Early Gene Products Research Articles

Identification and characterization of the 285L and K145R proteins of African swine fever virus

African swine fever (ASF) is a lethal disease of domestic pigs and wild boar, against which no vaccines are available to date. The large dsDNA genome of African swine fever virus (ASFV) contains up to 167 ORFs predicted to encode proteins. The functions and antigenic properties of many of these proteins are still unknown, which impedes vaccine development. Based on the results of mass spectrometry-based proteome analyses of ASFV-infected cells, two highly abundant but previously uncharacterized viral proteins, p285L and pK145R, were investigated in detail. To this end, monospecific rabbit antisera and corresponding gene deletion mutants of ASFV were prepared. RNA and immunoblot analyses revealed that p285L is an early gene product expressed prior to viral DNA replication, whereas pK145R is a true late protein. The predicted membrane protein p285L could be localized in purified ASFV particles. In contrast, pK145R was not detectable in virions, but accumulated diffusely in the cytoplasm of infected cells. Deletion of 285L or K145R from the genome of a virulent ASFV strain from Armenia did not significantly affect spread and productive growth in a permissive wild boar lung cell line, nor in primary macrophage cultures. Future studies must elucidate, whether p285L and pK145R, although non-essential for in vitro propagation of ASFV, are relevant for replication or virulence in swine. Furthermore, it remains to be investigated whether deletion of the abundant ASFV proteins p285L or pK145R might support serological differentiation from wild-type-infected animals.

Cortical network dynamics are coupled with cognitive aging in rats.

Changes in neuronal network activity and increased interindividual variability in memory are among the most consistent features of growing older. Here, we examined the relationship between these hallmarks of aging. Young and aged rats were trained on a water maze task where aged individuals reliably display an increased range of spatial memory capacities relative to young. Two weeks later, neuronal activity was induced pharmacologically with a low dose of pilocarpine and control animals received vehicle. Activity levels were proxied by quantifying the immediate early gene products Arc and c-Fos. While no relationship was observed between basal, resting activity, and individual differences in spatial memory in any brain region, pilocarpine-induced marker expression was tightly coupled with memory in all areas of the prefrontal cortex (PFC) and hippocampus examined. The nature of this association, however, differed across regions and in relation to age-related cognitive outcome. Specifically, in the medial PFC, induced activity was greatest in aged rats with cognitive impairment and correlated with water maze performance across all subjects. In the hippocampus, the range of induced marker expression was comparable between groups and similarly coupled with memory in both impaired and unimpaired aged rats. Together the findings highlight that the dynamic range of neural network activity across multiple brain regions is a critical component of neurocognitive aging.

Selective response of the nucleus taeniae of the amygdala to a naturalistic social stimulus in visually naive domestic chicks

The detection of animate beings at the onset of life is important for phylogenetically distant species, such as birds and primates. Naïve chicks preferentially approach a stimulus resembling a conspecific (a stuffed fowl) over a less naturalistic one (a scrambled version of the stuffed fowl, presenting the same low-level visual features as the fowl in an unnatural configuration). The neuronal mechanisms underlying this behavior are mostly unknown. However, it has been hypothesized that innate social predispositions may involve subpallial brain areas including the amygdala. Here we asked whether a stuffed hen would activate areas of the arcopallium/amygdala complex, in particular the nucleus taeniae of the amygdala (TnA) or septum. We measured brain activity by visualizing the immediate early gene product c-Fos. After exposure to the hen, TnA showed higher density of c-Fos expressing neurons, compared to chicks that were exposed to the scrambled stimulus. A similar trend was present in the lower portion of the arcopallium, but not in the upper portion of the arcopallium or in the septum. This demonstrates that at birth the TnA is already engaged in responses to social visual stimuli, suggesting an important role for this nucleus in the early ontogenetic development of social behavior.

