Elements in Serum, Muscle, Liver, and Kidney of Rabbits Fed Macroalgae-Supplemented Diets.

The addition of marine macroalgae to animal feed has garnered interest due to the demonstrated benefits of gut health in many livestock species. Most macroalgae have a higher mineral content than terrestrial vegetables, making them an attractive, sustainable source of minerals. However, some macroalgae contain elevated concentrations of iodine and arsenic, which may be transferred to the meat of livestock fed with macroalgae. This study evaluated the mineral profile of rabbit serum, muscle, liver, and kidney of rabbits fed diets supplemented with different marine macroalgae, with the goal of improving post-weaning gut health and reducing reliance on antibiotics. We found increased deposition of iodine in muscle, liver, and kidney due to macroalgae supplementation, which is particularly promising for regions with low iodine endemicity. Higher, though relatively low arsenic concentrations, compared to those in other animal meats and food sources, were also detected in the muscle, liver, and kidney of macroalgae-fed rabbits. The absence of apparent interactions with other micronutrients, particularly selenium, suggests that the inclusion of macroalgae in rabbit diets will not affect the overall mineral content. Enhanced bioavailability of elements such as phosphorus and iron may provide additional benefits, potentially reducing the need for mineral supplementation.

De Novo Human Angiotensin-Converting Enzyme 2 Decoy NL-CVX1 Protects Mice From Severe Disease After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

The emergence of novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need to investigate alternative approaches to prevent infection and treat patients with coronavirus disease 2019. Here, we report the preclinical efficacy of NL-CVX1, a de novo decoy that blocks virus entry into cells by binding with nanomolar affinity and high specificity to the receptor-binding domain of the SARS-CoV-2 spike protein. Using a transgenic mouse model of SARS-CoV-2 infection, we showed that a single prophylactic intranasal dose of NL-CVX1 conferred complete protection from severe disease following SARS-CoV-2 infection. Multiple therapeutic administrations of NL-CVX1 also protected mice from succumbing to infection. Finally, we showed that infected mice treated with NL-CVX1 developed both anti-SARS-CoV-2 antibodies and memory T cells and were protected against reinfection a month after treatment. Overall, these observations suggest NL-CVX1 is a promising therapeutic candidate for preventing and treating severe SARS-CoV-2 infections.

KSHV LANA acetylation-selective acidic domain reader sequence mediates virus persistence.

Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA's acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.

Non-celiac gluten sensitivity: Clinical presentation, etiology and differential diagnosis. / Sensibilidad al gluten no celiaca: etiología, diagnóstico diferencial y presentación clínica.

Non-celiac gluten sensitivity (NCGS) is the latest pathology incorporated into the group of gluten-related disorders. This review addresses the evidence on its etiology, differential diagnosis and symptomatology. Although NCGS is defined by a reaction to gluten, other possible etiopathogenic mechanisms have been described, such as an inadequate response to other components of wheat or to FODMAPs, with the term non-celiac sensitivity to wheat recently being extended. There are contradictory results on the validity of the diagnostic protocol of the Salerno experts. Evidence on diagnostic biomarkers for NCGS is scarce, although some studies indicate the following: antigliadin antibodies, zonulin, ALCAT test, micro-RNA, incRNA and certain cytokines. In NCGS, abdominal pain and fatigue are the most common symptoms. In addition, altered nutritional status is common. In conclusion, more research on NCGS is needed to improve understanding of its etiopathogenesis and clinical features.

Concentrations of Essential Trace and Toxic Elements Associated with Production and Manufacturing Processes in Galician Cheese.

The objective of this study was to determine the trace element composition and the toxic metal residues in Galician cow's milk cheese produced in different systems (artisan, industrial, and organic). Fourteen elements (As, Cd, Co, Cr, Cu, Fe, Hg, I, Mn, Mo, Ni, Pb, Se, and Zn) were determined in 58 representative samples of Galician cheeses by inductively coupled plasma mass spectrometry. The toxic elements were present at low concentrations, similar to those reported for other unpolluted geographical areas. The essential elements were also within the normal range in cheeses. There were no statistically significant differences between smoked and unsmoked cheeses for any of the elements. Chemometric analyses (principal component analysis and cluster analysis) revealed that the industrial cheeses produced in Galicia using the milk from intensive dairy farms were different, in terms of elemental content, from artisan and organic cheeses, in which the elemental contents were similar.

