An Introduction to JCV Polyomavirus
Over fifty percent of the world's adults have been infected by the JC polyomavirus ((JCV); Kean et al, 2009 ). Unfortunately, for those individuals who become immunocompromised, there is an increased probability that JCV will progress from subclinical infections to reactivation. The "immunocompromised population" is large and includes AIDS patients, transplant recipients, those with hematological malignancies and patients treated with immunomodulatory medications. Reactivation of JCV in the immunocompromised population is associated with Progressive Multifocal Leukoencephalopathy (PML): a demyelinating disease of the central nervous system (reviewed in Gheuens et al, 2013 ). JCV is also known to cause additional syndromes following infections of neurons and meningeal cells (reviewed in Miskin and Koralnik, 2015 ). A possible association of JCV with human brain and non-central nervous system tumors has also been reported. Specific treatments for JCV associated diseases do not exist.
A Summary of our Current Research on JCV
For a number of reasons, including inhibitor development (see below), we want to understand the initiation of JCV DNA replication at the molecular level. Therefore, in collaboration with members of the Bohm laboratory at Tufts University School of Medicine, we are analyzing the structures of protein domains involved in the initiation of JCV DNA replication (eg, Meinke et al, 2014 ).
Characterization of cellular factors needed for JCV DNA Replication
The regulatory region within the JCV genome is termed the noncoding control region (NCCR). The binding of cellular factors to the NCCR, most of which have yet to be identified, determines the tropism of JCV for glial cells in the brain. Along with the virally encoded T-antigen (T-ag), these same cellular factors control the initiation of viral DNA replication. Therefore, a central theme of our research is the identification and characterization of the cellular factors that bind to the NCCR during the initiation of JCV DNA replication (eg, Shin et al, 2014 ).
Recent progress in this endeavor includes our increasing evidence that NFI-A, a member of the NFI family of transcription factors, binds to the NCCR and is required for JCV DNA replication. This is an interesting result since NFI-A is also necessary and sufficient to initiate glial cell development. Moreover, the tissue that contains the highest level of NFI-A mRNA is the cerebellum, the site of most PML lesions. Collectively, these observations raise the possibility that one very important reason why JCV preferentially replicates in glial cells in the cerebellum is the elevated levels of NFI-A protein in this tissue. Therefore, we are continuing to investigate NFI-A and its roles in JCV DNA replication. Additional cellular factors that bind to the NCCR are being identified using a novel approach that involves newly available RNAseq data.
Isolation of inhibitors of JCV DNA replication
An additional major theme of our research is the discovery of small molecular weight inhibitors of JCV DNA replication. These structural based design studies are being conducted in collaboration with members of the Kritzer Laboratory in the Chemistry Department at Tufts University.
Characterization of monoclonal antibodies that selectively target the origin binding domains of polyomavirus T-antigens
In collaboration with members of the Bohm and Jefferson laboratories, we are raising monoclonal antibodies that selectively recognize the T-ags encoded by different human polyomaviruses. These monoclonal antibodies will be used for a number of purposes, such as detecting the replication of these viruses.
Overview of our Research into the Mechanisms Needed For the Initiation DNA Replication
Using the SV40 model system, several laboratories have investigated the mechanisms needed to initiate polyomavirus DNA replication. These viruses contribute only a single protein to the initiation process, the previously discussed T-ag; the host provides all the other replication factors. Therefore, given its central role in polyomavirus DNA replication, we were among those who focused on T-ag's multiple functions during the initiation process.
T Antigen and its Interactions with DNA
The question, "how are origins of replication recognized by initiator proteins" is of general interest. In collaboration with members of the Bohm laboratory, we determined the co-structure of the origin-binding domain (OBD) of T-ag bound to its binding sites on the SV40 origin (Meincke et al, 2007 ). This study revealed how T-ag recognizes its GAGGC binding sites within the origin of replication.
Furthermore, in Meinke et al, 2006 , we reported that the OBD can also form a left-handed “spiral hexamer” (Fig 1). An intriguing feature of the spiral hexamer is the presence of a gap between the first and last subunits. This gap may play an important role in the subsequent extrusion of ssDNA from the origin region. It is emphasized that the spiral structure is thought to form after the initial site-specific binding events.
Figure 1. The crystal structure of the T-ag-OBD; the six different monomers are colored separately. The overall structure is that of a hexameric left-handed spiral. Relative to one monomer (e.g. yellow) the neighboring monomer (purple) is rotated by sixty degrees about the six-fold axis, and translated ~5.9 angstroms along the C-axis. Thus, the two ends of the spiral are separated by a gap, between the yellow and pink subunits, that is ~29.5 angstroms wide (5.9 x 5).
Li et al, 2003 reported the structure of the T-ag helicase domain. Using this structure we investigated an additional protein/DNA interaction needed for recognition of the viral origin. Studies described in Reese et al, 2004 led to the realization that the “beta-hairpin” motif in the T-ag helicase domain (red residues in Fig. 2) plays critical roles in origin recognition and DNA melting. Based on extensive sequence homology, Reese et al. proposed that this motif is present in many other DNA helicases. Indeed, it is now clear that “beta-hairpins” are essential for both origin recognition and in the ATP dependent movement of ssDNA through helicase domains (Gai et al, 2004 ; Kumar et al, 2007 ).
Figure 2. A model of a T-ag monomer assembled on a sub-fragment of the core origin highlighting the interaction between the beta-hairpin, in red, with the region of the origin (i.e., the early palindrome (EP)) that is initially melted (Reese et al, 2004 ). The helicase domain is in blue. The interaction of the T-ag OBD (purple residues) with the GAGGC pentanucleotides (P1-P4) is also critical for origin recognition.
Once T-antigen has located the origin, and melted the “early-palindrome” region via the "beta-hairpin", it oligomerizes into a double hexamer. Using mutant forms of T-antigen, we investigated this process and proposed a model (Kumar et al, 2007 ) that is reprinted in Fig 3. An essential feature of this model is that single stranded DNA generated by the “beta-hairpin” is a prerequisite for the subsequent assembly of the helicase domain. Assembly around single-stranded DNA helps to account for the 3’ to 5’ movement of the helicase once formed.
Figure 3. A model depicting "beta-hairpin dependent melting of the "EP" region of the origin and subsequent oligomerization of the helicase domain on single stranded DNA.
A model accounting for T-ag's multiple roles during the initiation process
Owing to additional biochemical, genetic and structural studies we now have a model that accounts for T-ag's multiple roles during the initiation of DNA replication (Fig 4). Details of this model are reviewed in Meinke and Bullock, 2012).
Figure 4. A model accounting for how T-ag transitions from site-specific DNA binding to functioning as a helicase at replication forks (derived from Meinke and Bullock (2012)). According to this model, "spiral formation" by the OBD is critical to the initiation process. For example, notice in step 5 that single stranded DNA is depicted going through the "gap" that is created by the spiral.
It will be of interest to determine if this model accounts for the initiation events catalyzed by other double hexamer helicases, such as the Mcm2-7 complex (Ticau et al, 2015 ).