Professor Cornel Fraefel, from the Institute of Virology at the University of Zurich, discusses viral co-infections.
Viruses consist of a nucleic acid genome, which is enclosed by a protein shell and, sometimes, an additional lipid envelope. The protein and lipid layers are responsible for protecting the viral genome and facilitating its transfer from infected, virus producing cells to as yet uninfected target cells. Once inside the cell, viruses hijack the cellular infrastructure to promote their own replication. Provided that the cell is able to sustain the complete viral replication cycle (permissive), progeny virus particles are then assembled and exit the cell to start a new infection cycle in other cells of the same tissue or host, or in cells of a different host. For some viruses, the availability of a permissive cell is not sufficient to allow its replication. For example, adeno-associated viruses, hepatitis D viruses, and sputnik viruses require for productive replication the simultaneous presence of a helper virus in the host cell. The helper virus provides essential helper proteins and also conditions the host cell to support virus replication, e.g. by interfering with cell cycle progression, cellular DNA damage response signalling, and host defense mechanisms. Viruses that depend on a helper virus for replication provide invaluable models for studying the interactions between two different viruses and the co-infected cells on a molecular, mechanistic level in cell culture.
Since their discovery as independent biological agents a little more than a hundred years ago, hundreds of different viruses have been identified and characterised concerning their biology, epidemiology, and pathogenic potential. However, it is estimated that hundreds of thousands more different viruses exist in vertebrates alone. The rapidly growing number of known viruses in recent years has been supported by technological advances, next generation sequencing (NGS) in particular, which allows to identify and quantify all nucleic acids of host and infectious-agent origin in a sample. Their small size does in general not support direct visualisation of viruses by light microscopy, which has contributed to their late discovery. Interestingly however, it was in fact the small viruses, such as the tobacco mosaic virus and the foot-and mouth disease virus, that were detected first, using filtration experiments, while giant viruses with the size of small bacteria, such as the mimi- and pandora-viruses which infect amoeba, were detected only very recently with the advent of the NGS technologies. In coming years, NGS will likely lead to the identification of a legion of new viruses and may reveal many more infectious agents that are less complex than viruses, including viroids (small, infectious RNA molecules), virusoids (small RNA molecules in a protein coat provided by a helper virus), and satellite viruses (small RNA molecules which encode their own protein coat in a helpervirus-dependent fashion). However, while NGS can reveal the viromes and microbiomes of the hosts (see Box Out), we do not presently have the tools to investigate the effects of a specific virome/microbiome composition and of the diverse interactions between individual infectious agents on health and disease of the host organism. Some of these interactions may prove beneficial for the host while others may predict a more severe disease progression. The knowledge of the microbiome composition of a patient may also directly affect the strategy of the therapeutic intervention.
Over the past decade, our laboratory has investigated the molecular mechanisms of interaction between adeno-associated virus 2 (AAV2) and its helper viruses, specifically herpes simplex virus type 1 (HSV-1). These studies revealed fascinating viral strategies to cope with competition for cellular resources in co-infected cell cultures. The experimental tools developed and knowledge gained in the course of these investigations may also support the goal of assessing more complex mechanisms of pathogen-pathogen interactions on the level of the co-infected host. Towards the same goal, we have recently started to determine the virome compositions of clinical samples by NGS, which may facilitate the identification and characterisation of yet unknown virus-virus interactions that have an impact on health and disease.
Adeno-associated virus 2
AAV2 is a non-pathogenic, human parvovirus with a small, single-stranded DNA genome of 4.68kb enclosed in an icosahedral capsid with a diameter of approximately 20nm (for a review see reference (1)). AAV2 belongs to the genus iDependoparvovirus and, as the name suggests, depends on the simultaneous presence of a helper virus for productive replication (see Fig. 1). In absence of a helper virus, AAV2 establishes a latent infection by maintaining its genome as an episome or integrating it into the host cell genome, preferentially at the so-called AAVS1 site on chromosome 19 (2). The AAV2 genome contains only two clusters of genes, rep and cap, flanked by inverted terminal repeats (ITRs), which contain the viral origin of DNA replication and the DNA packaging signal. The cap genes encode the capsid proteins VP1, VP2, and VP3, as well as the assembly-activating protein AAP. The rep gene encodes four Rep isoforms, Rep40, Rep52, Rep68, and Rep78, which are involved in DNA replication, packaging and genomic integration (for a review see reference (3)).
A number of different viruses can provide helper functions for AAV2 replication, including adenovirus 2 (4), human papillomavirus 16 (5), and herpes simplex virus type 1 (HSV-1; (6)). While AAV2 depends on specific functions provided by the helper virus, it has developed specialised mechanisms to inhibit the replication of its helper viruses, likely to compete for cellular resources and helper viral proteins. The exploitation and control of HSV-1 by AAV2 is particularly multifold. AAV2 scavenges essential HSV-1 proteins such as ICP8 and the helicase/primase complex and profits from the helper virus-mediated conditioning of cellular DNA damage responses, interference with cell cycle progression, and blocking of innate host defense mechanisms. At the same time, AAV2 blocks HSV-1 replication in a Rep protein- and cell cycle-dependent manner. Some of these strategies are outlined below in more detail.
AAV2 inhibits HSV-1 DNA replication
The AAV2 mediated inhibition of adenovirus replication has been shown to be, at least in part, due to the AAV2 Rep protein-mediated inhibition of the protein kinases PKA and PRKX, both members of the cyclic AMP (cAMP) signal transduction pathway (7, 8). Interestingly, AAV2 Rep inhibits also HSV-1 replication but through a different mechanism.