Spontaneous and light-induced lateralization of immediate early genes expression in domestic chicks

Exposure of domestic chicks’ eggs to light during embryo incubation stimulates asymmetrically the two eye-systems, reaching selectively the right eye (left hemisphere) and inducing asymmetries at the behavioral and neural level. Surprisingly, though, some types of lateralization have been observed also in dark incubated chicks, especially at the behavioral level. Here we investigate the mechanisms subtending the development of lateralization, in the presence and in the absence of embryonic light exposure. We measured the baseline level of expression for the immediate early gene product c-Fos, used as an indicator of the spontaneous level of neural activity and plasticity in four areas of the two hemispheres (preoptic area, septum, hippocampus and intermediate medial mesopallium). Additional DAPI staining measured overall cell density (regardless of c-Fos expression), ruling out any confound due to underlying asymmetries in cell density between the hemispheres. In different brain areas, c-Fos expression was lateralized either in light- (septum) or in dark-incubated chicks (preoptic area). Light exposure increased c-Fos expression in the left hemisphere, suggesting that c-Fos expression could participate to the known effects of light stimulation on brain asymmetries. Interestingly, this effect was visible few days after the end of the light exposure, revealing a delayed effect of light exposure on c-Fos baseline expression in brain areas outside the visual pathways. In the preoptic area of dark incubated chicks, we found a rightward bias for c-Fos expression, revealing that lateralization of the baseline level of activity and plasticity is present in the developing brain also in the absence of light exposure.

An Epigenetic Journey: Epstein-Barr Virus Transcribes Chromatinized and Subsequently Unchromatinized Templates during Its Lytic Cycle.

The Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication in cis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.

The cardiomyocyte “redox rheostat”: Redox signalling via the AMPK-mTOR axis and regulation of gene and protein expression balancing survival and death

Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H2O2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H2O2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H2O2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H2O2 levels are cytoprotective. We identified p21Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a “redox rheostat” in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death.

Social odour activates the hippocampal formation in zebra finches (Taeniopygia guttata).

Experiments from our research group have demonstrated that the olfactory sense of birds, which has been considered as unimportant for a long time, plays a prominent role as communication channel in social behaviour. Odour cues are used e.g. by zebra finch chicks to recognize the mother, by adult birds to distinguish their own eggs from others, or to recognize kin. While there is quite a lot of evidence for the importance of odour for social behaviour, it is not known as yet which brain areas may be involved in the processing of socially relevant odours. We therefore compared the brain activation pattern of zebra finch males exposed to their own offspring odour with that induced by a neutral odour stimulus. By measuring head saccade changes as behavioural reaction and using the expression of the immediate early gene product c-Fos as brain activity marker, we show here that the activation pattern, namely the activity difference between the left and the right hemisphere, of several hippocampal areas in zebra finch males is altered by the presentation of the odour of their own nestlings. In contrast, the nucleus taeniae of the amygdala (TnA) exhibits a tendency of a reduction of c-Fos activation in both hemispheres as a consequence of exposure to the nestling odour. We conclude that the hippocampus is involved in odour based processing of social information, while the role of TnA remains unclear.

Activity-induced MEMRI cannot detect functional brain anomalies in the APPxPS1-Ki mouse model of Alzheimer\u2019s disease

Alzheimer’s disease (AD) is the most common cause of dementia. Aside neuropathological lesions, abnormal neuronal activity and brain metabolism are part of the core symptoms of the disease. Activity-induced Manganese-Enhanced Magnetic Resonance Imaging (MEMRI) has been proposed as a powerful approach to visualize evoked brain activity in rodents. Here, we evaluated the relevance of MEMRI in measuring neuronal (dys-)function in the APPxPS1 knocked-in (KI) mouse model of AD. Brain anomalies were firstly demonstrated in APPxPS1-Ki mice using cognitive testing (memory impairment) and histological mapping of immediate early gene products (decreased density of fos-positive neurons). Paradoxically, MEMRI analyses were not able to confirm the occurrence of neuronal hypoactivities in vivo. We then performed a neuropathological analysis that highlighted an abnormal increased permeability of the blood-brain barrier (BBB) in APPxPS1-Ki mice. We hypothesized that diffuse weakening of the BBB results in an uncontrolled diffusion of the MR contrast agent and a lack of correlation between manganese accumulation and neuronal activity. These results bring to light a limitation of the activity-induced MEMRI approach when applied to the APPxPS1-Ki mouse model as well as other mouse models harboring a compromised BBB.