Seasonal Variation of the Proximate Composition, Mineral Content, Fatty Acid Profiles and Other Phytochemical Constituents of Selected Brown Macroalgae.

The main objective was to determine the chemical, phytochemical, fatty acid and mineral profiles of three commercially relevant brown macroalgae (Laminaria digitata, Laminaria hyperborea and Ascophyllum nodosum) collected each season for two years off the west coast of Ireland. All the chemical, phytochemical, fatty acid and minerals analysed varied significantly depending on the macroalgal species, season and year of collection. Overall, the protein contents of macroalgae were negatively correlated with carbohydrate content. Protein (2-11%) was at its highest during winter and/or spring, decreasing to a minimum during summer and/or autumn. The three macroalgal species analysed in this study had clearly differentiated fatty acid profiles. The concentration of fatty acids was higher in A. nodosum compared with both Laminaria species. The mineral profile of the three macroalgal species was rich in essential metals, particularly Ca, Mg and P, while the levels of I were approximately 9- to 10-fold higher in both Laminaria spp. compared with A. nodosum. The levels of toxic metals (Cd, Hg and Pb) in all the macroalgal species studied were low in the current study; while the levels of total As were high (49-64 mg/kg DW macroalgae) compared with previous reports.

VIDEO: Dynamic Ultrasound Evaluation for Soft-Tissue Injuries of the Extremities: Hand, Wrist, and Ankle.

OBJECTIVE. The purpose of this article is to show the sonographic anatomy of the extremities relevant to various ligamentous, tendinous, and articular injuries occurring in the hand, wrist, and ankle. A brief discussion and depiction of the specific elements relevant to the pathophysiologic mechanism of these entities is followed by demonstrations of the dynamic ultrasound techniques that can be used to diagnose these injuries. The schematics and video clips illustrate the normal and pathologic features of these injuries. The first two videos discuss soft-tissue injuries to the hand and wrist, and the third addresses ankle injuries. CONCLUSION. After clinical assessment, dynamic ultrasound examination is a useful tool for diagnosing and assessing the degree of severity of several soft-tissue injuries to the extremities, some of which can be detected only during active movement. Familiarity with these specific dynamic techniques will enhance the value of the ultrasound examination.

Breed performance in organic dairy farming in Northern Spain.

Organic farm management combines best environmental practices, a high level of biodiversity, preservation of natural resources and high animal welfare standards. To meet these criteria, farmers must have livestock well adapted to local organic conditions and information about how different breeds and crosses perform under different conditions. The objective of this study was to compare the performance of different pure breeds and cross-breeds of cattle in organic dairy systems in Northern Spain. The data analysed were obtained from monthly records kept between 2010 and 2016 on organic farms registered in the regional milk recording system. Analysis of various traits indicated that the Holstein-Friesian breed suits the organic production system in the study region. Although the reproductive performance of Holstein-Friesian cows was poorer (in terms of number of services per conception) than that of cross-breed and Brown Swiss cows, the Holstein-Friesian produced more milk and lived longer. In addition, there was no difference in calving type or calving ease between the different breed groups. The better milk fat and protein yields produced by the crosses may be useful traits for farmers interested in milk transformation. The advantage of continuing to use Holstein-Friesian cattle is that the breed is predominant worldwide, and the genealogy is well documented. If Holstein-Friesian cattle continue to be used, the main priority will be to search for well-adapted bulls (particularly for pasture-based conditions) and to elaborate a genetic merit index for organic and pasture-based systems with the aim of predicting and minimizing genotype × environment interactions.

In Vivo Persistence of Chimeric Virus after Substitution of the Kaposi's Sarcoma-Associated Herpesvirus LANA DNA Binding Domain with That of Murid Herpesvirus 4.

