Tiffany A. Russell

Lytic Gene Expression Is Frequent in HSV-1 Latent Infection and Correlates with the Engagement of a Cell- Intrinsic Transcriptional Response

Herpes simplex viruses (HSV) are significant human pathogens that provide one of the best-described examples of viral latency and reactivation. HSV latency occurs in sensory neurons, being characterized by the absence of virus replication and only fragmentary evidence of protein production. In mouse models, HSV latency is especially stable but the detection of some lytic gene transcription and the ongoing presence of activated immune cells in latent ganglia have been used to suggest that this state is not entirely quiescent. Alternatively, these findings can be interpreted as signs of a low, but constant level of abortive reactivation punctuating otherwise silent latency. Using single cell analysis of transcription in mouse dorsal root ganglia, we reveal that HSV-1 latency is highly dynamic in the majority of neurons. Specifically, transcription from areas of the HSV genome associated with at least one viral lytic gene occurs in nearly two thirds of latently-infected neurons and more than half of these have RNA from more than one lytic gene locus. Further, bioinformatics analyses of host transcription showed that progressive appearance of these lytic transcripts correlated with alterations in expression of cellular genes. These data show for the first time that transcription consistent with lytic gene expression is a frequent event, taking place in the majority of HSV latently-infected neurons. Furthermore, this transcription is of biological significance in that it influences host gene expression. We suggest that the maintenance of HSV latency involves an active host response to frequent viral activity.

Engineering herpes simplex viruses by infection–transfection methods including recombination site targeting by CRISPR/Cas9 nucleases

Herpes simplex viruses (HSVs) are frequent human pathogens and the ability to engineer these viruses underpins much research into their biology and pathogenesis. Often the ultimate aim is to produce a virus that has the desired phenotypic change and no additional alterations in characteristics. This requires methods that minimally disrupt the genome and, for insertions of foreign DNA, sites must be found that can be engineered without disrupting HSV gene function or expression. This study advances both of these requirements. Firstly, the use of homologous recombination between the virus genome and plasmids in mammalian cells is a reliable way to engineer HSV such that minimal genome changes are made. This has most frequently been achieved by cotransfection of plasmid and isolated viral genomic DNA, but an alternative is to supply the virus genome by infection in a transfection-infection method. Such approaches can also incorporate CRISPR/Cas9 genome engineering methods. Current descriptions of infection-transfection methods, either with or without the addition of CRISPR/Cas9 targeting, are limited in detail and the extent of optimization. In this study it was found that transfection efficiency and the length of homologous sequences improve the efficiency of recombination in these methods, but the targeting of the locus to be engineered by CRISPR/Cas9 nucleases has an overriding positive impact. Secondly, the intergenic space between UL26 and UL27 was reexamined as a site for the addition of foreign DNA and a position identified that allows insertions without compromising HSV growth in vitro or in vivo.

Distinct APC subtypes drive spatially segregated CD4+ and CD8+ T-cell effector activity during skin infection with HSV-1.

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4+ and CD8+ T-cells during skin infection with HSV-1. IFN-γ-producing CD4+ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8+ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4+ T cells, CD8+ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8+ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4+ and CD8+ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).

Local proliferation maintains a stable pool of tissue-resident memory T cells after antiviral recall responses

Although tissue-resident memory T cells (TRM cells) are critical in fighting infection, their fate after local pathogen re-encounter is unknown. Here we found that skin TRM cells engaged virus-infected cells, proliferated in situ in response to local antigen encounter and did not migrate out of the epidermis, where they exclusively reside. As a consequence, secondary TRM cells formed from pre-existing TRM cells, as well as from precursors recruited from the circulation. Newly recruited antigen-specific or bystander TRM cells were generated in the skin without displacement of the pre-existing TRM cell pool. Thus, pre-existing skin TRM cell populations are not displaced after subsequent infections, which enables multiple TRM cell specificities to be stably maintained within the tissue.Mackay, Mueller and colleagues show that tissue-resident memory T cells proliferate in situ in response to local antigen and persist during subsequent antigen encounters.

Analysis of A47, an Immunoprevalent Protein of Vaccinia Virus, Leads to a Reevaluation of the Total Antiviral CD8+ T Cell Response

ABSTRACT Vaccinia virus (VACV) is the prototypic orthopoxvirus and was the live vaccine used to eradicate smallpox. In addition, VACV is a possible vector for recombinant vaccines. Despite these reasons for study, the roles of many VACV genes are unknown, and some fundamental aspects, such as the total size of immune responses, remain poorly characterized. VACV gene A47L is of interest because it is highly transcribed, has no sequence similarity to any nonpoxvirus gene, and contains a larger-than-expected number of CD8+ T cell epitopes. Here it is shown that A47L is not required for growth in vitro and does not contribute to virulence in mice. However, we confirmed that this one protein primes CD8+ T cells to three different epitopes in C57BL/6 mice. In the process, one of these epitopes was redefined and shown to be the most dominant in A47 and one of the more highly ranked in VACV as a whole. The relatively high immunogenicity of this epitope led to a reevaluation of the total CD8+ T cell response to VACV. By the use of two methods, the true size of the response was found to be around double previous estimates and at its peak is on the order of 60% of all CD8+ T cells. We speculate that more CD8+ T cell epitopes remain to be mapped for VACV and that underestimation of responses is unlikely to be unique to VACV, so there would be merit in revisiting this issue for other viruses.