Temperature-sensitive origin-binding protein as a tool for investigations of herpes simplex virus activities in vivo.

While it is fairly clear that herpes simplex virus (HSV) DNA replication requires at least seven virus-encoded proteins in concert with various host cell factors, the mode of this process in infected cells is still poorly understood. Using HSV-1 mutants bearing temperature-sensitive (ts) lesions in the UL9 gene, we previously found that the origin-binding protein (OBP), a product of the UL9 gene, is only needed in the first 6 hours post-infection. As this finding was just a simple support for the hypothesis of a biphasic replication mode, we became convinced through these earlier studies that the mutants tsR and tsS might represent suitable tools for more accurate investigations in vivo. However, prior to engaging in highly sophisticated research projects, knowledge of the biochemical features of the mutated versions of OBP appeared to be essential. The results of our present study demonstrate that (i) tsR is most appropriate for cell biological studies, where only immediate early and early HSV gene products are being expressed without the concomital viral DNA replication, and (ii) tsS is a prime candidate for the analysis of HSV DNA replication processes because of its reversibly thermosensitive OBP-ATPase, which allows one to switch on the initiation of DNA synthesis precisely.

Genetic Targeting and Chemogenetic Inhibition of Newborn Neurons.

The aim of this study was to develop a method to silence a very specific set of cells in a spatially and temporally refined manner. Here, an approach is presented that combines the use of a transgenic mouse line, expressing cre recombinase under a nestin promoter, with lentiviral delivery of a floxed, ivermectin (IVM)-gated chloride channel construct to the dentate gyrus. This approach was used to express an IVM-sensitive chloride channel in newly born granule cells in adult mouse brains, and its ability to silence neuronal activity was tested by analyzing the effect on immediate early gene expression in vitro in cre-transgenic primary neuronal cultures. IVM treatment of cells expressing the chloride channel prevented gabazine-induced expression of the immediate early gene product EGR1, while cells expressing a control inactive channel or no channel retained their EGR1 response. Thus, a genetic strategy is presented for targeting a specific neurogenic niche for transgene expression in the adult mouse brain, and proof of principle is shown that it can be used in vitro as a method for silencing neuronal activity.

Neural activity in the social decision-making network of the brown anole during reproductive and agonistic encounters

Animals have evolved flexible strategies that allow them to evaluate and respond to their social environment by integrating the salience of external stimuli with internal physiological cues into adaptive behavioral responses. A highly conserved social decision-making network (SDMN), consisting of interconnected social behavior and mesolimbic reward networks, has been proposed to underlie such adaptive behaviors across all vertebrates, although our understanding of this system in reptiles is very limited. Here we measure neural activation across the SDMN and associated regions in the male brown anole (Anolis sagrei), within both reproductive and agonistic contexts, by quantifying the expression density of the immediate early gene product Fos. We then relate this neural activity measure to social context, behavioral expression, and activation (as measured by colocalization with Fos) of different phenotypes of ‘source’ node neurons that produce neurotransmitters and neuropeptides known to modulate SDMN ‘target’ node activity. Our results demonstrate that measures of neural activation across the SDMN network are generally independent of specific behavioral output, although Fos induction in a few select nodes of the social behavior network component of the SDMN does vary with social environment and behavioral output. Under control conditions, the mesolimbic reward nodes of the SDMN actually correlate little with the social behavior nodes, but the interconnectivity of these SDMN components increases dramatically within a reproductive context. When relating behavioral output to specific source node activation profiles, we found that catecholaminergic activation is associated with the frequency and intensity of reproductive behavior output, as well as with aggression intensity. Finally, in terms of the effects of source node activation on SDMN activity, we found that Ile8-oxytocin (mesotocin) populations correlate positively, while Ile3-vasopressin (vasotocin), catecholamine, and serotonin populations correlate negatively with SDMN activity. Taken together, our findings present evidence for a highly dynamic SDMN in reptiles that is responsive to salient cues in a social context-dependent manner.

