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AG Sodeik

The Cell Biology of Viral Infections: Herpes Simplex Virus - a hitch hiker’s guide to the Cell.

 

Our group is interested in the cell biology of alphaherpesviruses. How does a virus get access to a cell, disassemble to release its genome for transcription and replication, and how is - within a couple of hours - the viral program switched to virus assembly and egress?

 

Our focus is to analyze all virus-host interactions of Herpes Simplex Virus Type 1 (HSV1) in cell types that it also interacts with in the human host: epithelial, dendritic and neuronal cells. Towards this end, we investigate the virus-host cell interactions that are required for efficient HSV1 cell entry, nuclear targeting of the viral genome, viral gene expression, virus assembly, egress and spread.  We use HSV1 mutants as well as state-of-the-art biochemical, life-cell imaging approaches and electron mciroscopy to characterize how HSV1 activates cellular signal transduction cascades to utilize the actin cytoskeleton, the microtubule transport machinery, the nuclear import machinery, and the membranes of the secretory pathway during HSV1 cell entry as well as assembly.

 

 

Research summary

 

HSV1 is a neurotropic virus with a double-stranded DNA genome of 152 kb that initially replicates in the skin and mukosa of the mouth. Progeny virus enters sensory neurons and is transported to the nuclei located in the trigeminal ganglion, where a latent infection is established. Reactivation of the latent genomes results in re-infection of the epithelial tissue (Herpes labialis), or in rare cases to life-threatening diseases by further spread into the brain (Herpes encephalitis).

 

HSV1 enters cells by fusion of the viral envelope with the plasma membrane. The incoming viral capsids are propelled along microtubules (MTs) to the nucleus by dynein, a MT motor. They are ultimately targeted to the nuclear pore complexes (NPCs) where the viral genome is uncoated for viral transcription and replication in the nucleoplasm. During virus assembly and egress, cytosolic capsids are also transported along MTs to the membranes of secondary envelopment.

 

We have developed several in vivo and in vitro motility assays to analyze the cell biology of HSV1, and to characterize cytosolic host factors as well as structural viral proteins important in HSV1 entry and egress. Besides wild type we use mutant viruses in which the small capsid protein VP26 has been tagged with autofluorescent protein domains. Individual GFP-VP26 labeled capsids entering or leaving living cells can then be analyzed by digital time-lapse microscopy.

 

To dissect the interaction between the viral capsids and the MTs or NPCs biochemically, we have developed in vitro binding assays. The simplest consists of isolated viral capsids and a cytosolic extract. When the cytosol-capsid mixture is incubated with MTs or isolated nuclei, capsids can bind to the MTs or the NPCs. Recombinant candidate proteins, peptides or antibodies against cytosolic as well as viral proteins are then tested for their ability to interfere with virus-host interactions.

 

The ultimate goal of our research is to understand the molecular determinants of cytosolic viral capsid transport. Along these lines, we have developed an in vitro motility assay containing isolated GFP-VP26-capsids, Cy3-labelled MTs, and cytosol. By digital time-lapse microscopy, we observe individual GFP-VP26-capsids moving unidirectional and with constant speed along MTs.

 

Using a functional proteomics approach, we mutate several structural viral proteins. We are mainly interested in tegument proteins localized between the viral envelope and the capsid, since they include viral receptors for cytosolic host factors. Using a recombinant BAC-vector (bacterial artificial chromosome) encoding the entire HSV1 genome, we have generated specific mutants of HSV1 that we test for their ability to move along MTs as well as for the directionality of such transport. In contrast to other cargo transported by MTs, the known protein composition of this one can be manipulated in a controlled manner. Dependent on the stage of the viral life cycle and the cellular context, herpes virus capsids move to the cell centre or periphery by being transported to the MT minus- or plus-ends. We will use our system to identify putative motor receptors and to analyze the factors controlling the directionality of MT transport and the activation of different MT motors.

 

The in vitro assays that we have developed provide tools to analyze other human pathogenic herpes viruses for which so far no good cell culture models have been established such as cytomegalovirus or Kaposi sarcoma-associated herpesvirus. Our studies will hopefully in the long run identify viral-host interactions suitable for drug discovery and new antiviral therapy.