The latency-associated nuclear antigen from Kaposi's sarcoma-associated herpesvirus (KSHV), kLANA, and its homolog from the murid herpesvirus 4 (MuHV-4), mLANA, are essential for viral latency. kLANA is nearly four times the size of mLANA, mainly due to an extensive central repeat region that is absent in mLANA. Both proteins harbor a C-terminal DNA binding domain (DBD). The DBD binds the terminal repeat (TR) DNA sequences of the viral genome to mediate persistence. Despite structural conservation, the kLANA and mLANA DBDs differ in sequence and mode of oligomerization. kLANA DBD oligomers are flexible and bent, while mLANA DBD oligomers bind DNA in a rigid, linear conformation. We previously reported that kLANA and mLANA acted reciprocally on TR sequences. Furthermore, a MuHV-4 expressing kLANA instead of mLANA (v-kLANA) established latency in mice, albeit at a lower magnitude than the wild-type (WT) virus. Here, we asked if kLANA can accommodate the mLANA DBD and generated a fusion protein which contains kLANA but with the mLANA C-terminal region in place of that of kLANA. We report a recombinant MuHV-4 (v-KM) encoding this LANA fusion protein instead of mLANA. The fusion protein was expressed in lytic infection in vitro and assembled nuclear LANA dots in infected splenocytes. Results demonstrated that kLANA functionally accommodated mLANA's mode of DNA binding, allowing MuHV-4 chimeric virus to establish latency in vivo Notably, v-KM established latency in germinal center B cells more efficiently than did v-kLANA, although levels were reduced compared to WT MuHV-4.IMPORTANCE KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA.

Gammaherpesvirus Colonization of the Spleen Requires Lytic Replication in B Cells.

Gammaherpesviruses infect lymphocytes and cause lymphocytic cancers. Murid herpesvirus-4 (MuHV-4), Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus all infect B cells. Latent infection can spread by B cell recirculation and proliferation, but whether this alone achieves systemic infection is unclear. To test the need of MuHV-4 for lytic infection in B cells, we flanked its essential ORF50 lytic transactivator with loxP sites and then infected mice expressing B cell-specific Cre (CD19-Cre). The floxed virus replicated normally in Cre- mice. In CD19-Cre mice, nasal and lymph node infections were maintained; but there was little splenomegaly, and splenic virus loads remained low. Cre-mediated removal of other essential lytic genes gave a similar phenotype. CD19-Cre spleen infection by intraperitoneal virus was also impaired. Therefore, MuHV-4 had to emerge lytically from B cells to colonize the spleen. An important role for B cell lytic infection in host colonization is consistent with the large CD8+ T cell responses made to gammaherpesvirus lytic antigens during infectious mononucleosis and suggests that vaccine-induced immunity capable of suppressing B cell lytic infection might reduce long-term virus loads.IMPORTANCE Gammaherpesviruses cause B cell cancers. Most models of host colonization derive from cell cultures with continuous, virus-driven B cell proliferation. However, vaccines based on these models have worked poorly. To test whether proliferating B cells suffice for host colonization, we inactivated the capacity of MuHV-4, a gammaherpesvirus of mice, to reemerge from B cells. The modified virus was able to colonize a first wave of B cells in lymph nodes but spread poorly to B cells in secondary sites such as the spleen. Consequently, viral loads remained low. These results were consistent with virus-driven B cell proliferation exploiting normal host pathways and thus having to transfer lytically to new B cells for new proliferation. We conclude that viral lytic infection is a potential target to reduce B cell proliferation.

Cross-species conservation of episome maintenance provides a basis for in vivo investigation of Kaposi's sarcoma herpesvirus LANA.