Arc/Arg3.1 has an activity-regulated interaction with PICK1 that results in altered spatial dynamics

Activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) is an immediate early gene product that is transcribed in dendritic spines and, to date, has been best characterized as a positive regulator of AMPAR endocytosis during long-term depression (LTD) through interaction with endocytic proteins. Here, we show that protein interacting with C terminal kinase 1 (PICK1), a protein known to bind to the GluA2 subunit of AMPARs and associated with AMPAR trafficking, was pulled-down from brain homogenates and synaptosomes when using Arc as immobilized bait. Fluctuation and FLIM-FRET-Phasor analysis revealed direct interaction between these proteins when co-expressed that was increased under depolarizing conditions in live cells. At the plasma membrane, Arc-mCherry oligomerization was found to be concentration dependent. Additionally, co-expression of Arc-mCherry and EGFP-PICK1 followed by depolarizing conditions resulted in significant increases in the number and size of puncta containing both proteins. Furthermore, we identified the Arc binding region to be the first 126 amino acids of the PICK1 BAR domain. Overall, our data support a novel interaction and model where PICK1 mediates Arc regulation of AMPARs particularly under depolarizing conditions.

Dopaminergic neurons are preferentially responsive to advertisement calls and co-active with social behavior network nuclei in sneaker male midshipman fish

Vocal species use acoustic signals to facilitate diverse behaviors such as mate attraction and territorial defense. However, little is known regarding the neural substrates that interpret such divergent conspecific signals. Using the plainfin midshipman fish model, we tested whether specific catecholaminergic (i.e., dopaminergic and noradrenergic) nuclei and nodes of the social behavior network (SBN) are differentially responsive following exposure to playbacks of divergent social signals in sneaker males. We chose sneaker (type II) males since they attempt to steal fertilizations from territorial type I males who use an advertisement call (hum) to attract females yet are also subjected to vocal agonistic behavior (grunts) by type I males. We demonstrate that induction of cFos (an immediate early gene product and proxy for neural activation) in two forebrain dopaminergic nuclei is greater in sneaker males exposed to hums but not grunts compared to ambient noise, suggesting hums preferentially activate these nuclei, further asserting dopamine as an important regulator of social-acoustic behaviors. Moreover, acoustic exposure to social signals with divergent salience engendered contrasting shifts in functional connectivity between dopaminergic nuclei and nodes of the SBN, supporting the idea that interactions between these two circuits may underlie adaptive decision-making related to intraspecific male competition.

EBV persistence without its EBNA3A and 3C oncogenes in vivo.

The oncogenic Epstein Barr virus (EBV) infects the majority of the human population and usually persists within its host for life without symptoms. The EBV oncoproteins nuclear antigen 3A (EBNA3A) and 3C (EBNA3C) are required for B cell transformation in vitro and are expressed in EBV associated immunoblastic lymphomas in vivo. In order to address the necessity of EBNA3A and EBNA3C for persistent EBV infection in vivo, we infected NOD-scid γcnull mice with reconstituted human immune system components (huNSG mice) with recombinant EBV mutants devoid of EBNA3A or EBNA3C expression. These EBV mutants established latent infection in secondary lymphoid organs of infected huNSG mice for at least 3 months, but did not cause tumor formation. Low level viral persistence in the absence of EBNA3A or EBNA3C seemed to be supported primarily by proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression without elevated lytic replication. In vitro, EBNA3A and EBNA3C deficient EBV infected B cells could be rescued from apoptosis through CD40 stimulation, mimicking T cell help in secondary lymphoid tissues. Thus, even in the absence of the oncogenes EBNA3A and 3C, EBV can access a latent gene expression pattern that is reminiscent of EBV persistence in healthy virus carriers without prior expression of its whole growth transforming program.