Our early studies revealed that AAV2 and HSV-1 DNA replication occurs in distinct nuclear compartments (9, 10), while AAV2 has been shown to be recruited into adenovirus replication compartments (RCs) (11). In AAV2 and HSV-1 co-infections, helper virus replication appeared to be inhibited, as HSV-1 RCs consistently remained small in cells that contained mature AAV2 RCs (10). As the HSV-1 ICP8 protein, which is essential for the replication of both viruses, was found to co-localise with AAV2 RCs while HSV-1 RCs were devoid of ICP8, we initially hypothesised that the AAV2-mediated inhibition of HSV-1 replication was due to the recruitment of essential helper virus proteins to AAV2 RCs (10). However, follow-up experiments demonstrated that AAV2 Rep proteins 68 and 78 alone, in absence of AAV2 DNA replication, can block HSV-1 DNA replication and that the Rep helicase and DNA binding domains, but not the nuclease domain, are essential for the inhibition (12, 13).
Rep helicase activity
As the AAV2 mediated inhibition of HSV-1 replication was found to occur at the level of DNA synthesis and to depend on the DNA binding and helicase but not the nuclease activities of Rep, we investigated the hypothesis that Rep can directly bind to putative consensus Rep binding sites on the HSV-1 genome.
The replication strategy of AAV2 is based on a unique rolling-hairpin mechanism in which the inverted terminal repeats at the 3’ end of the genome form a hairpin structure that provides a primer for second-strand synthesis. This results in a closed-end intermediate at the 3’-ITR which is resolved by the Rep68/78 proteins that bind to the Rep binding site (RBS) and introduce a site- and strand-specific nick at the so-called terminal resolution signal (TRS), thereby creating a primer that allows for the completion of the DNA replication process (14) (for a review see: (1)).
We demonstrated in silico (sequence alignment), in vitro (electrophoretic mobility shift assays), and in vivo (chromosome immune precipitation analyses) that consensus RBS indeed exist on the HSV-1 genome and that the Rep proteins can bind to these sites; however, consensus TRS were not found in close proximity. We also showed that the Rep 68/78 helicase domain can inhibit the replication of any DNA when binding is facilitated but a TRS is not present. We therefore hypothesised that the binding of Rep to consensus RBS and the helicase activity may generate a situation resembling a stalled replication fork, in particular when the Rep helicase activity is uncoupled from the replication activity because of the absence of a functional TRS ((13)).
AAV2 efficiently blocks the replication of HSV-1 which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy prompts the question how AAV2 and HSV-1 can co-exist in a cell population. To address this, we investigated AAV2 gene expression and AAV2 DNA replication in HeLa Fucci cells, which allow monitoring cell cycle progression by live-cell fluorescence microscopy ((15)); in G1-phase, the cells appear red-fluorescent while in S- and G2-phases they appear green-fluorescent. Viral gene expression and DNA replication in these cells was monitored by using recombinant viruses that encode blue-fluorescent proteins per se, to allow monitoring gene expression, or fused with viral DNA binding proteins, to support visualising the formation of viral DNA replication compartments.
These experiments showed that AAV2 (rep) gene expression and AAV2 DNA replication occur primarily in S/G2 cells (see Fig. 2). HSV-1, which can normally replicate in all phases of the cell cycle in the absence of AAV2, was restricted to G1 cells in the presence of AAV2 ((15)). As the Rep proteins are essential for both the replication of the AAV2 DNA and the inhibition of HSV-1 DNA replication, we concluded that cell cycle-dependent rep expression is responsible for the observed cell cycle-dependent AAV2 replication and inhibition of HSV-1 replication. The licensing factor that allows AAV2 (rep) gene expression and DNA replication in S/G2 phase or the inhibitor that blocks it in G1 remains to be identified. Nevertheless, the ‘segregation’ of the host cell population between the two different viruses is a novel concept of viral adaptation and a fascinating strategy to minimise competition allowing both, AAV2 and HSV-1 to productively replicate, although in distinct sets of cells ((15); see Fig. 3).
Many viruses are efficiently eliminated or controlled by host response mechanisms. However, when the co-evolution of a virus and its host is in an early stage, virus infection can have severe consequences for the host. Indeed, viruses are among the main driving forces of host evolution, and they constantly challenge and coach the host immune system. The example of the interaction of AAV2 with its helper viruses demonstrates that non-pathogenic viruses can also directly limit the replication of highly pathogenic viruses such as herpes, adeno, and papilloma viruses. In this sense, one may speculate that some viruses have protective roles for the host and that the microbiome composition of a patient has a direct impact on severity of the symptoms, disease progression, treatment and prognosis. Some of these hypotheses can be addressed now, as NGS allows us to determine microbiome compositions as well as the genetic background and transcriptome (see Box Out) of a patient, in particular when combined with clinical parameters and knowledge gained from analyzing the mechanisms of pathogen-pathogen interactions on the molecular level in cell culture.
I would like to acknowledge all present and past members of my laboratory who contributed to the data presented in this article. I would also like to acknowledge Dr Diogo Guerra (Medical and Veterinary Illustrations) who helped with the preparation of the figures
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Institute of Virology
University of Zurich
This article will appear in SciTech Europa Quarterly issue 26, which will be published in March, 2018.