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  1. Evaluation Summary:

    This is a very significant new model for papillomavirus infection but all the reviewers have major reservations about the data as they stand, and the quality of some of the data. The manuscript is not publishable without better/more data and a re-write. The work has potential but there is quite a bit of essential experimental work required to make this manuscript sound.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    In the article by Yilmaz et al., the authors generated a system that will allow researchers to both identify and track the target cells of papillomavirus infection. Importantly, their approach enables lineage tracing to be performed in an in vivo setting, which more closely mimics the conditions present and required during a natural papillomavirus infection of its host. Using the recently discovered murine papillomavirus (MmuPV1), which infects common strains of the laboratory mouse Mus musculus, the authors genetically engineered the MmuPV1 genome to contain a lox-Cre-lox sequence. As part of their approach, the authors hypothesize that application of the plasmid DNA to murine skin of Rosa26-LSL-YFP reporter mice will achieve two major outcomes: 1) the Cre recombinase self-deletes the lox-Cre-lox sequence, which subsequently allows recircularization of the MmuPV1 circular dsDNA genome and ultimately, active viral infection of keratinocytes, and 2) Cre-induced excision of the LSL sequence upstream of the YFP reporter present in the murine skin, thus 'tagging' the infected target cell and its progeny and allowing longitudinal lineage tracing of this infected population.

    Strengths:
    1. The MmuPV1-lox-Cre-lox virus engineered by the authors represents a significant technological advancement in the MmuPV1 infection model system that will facilitate many fundamental and exciting experimental opportunities in the future.
    2. The authors provide sufficient verification of delivery and recircularization of the MmuPV1-lox-Cre-lox virus using both in vitro and in vivo.
    3. The time course nature of their in vivo studies provides important insight into the infection dynamics of the MmuPV1-lox-Cre-lox infection model.

    Weaknesses:
    1. The authors provide strong evidence of MmuPV1-lox-Cre-lox delivery, Cre expression/loss, and genome recircularization; however, their evidence of a productive MmuPV1 viral infection as a result can be strengthened.

    Overall, the authors achieved their aims and provide sufficient data to support their conclusions. In the future, this new model system will be of significant interest to those in the HPV field and will likely have a demonstrable impact on our understanding of HPV pathogenesis, transmission, and the development of HPV-associated malignancies.

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  3. Reviewer #2 (Public Review):

    The authors have used the recently described mouse PV infection model to study early molecular and cellular events of PV infection at the site of virus inoculation. Cellular "tags" (YFP) are used to precisely track epithelium infected with MmuPV1.

    The approach was to use a genetically tagged virus construct and a transgenic mouse model that generates a reported signal (YFP) at the sites of infection.

    Novel findings are presented in this study including:
    (i) Viability and utility of the approach allowing the tracing of the sites of primary infection and the expansion of the infected cells in an authentic papillomavirus infection model.
    (ii) Careful assessment to show that the expression of the transgene (YFP) is matched in cells with authentic viral activity (viral RNA).
    (iii) Initial identification of several host-associated factors that have been identified in MmuPV1-infected mouse tissues.

    The described research and model system expands nicely the opportunities to trace virally-infected cells in vivo and to isolate virally-infected from uninfected cells in these tissues to assess their genetic and cellular changes at the single cell level.

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  4. Reviewer #3 (Public Review):

    This paper introduces a novel genetic model to study lineage tracing of papillomavirus infected cells in the mouse. The model has the potential to be highly useful in study of the biology of the intimate interaction between the epithelium and the papillomavirus life cycle. This sort of model could revolutionise papillomavirus life cycle study and is particularly relevant to cutaneous human papillomavirus infections where there are no good life cycle models. The authors aim to prove that their model can accurately recapitulate the MmuPV1 life cycle and that the reconstituted virus has phenotypic effects on the infected keratinocyte.

    The major strength of the study is the creation of the in vivo model to generate infectious MmuPV1. However, a weakness of the study is that the data do not go far enough to be entirely convincing at this stage. The data analysing the Cre recombinase outcomes are not sufficiently detailed or robust to allow the reader to be certain that the recombination event has been completely successful. In addition, further data on the effects of the recombination event on viral gene expression and on the infected epithelium would be required to fully support the authors conclusions.

    It is very interesting that the authors find a decrease in MHC class I presentation on the surface of infected cells. However, the finding could be taken further to understand the mechanism by which MmuPV1 achieves this. This would represent a major contribution to the MmuPV1 field which may have implications for both oncogenic and non-oncogenic human papillomaviruses.

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