High salt intake enhances swim stress-induced PVN vasopressin cell activation and active stress coping

PurposeStress contributes to many psychiatric disorders; however, responsivity to stressors can vary depending on previous or current stress exposure. Relatively innocuous heterotypic (differing in type) stressors can summate to result in exaggerated neuronal and behavioral responses. Here we investigated the ability of prior high dietary sodium chloride (salt) intake, a dehydrating osmotic stressor, to enhance neuronal and behavioral responses of mice to an acute psychogenic swim stress (SS). Further, we evaluated the contribution of the osmo-regulatory stress-related neuropeptide arginine vasopressin (VP) in the hypothalamic paraventricular nucleus (PVN), one of only a few brain regions that synthesize VP. The purpose of this study was to determine the impact of high dietary salt intake on responsivity to heterotypic stress and the potential contribution of VPergic-mediated neuronal activity on high salt-induced stress modulation, thereby providing insight into how dietary (homeostatic) and environmental (psychogenic) stressors might interact to facilitate psychiatric disorder vulnerability. ApproachSalt loading (SL) with 4% saline for 7 days was used to dehydrate and osmotically stress mice prior to exposure to an acute SS. Fluid intake and hematological measurements were taken to quantify osmotic dehydration, and serum corticosterone levels were measured to index stress axis activation. Immunohistochemistry (IHC) was used to stain for the immediate early gene product c-Fos to quantify effects of SL on SS-induced activation of neurons in the PVN and extended amygdala – brain regions that are synaptically connected and implicated in responding to osmotic stress and in modulation of SS behavior, respectively. Lastly, the role of VPergic PVN neurons and VP type 1 receptor (V1R) activity in the amygdala in mediating effects of SL on SS behavior was evaluated by quantifying c-Fos activation of VPergic PVN neurons and, in functional experiments, by nano-injecting the V1R selective antagonist dGly[Phaa1,d-tyr(et), Lys, Arg]-VP bilaterally into the amygdala prior to the SS. FindingsSL increased serum osmolality (P < 0.01), which positively correlated with time spent mobile during, and time spent grooming after a SS (P < 0.01, P < 0.01), and SL increased serum corticosterone levels (P < 0.01). SL alone increased c-Fos immunoreactivity among PVN neurons (P = .02), including VP positive neurons (P < 0.01). SL increased SS-induced c-Fos activation of PVN neurons as well (P < 0.01). In addition, SL and SS each increased the total number of PVN neurons that were immunoreactive for VP (P < 0.01). An enhancing effect of SL and SS was observed on c-Fos positive cell counts in the central (P = .02) and basolateral (P < 0.01) nuclei of the amygdala and bilateral nano-injections of V1R antagonist into the amygdala reduced time spent mobile both in salt loaded and control mice during SS (P < 0.05, P < 0.05). SummaryTaken together, these data indicate that neuronal and behavioral responsivity to an acute psychogenic stressor is potentiated by prior exposure to high salt intake. This synergistic effect was associated with activation of PVN VP neurons and depended, in part, on activity of V1 receptors in the amygdala. Findings provide novel insight into neural mechanisms whereby prior exposure to a homeostatic stressor such as osmotic dehydration by excessive salt intake increases responsivity to a perceived stress. These experiments show that high dietary salt can influence stress responsivity and raise the possibility that excessive salt intake could be a contributing factor in the development of stress-related psychiatric disorders.

Brain Activation Patterns in Response to Conspecific and Heterospecific Social Acoustic Signals in Female Plainfin Midshipman Fish, Porichthys notatus

While the peripheral auditory system of fish has been well studied, less is known about how the fish’s brain and central auditory system process complex social acoustic signals. The plainfin midshipman fish, Porichthys notatus, has become a good species for investigating the neural basis of acoustic communication because the production and reception of acoustic signals is paramount for this species’ reproductive success. Nesting males produce long-duration advertisement calls that females detect and localize among the noise in the intertidal zone to successfully find mates and spawn. How female midshipman are able to discriminate male advertisement calls from environmental noise and other acoustic stimuli is unknown. Using the immediate early gene product cFos as a marker for neural activity, we quantified neural activation of the ascending auditory pathway in female midshipman exposed to conspecific advertisement calls, heterospecific white seabass calls, or ambient environment noise. We hypothesized that auditory hindbrain nuclei would be activated by general acoustic stimuli (ambient noise and other biotic acoustic stimuli) whereas auditory neurons in the midbrain and forebrain would be selectively activated by conspecific advertisement calls. We show that neural activation in two regions of the auditory hindbrain, i.e., the rostral intermediate division of the descending octaval nucleus and the ventral division of the secondary octaval nucleus, did not differ via cFos immunoreactive (cFos-ir) activity when exposed to different acoustic stimuli. In contrast, female midshipman exposed to conspecific advertisement calls showed greater cFos-ir in the nucleus centralis of the midbrain torus semicircularis compared to fish exposed only to ambient noise. No difference in cFos-ir was observed in the torus semicircularis of animals exposed to conspecific versus heterospecific calls. However, cFos-ir was greater in two forebrain structures that receive auditory input, i.e., the central posterior nucleus of the thalamus and the anterior tuberal hypothalamus, when exposed to conspecific calls versus either ambient noise or heterospecific calls. Our results suggest that higher-order neurons in the female midshipman midbrain torus semicircularis, thalamic central posterior nucleus, and hypothalamic anterior tuberal nucleus may be necessary for the discrimination of complex social acoustic signals. Furthermore, neurons in the central posterior and anterior tuberal nuclei are differentially activated by exposure to conspecific versus other acoustic stimuli.