Lytic Promoters Express Protein during Herpes Simplex Virus Latency

Herpes simplex virus (HSV) has provided the prototype for viral latency with previously well-defined acute or lytic and latent phases. More recently, the deep quiescence of HSV latency has been questioned with evidence that lytic genes can be transcribed in this state. However, to date the only evidence that these transcripts might be translated has come from immunological studies that show activated T cells persist in the nervous system during latency. Here we use a highly sensitive Cre-marking model to show that lytic and latent phases are less clearly defined in two significant ways. First, around half of the HSV spread leading to latently infected sites occurred beyond the initial acute infection and second, we show direct evidence that lytic promoters can drive protein expression during latency.

Strikingly poor CD8+ T-cell immunogenicity of vaccinia virus strain MVA in BALB/c mice.

Vaccinia virus (VACV) strain MVA is a highly attenuated vector for vaccines that is being explored in clinical trials. We compared the CD8+ T‐cell immunogenicity of MVA with that of a virulent laboratory strain of VACV (strain WR) in BALB/c mice by examining epitope‐specific responses as well as estimating the total number of activated CD8+ T cells, irrespective of specificity. We found that MVA elicited total CD8+ T‐cell responses that were reduced by at least 20‐fold compared with strain WR in BALB/c mice. In C57Bl/6 mice, we also found a substantial difference in immunogenicity between these VACV strains, but it was more modest at around fivefold. Of note, the size of responses to the virulent WR virus was similar in both strains of mice suggesting that BALB/c mice can mount robust CD8+ T‐cell responses to VACV. Although the data for total responses clearly showed that MVA overall is poorly immunogenic in BALB/c mice, we found one epitope for which strong responses were made irrespective of virus strain. Therefore, in the context of a vaccine, some recombinant epitopes may have similar immunogenicity when expressed from MVA and other strains of VACV, but we would expect these to be exceptions. These data show clearly the substantial difference in immunogenicity between MVA and virulent VACV strains and suggest that the impact of host genetics on responses to attenuated vaccine vectors like MVA requires more consideration.

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion

Significance T cells are required for control of many intracellular infections, and a critical component of T cell immunity is the proliferative expansion of effector T cells upon stimulation. Using a forward-based genetic screen, we identify the mouse Etaa1 gene as critically important for T cell proliferative expansion after vaccination and during infection. Consistent with recent findings that ETAA1 prevents DNA damage during proliferation, our data demonstrate elevated DNA damage within Etaa1-deficient effector T cells, which likely leads to cell death. This phenotype is restricted to effector T cell proliferation, with T cell development and other immune parameters remaining normal. Thus, ETAA1 may represent a novel drug target to selectively suppress pathological T cell responses in transplantation or autoimmunity. T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8+ and CD4+ T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.

Increasing antigen presentation on HSV-1-infected cells increases lesion size but does not alter neural infection or latency

CD8+ T cells have a role in the control of acute herpes simplex virus (HSV) infection and may also be important in the maintenance of latency. In this study we have explored the consequences of boosting the efficacy of CD8+ T cells against HSV by increasing the amount of an MHC I-presented epitope on the surface of infected cells. To do this we used HSVs engineered to express an extra copy of the immunodominant CD8+ T cell epitope in C57Bl/6 mice, namely gB498 (SSIEFARL). Despite greater presentation of gB498 on infected cells, CD8+ T cell responses to these viruses in mice were similar to those elicited by a control virus. Further, the expression of extra gB498 did not significantly alter the extent or stability of latency in our mouse model, and virus loads in skin and sensory ganglia of infected mice were not affected. Surprisingly, mice infected with these viruses developed significantly larger skin lesions than those infected with control viruses and notably, this phenotype was dependent on MHC haplotype. Therefore increasing the visibility of HSV-infected cells to CD8+ T cell attack did not impact neural infection or latency, but rather enhanced pathology in the skin.

Effective Priming of Herpes Simplex Virus-Specific CD8+ T Cells In Vivo Does Not Require Infected Dendritic Cells

ABSTRACT Resolution of virus infections depends on the priming of virus-specific CD8+ T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8+ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8+ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation, while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrated in vivo that this requirement for cross-presenting DC was not related to their infection but instead reflected their capacity to cross-present HSV-derived antigen. Taking the results together, this study shows that infected DC are not required for effective CD8+ T cell priming during a peripheral virus infection. IMPORTANCE The ability of some DC to present viral antigen to CD8+ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. The work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo. Our finding that HSV-specific CD8+ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8+ T cell priming during a peripheral virus infection.