeno-associated virus genome.

Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3' inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.

Herpes Simplex Virus 1 Coinfection Modifies Adeno-associated Virus Genome End Recombination.

Wild-type adeno-associated virus (AAV) can only replicate in the presence of helper factors, which can be provided by coinfecting helper viruses such as adenoviruses and herpesviruses. The AAV genome consists of a linear, single-stranded DNA (ssDNA), which is converted into different molecular structures within the host cell. Using high-throughput sequencing, we found that herpes simplex virus 1 (HSV-1) coinfection leads to a shift in the type of AAV genome end recombination. In particular, open-end inverted terminal repeat (ITR) recombination was enhanced, whereas open-closed ITR recombination was reduced in the presence of HSV-1. We demonstrate that the HSV-1 protein ICP8 plays an essential role in HSV-1-mediated interference with AAV genome end recombination, indicating that the previously described ICP8-driven mechanism of HSV-1 genome recombination may be underlying the observed changes. We also provide evidence that additional factors, such as products of true late genes, are involved. Although HSV-1 coinfection significantly changed the type of AAV genome end recombination, no significant change in the amount of circular AAV genomes was identified. IMPORTANCE Adeno-associated virus (AAV)-mediated gene therapy represents one of the most promising approaches for the treatment of genetic diseases. Currently, various GMP-compatible production methods can be applied to manufacture clinical-grade vector, including methods that employ helper factors derived from herpes simplex virus 1 (HSV-1). Yet, to date, we do not fully understand how HSV-1 interacts with AAV. We observed that HSV-1 modulates AAV genome ends similarly to the genome recombination events observed during HSV-1 replication and postulate that further improvements of the HSV-1 production platform may enhance packaging of the recombinant AAV particles.

Polycistronic Herpesvirus Amplicon Vectors for Veterinary Vaccine Development.

Heterologous virus-vectored vaccines, particularly those based on canarypox virus vectors, have established a firm place in preventive veterinary medicine. However, herpesvirus-based vaccines have paved the way for DIVA vaccines (discrimination of infected against vaccinated animals), which are particularly desirable for highly contagious livestock diseases that are otherwise combatted by culling of infected animals.In this chapter, we describe the design, the preparation, and the testing of a polycistronic herpesvirus amplicon vaccine against rotaviruses with a particular emphasis on generating heterologous virus-like particles for immunization. After the design, the procedure consists of three steps, first, transient expression of the construct in cell cultures, second, expression and antibody response in a mouse model, and third, application of the system to the desired host species. As a whole, the present information will facilitate the design of novel vaccines of veterinary interest from the designing process until pre-licensing.