Astrocyte Hypertrophy and Microglia Activation in the Rat Auditory Midbrain Is Induced by Electrical Intracochlear Stimulation.

Neuron–glia interactions contribute to tissue homeostasis and functional plasticity in the mammalian brain, but it remains unclear how this is achieved. The potential of central auditory brain tissue for stimulation-dependent cellular remodeling was studied in hearing-experienced and neonatally deafened rats. At adulthood, both groups received an intracochlear electrode into the left cochlea and were continuously stimulated for 1 or 7 days after waking up from anesthesia. Normal hearing and deafness were assessed by auditory brainstem responses (ABRs). The effectiveness of stimulation was verified by electrically evoked ABRs as well as immunocytochemistry and in situ hybridization for the immediate early gene product Fos on sections through the auditory midbrain containing the inferior colliculus (IC). Whereas hearing-experienced animals showed a tonotopically restricted Fos response in the IC contralateral to electrical intracochlear stimulation, Fos-positive neurons were found almost throughout the contralateral IC in deaf animals. In deaf rats, the Fos response was accompanied by a massive increase of GFAP indicating astrocytic hypertrophy, and a local activation of microglial cells identified by IBA1. These glia responses led to a noticeable increase of neuron–glia approximations. Moreover, staining for the GABA synthetizing enzymes GAD65 and GAD67 rose significantly in neuronal cell bodies and presynaptic boutons in the contralateral IC of deaf rats. Activation of neurons and glial cells and tissue re-composition were in no case accompanied by cell death as would have been apparent by a Tunel reaction. These findings suggest that growth and activity of glial cells is crucial for the local adjustment of neuronal inhibition to neuronal excitation.

The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues.

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5’ RACE, 3’ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.

T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy.

HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART), these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18). Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that enhancing the cytolytic activity of Nef-specific T-cells may lead to reductions in infected cell frequencies, even in the absence of therapeutic latency reversal.

Comparison of Liver Detargeting Strategies for Systemic Therapy with Oncolytic Adenovirus Serotype 5.

Oncolytic viruses would ideally be of use for systemic therapy to treat disseminated cancer. To do this safely, this may require multiple layers of cancer specificity. The pharmacology and specificity of oncolytic adenoviruses can be modified by (1) physical retargeting, (2) physical detargeting, (3) chemical shielding, or (4) by modifying the ability of viral early gene products to selectively activate in cancer versus normal cells. We explored the utility of these approaches with oncolytic adenovirus serotype 5 (Ad5) in immunocompetent Syrian hamsters bearing subcutaneous HaK tumors. After a single intravenous injection to reach the distant tumors, the physically hepatocyte-detargeted virus Ad5-hexon-BAP was more effective than conditionally replicating Ad5-dl1101/07 with mutations in its E1A protein. When these control or Ad5 treated animals were treated a second time by intratumoral injection, prior exposure to Ad5 did not affect tumor growth, suggesting that anti-Ad immunity neither prevented treatment nor amplified anti-tumor immune responses. Ad5-dl1101/07 was next chemically shielded with polyethylene glycol (PEG). While 5 kDa of PEG blunted pro-inflammatory IL-6 production induced by Ad5-dl1101/07, this shielding reduced Ad oncolytic activity.