Many pathogens, including Kaposi's sarcoma herpesvirus (KSHV), lack tractable small animal models. KSHV persists as a multi-copy, nuclear episome in latently infected cells. KSHV latency-associated nuclear antigen (kLANA) binds viral terminal repeat (kTR) DNA to mediate episome persistence. Model pathogen murine gammaherpesvirus 68 (MHV68) mLANA acts analogously on mTR DNA. kLANA and mLANA differ substantially in size and kTR and mTR show little sequence conservation. Here, we find kLANA and mLANA act reciprocally to mediate episome persistence of TR DNA. Further, kLANA rescued mLANA deficient MHV68, enabling a chimeric virus to establish latent infection in vivo in germinal center B cells. The level of chimeric virus in vivo latency was moderately reduced compared to WT infection, but WT or chimeric MHV68 infected cells had similar viral genome copy numbers as assessed by immunofluorescence of LANA intranuclear dots or qPCR. Thus, despite more than 60 Ma of evolutionary divergence, mLANA and kLANA act reciprocally on TR DNA, and kLANA functionally substitutes for mLANA, allowing kLANA investigation in vivo. Analogous chimeras may allow in vivo investigation of genes of other human pathogens.

Supraspinatus Myotendinous Junction Injuries: MRI Findings and Prevalence.

OBJECTIVE: The purpose of this study is to describe the MRI findings and evaluate the prevalence of supraspinatus myotendinous injuries. MATERIALS AND METHODS: Among 1001 consecutive shoulders that underwent either conventional MRI or MR arthrography between January and December 2016, 843 shoulders were included. All MR images were retrospectively analyzed for identification and classification into the appropriate grade of acute or chronic rotator cuff myotendinous injuries. Other MRI findings, such as the presence of rotator cuff tendon insertional tears, and clinical information were also evaluated. RESULTS: At MRI, 0.47% (4/843) of shoulders had supraspinatus myotendinous injuries involving the anterior muscular bundle exclusively. Chronic grade III (n = 2), acute grade III (n = 1), and acute grade II (n = 1) injuries were identified in three men and one woman (mean age, 44 years) with a clinical history of trauma (n = 2) or of progressive shoulder pain (n = 2). A concurrent supraspinatus insertional tendon tear with either partial (n = 1) or full (n = 1) thickness was present in half the cases. Loss of tension of the myotendinous junction in grade III myotendinous junction injuries led to severe atrophy and fatty infiltration of the anterior supraspinatus. CONCLUSION: Supraspinatus myotendinous junction injuries are uncommon at MRI. These lesions invariably involve the anterior bundle of the supraspinatus muscle and may occur with a concomitant insertional tendon tear. High-grade chronic injuries lead to selective atrophy and fatty infiltration of the anterior supraspinatus muscle.

Organic or conventional dairy farming in northern Spain: Impacts on cow reproductive performance.

Organic farming has traditionally given preference to indigenous breeds that are well adapted to local conditions; however, current trends towards increased yields may produce less robust animals with potentially low fertility. The Holstein-Friesian breed dominates dairy sector, although there is a general concern that these cows may not be well adapted to organic systems. This study aims to evaluate the reproductive performance of organic dairy herds in northern Spain, by comparing organically reared Holstein-Friesian with their counterparts reared on conventional farms and also with other organically reared breeds and crosses. Reproductive parameters were obtained for individual cows on organic, conventional pasture-based and conventional zero-grazing farms. The reproductive performance of Holstein-Friesian cows was slightly better in organic systems than in pasture-based conventional systems, but not than in zero-grazing conventional systems. The comparison of reproductive performance of Holstein-Friesian from organic and conventional systems and with other organically reared breeds and crosses enabled us to demonstrate that it is not the organic system that constrains the reproductive performance of Holstein-Friesian, but that the manifestation of oestrus is less marked than in other breeds. Breeding selection considering reproductive traits and appropriate management of oestrous detection would improve the reproductive performance of Holstein-Friesian cows.

Breeding for organic dairy farming: what types of cows are needed?

