RESUMO
The adeno-associated virus (AAV) is a small, nonpathogenic parvovirus, which depends on helper factors to replicate. Those helper factors can be provided by coinfecting helper viruses such as adenoviruses, herpesviruses, or papillomaviruses. We review the basic biology of AAV and its most-studied helper viruses, adenovirus type 5 (AdV5) and herpes simplex virus type 1 (HSV-1). We further outline the direct and indirect interactions of AAV with those and additional helper viruses.
Assuntos
Adenoviridae/metabolismo , Dependovirus/crescimento & desenvolvimento , Vírus Auxiliares/metabolismo , Herpesvirus Humano 1/metabolismo , Replicação Viral/genética , Coinfecção/virologia , Dependovirus/genética , Humanos , Infecções por Parvoviridae/virologia , Proteínas Virais/genéticaRESUMO
The human herpesvirus family members, in particular herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2), are abundant and extremely contagious viruses with a high seroprevalence in the human population emphasizing the importance of studying their biology. Hence, the propagation and purification of virus stocks constitute a key element in laboratory work.
Assuntos
Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/crescimento & desenvolvimento , Herpesvirus Humano 2/isolamento & purificação , Animais , Chlorocebus aethiops , Humanos , Células VeroRESUMO
HSV-1 amplicon vectors have been used as platforms for the generation of genetic vaccines against both DNA and RNA viruses. Mice vaccinated with such vectors encoding structural proteins from both foot-and-mouth disease virus and rotavirus were partially protected from challenge with wild-type virus (D'Antuono et al., Vaccine 28:7363-7372, 2010; Laimbacher et al., Mol Ther 20:1810-1820, 2012; Meier et al., Int J Mol Sci 18:431, 2017), indicating that HSV-1 amplicon vectors are attractive tools for the development of complex and safe genetic vaccines.This chapter describes the preparation and testing of HSV-1 amplicon vectors that encode individual or multiple viral structural proteins from a polycistronic transgene cassette. We further put particular emphasis on generating virus-like particles (VLPs) in vector-infected cells. Expression of viral genes is confirmed by Western blot and immune fluorescence analysis and generation of VLPs in vector-infected cells is demonstrated by electron microscopy. Furthermore, examples on how to analyze the immune response in a mouse model and possible challenge experiments are described.
Assuntos
Vetores Genéticos , Herpesvirus Humano 1 , Transdução Genética , Vacinas Virais , Animais , Chlorocebus aethiops , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Camundongos , Células Vero , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.
Assuntos
DNA Nucleotidiltransferases/química , Dependovirus/enzimologia , Integrases/química , Biologia Computacional , Conjugação Genética , DNA/química , DNA Helicases/química , DNA Bacteriano/genética , DNA de Cadeia Simples , Endonucleases/química , Escherichia coli/metabolismo , Células HEK293 , Humanos , Plasmídeos , Domínios Proteicos , Proteínas Recombinantes/química , UltracentrifugaçãoRESUMO
In infected cells rotavirus (RV) replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT) network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used a MA104-Fucci system to identify three RV proteins (NSP3, NSP5, and VP2) involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular , Rotavirus/fisiologia , Pontos de Checagem da Fase S do Ciclo Celular , Transdução de Sinais , Replicação Viral/fisiologia , Animais , Ciclina B1/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/virologia , Cães , Células HEK293 , Humanos , Cinesinas/metabolismo , Macaca mulatta , Células Madin Darby de Rim Canino , Proteínas Virais/metabolismoRESUMO
Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate.IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells.
Assuntos
Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Dependovirus/crescimento & desenvolvimento , Vírus Auxiliares/crescimento & desenvolvimento , Herpesvirus Humano 1/crescimento & desenvolvimento , Interferência Viral , Proteínas Virais/metabolismo , Replicação Viral , Linhagem Celular , Coinfecção , Expressão Gênica , Humanos , Microscopia , Cultura de VírusRESUMO
Rotaviruses (RVs) are important enteric pathogens of newborn humans and animals, causing diarrhea and in rare cases death, especially in very young individuals. Rotavirus vaccines presently used are modified live vaccines that lack complete biological safety. Previous work from our laboratory suggested that vaccines based on in situ produced, non-infectious rotavirus-like particles (RVLPs) are efficient while being entirely safe. However, using either vaccine, active mucosal immunization cannot induce protective immunity in newborns due to their immature immune system. We therefore hypothesized that offspring from vaccinated dams are passively immunized either by transfer of maternal antibodies during pregnancy or by taking up antibodies from milk. Using a codon optimized polycistronic gene expression cassette packaged into herpesvirus particles, the simultaneous expression of the RV capsid genes led to the intracellular formation of RVLPs in various cell lines. Vaccinated dams developed a strong RV specific IgG antibody response determined in sera and milk of both mother and pups. Moreover, sera of naïve pups nursed by vaccinated dams also had RV specific antibodies suggesting a lactogenic transfer of antibodies. Although full protection of pups was not achieved in this mouse model, our observations are important for the development of improved vaccines against RV in humans as well as in various animal species.