Organic farming is believed by many to be an environmentally friendly production system that promotes the use of local forage while strongly limiting the input of chemicals, including allopathic treatments. As organic dairy farming has grown, farmers have realised that many available conventional breeds of cow are not well adapted to the new situations and that more 'robust' cows, able to function well in the constraining organic environment, are needed to yield acceptable longevity and productivity. In this review paper, the current breed diversity in organic dairy farming is analysed with the aim of identifying the types of cow that would best fulfil organic breeding goals. Unlike the conventional sector, organic dairy farming is very heterogeneous and no single type of cow can adapt well to all scenarios. There are advantages and disadvantages to the use of existing breeds (rustic Holstein-Friesian, other rustic breeds and crosses), and strong genotype × environment interactions demand different strategies for very diverse situations. Organic dairy farms producing milk for systems that recompense milk volume would benefit from using higher milk yielding cows, and rustic Holstein-Friesian cows may be the best option in such cases. Although most Holstein-Friesian cows are currently selected for use in conventional systems, this situation could be reversed by the implementation of an organic merit index that includes organic breeding goals. Farms producing milk either for systems that recompense milk solids or for transformation into dairy products would benefit from using breeds other than Holstein-Friesian or their crosses. Organic farmers who focus on rural tourism, farm schools or other businesses in which marketing strategies must be taken into account could benefit from using local breeds (when possible) or other rustic breeds that are highly valued by consumers.

Effector γδ T Cell Differentiation Relies on Master but Not Auxiliary Th Cell Transcription Factors.

γδ T lymphocytes are programmed into distinct IFN-γ-producing CD27(+) (γδ27(+)) and IL-17-producing CD27(-) (γδ27(-)) subsets that play key roles in protective or pathogenic immune responses. Although the signature cytokines are shared with their αß Th1 (for γδ27(+)) and Th17 (for γδ27(-)) cell counterparts, we dissect in this study similarities and differences in the transcriptional requirements of murine effector γδ27(+), γδ27(-)CCR6(-), and γδ27(-)CCR6(+) γδ T cell subsets and αß T cells. We found they share dependence on the master transcription factors T-bet and RORγt for IFN-γ and IL-17 production, respectively. However, Eomes is fully dispensable for IFN-γ production by γδ T cells. Furthermore, the Th17 cell auxiliary transcription factors RORα and BATF are not required for IL-17 production by γδ27(-) cell subsets. We also show that γδ27(-) (but not γδ27(+)) cells become polyfunctional upon IL-1ß plus IL-23 stimulation, cosecreting IL-17A, IL-17F, IL-22, GM-CSF, and IFN-γ. Collectively, our in vitro and in vivo data firmly establish the molecular segregation between γδ27(+) and γδ27(-) T cell subsets and provide novel insight on the nonoverlapping transcriptional networks that control the differentiation of effector γδ versus αß T cell subsets.

KSHV but not MHV-68 LANA induces a strong bend upon binding to terminal repeat viral DNA.

Latency-associated nuclear antigen (LANA) is central to episomal tethering, replication and transcriptional regulation of γ2-herpesviruses. LANA binds cooperatively to the terminal repeat (TR) region of the viral episome via adjacent LANA binding sites (LBS), but the molecular mechanism by which LANA assembles on the TR remains elusive. We show that KSHV LANA and MHV-68 LANA proteins bind LBS DNA using strikingly different modes. Solution structure of LANA complexes revealed that while kLANA tetramer is intrinsically bent both in the free and bound state to LBS1-2 DNA, mLANA oligomers instead adopt a rigid linear conformation. In addition, we report a novel non-ring kLANA structure that displays more flexibility at its assembly interface than previously demonstrated. We identified a hydrophobic pivot point located at the dimer-dimer assembly interface, which gives rotational freedom for kLANA to adopt variable conformations to accommodate both LBS1-2 and LBS2-1-3 DNA. Alterations in the arrangement of LBS within TR or at the tetramer assembly interface have a drastic effect on the ability of kLANA binding. We also show kLANA and mLANA DNA binding functions can be reciprocated. Although KSHV and MHV-68 are closely related, the findings provide new insights into how the structure, oligomerization, and DNA binding of LANA have evolved differently to assemble on the TR DNA.

Is lack of antibiotic usage affecting udder health status of organic dairy cattle?

This research communication aimed to compare somatic cell count (SCC), the main marker of udder health status, in organic farms not using antibiotics (O, n = 6), organic farms using antibiotics (OA, n = 7) and conventional farms (CA, n = 5) using antibiotic treatments, all of them at pasture. SCC was statistically significantly higher in O (173780) compared to CA (93325) and OA (107152). Milk yield had a significant diluting effect on SCC and differences between groups increased with parities. Stratified analysis of SCC depending on lactation number and % of monthly SCC test with different linear scores (LS) indicated that there is no difference in udder health in the primiparous heifers from the three groups of farms, but it deteriorates in older cows because of chronic infections in O (possibly due to lack of antibiotic use). Our results suggest that the non-use of antibiotics had an effect in udder health leading to higher occurrence of clinical and subclinical mastitis. Therefore, preventive management practices for mastitis control are essential in organic farms.

Use of homeopathy in organic dairy farming in Spain.

Organic farming principles promote the use of unconventional therapies as an alternative to chemical substances (which are limited by organic regulations), with homeopathy being the most extensive. Traditionally, Spain has had little faith in homeopathy but its use in organic farming is growing. Fifty-six Spanish organic dairy farmers were interviewed to obtain what we believe to be the first data on the use of homeopathy in organic dairy cattle in Spain. Only 32% of farms use some sort of alternative therapy (16.1% homeopathy, 10.7% phytotherapy and 5.3% using both therapies) and interestingly, a clear geographical pattern showing a higher use towards the East (similar to that in the human population) was observed. The main motivation to use homeopathy was the need to reduce chemical substances promoted by organic regulations, and the treatment of clinical mastitis being the principle reason. The number of total treatments was lower in farms using homeopathy compared with those applying allopathic therapies (0.13 and 0.54 treatments/cow/year respectively) and although the bulk SCC was significantly higher (p < 0.001) in these farms (161,826 and 111,218 cel/ml, respectively) it did not have any negative economical penalty for the farmer and milk quality was not affected complying with the required standards; on the contrary homeopathic therapies seems to be an alternative for reducing antibiotic treatments, allowing farmers to meet the organic farming principles.

Crystal structure of the gamma-2 herpesvirus LANA DNA binding domain identifies charged surface residues which impact viral latency.

Latency-associated nuclear antigen (LANA) mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 Šresolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s).

Role of Src homology domain binding in signaling complexes assembled by the murid γ-herpesvirus M2 protein.

γ-Herpesviruses express proteins that modulate B lymphocyte signaling to achieve persistent latent infections. One such protein is the M2 latency-associated protein encoded by the murid herpesvirus-4. M2 has two closely spaced tyrosine residues, Tyr(120) and Tyr(129), which are phosphorylated by Src family tyrosine kinases. Here we used mass spectrometry to identify the binding partners of tyrosine-phosphorylated M2. Each M2 phosphomotif is shown to bind directly and selectively to SH2-containing signaling molecules. Specifically, Src family kinases, NCK1 and Vav1, bound to the Tyr(P)(120) site, PLCγ2 and the SHP2 phosphatase bound to the Tyr(P)(129) motif, and the p85α subunit of PI3K associated with either motif. Consistent with these data, we show that M2 coordinates the formation of multiprotein complexes with these proteins. The effect of those interactions is functionally bivalent, because it can result in either the phosphorylation of a subset of binding proteins (Vav1 and PLCγ2) or in the inactivation of downstream targets (AKT). Finally, we show that translocation to the plasma membrane and subsequent M2 tyrosine phosphorylation relies on the integrity of a C-terminal proline-rich SH3 binding region of M2 and its interaction with Src family kinases. Unlike other γ-herpesviruses, that encode transmembrane proteins that mimic the activation of ITAMs, murid herpesvirus-4 perturbs B cell signaling using a cytoplasmic/membrane shuttling factor that nucleates the assembly of signaling complexes using a bilayered mechanism of phosphotyrosine and proline-rich anchoring motifs.