The replacement histone H2A.Z is variously reported as being linked to gene expression and preventing the spread of heterochromatin in yeast, or concentrated at heterochromatin in mammals. To resolve this apparent dichotomy, affinity-purified antibodies against the N-terminal region of H2A.Z, in both a triacetylated and non-acetylated state, are used in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 5′ end of active genes, both tissue specific and housekeeping but is absent from inactive genes, while the unacetylated form is absent from both active and inactive genes. A concentration of H2A.Z is also found at insulators under circumstances implying a link to barrier activity but not to enhancer blocking. Although acetylated H2A.Z is widespread throughout the interphase genome, at mitosis its acetylation is erased, the unmodified form remaining. Thus, although H2A.Z may operate as an epigenetic marker for active genes, its N-terminal acetylation does not.

Cytomegaloviruses (CMVs) establish chronic infections that spread from a primary entry site to secondary vascular sites, such as the spleen, and then to tertiary shedding sites, such as the salivary glands. Human CMV (HCMV) is difficult to analyze, because its spread precedes clinical presentation. Murine CMV (MCMV) offers a tractable model. It is hypothesized to spread from peripheral sites via vascular endothelial cells and associated monocytes. However, viral luciferase imaging showed footpad-inoculated MCMV first reaching the popliteal lymph nodes (PLN). PLN colonization was rapid and further spread was slow, implying that LN infection can be a significant bottleneck. Most acutely infected PLN cells were CD169 ؉ subcapsular sinus macrophages (SSM). Replication-deficient MCMV also reached them, indicating direct infection. Many SSM expressed viral reporter genes, but few expressed lytic genes. SSM expressed CD11c, and MCMV with a cre-sensitive fluorochrome switch showed switched infected cells in PLN of CD11c-cre mice but yielded little switched virus. SSM depletion with liposomal clodronate or via a CD169-diphtheria toxin receptor transgene shifted infection to ER-TR7 ؉ stromal cells, increased virus production, and accelerated its spread to the spleen. Therefore, MCMV disseminated via LN, and SSM slowed this spread by shielding permissive fibroblasts and poorly supporting viral lytic replication. IMPORTANCE HCMV chronically infects most people, and it can cause congenital disability and harm the immunocompromised. A major goal of vaccination is to prevent systemic infection. How this is established is unclear. Restriction to humans makes HCMV difficultto analyze. We show that peripheral MCMV infection spreads via lymph nodes. Here, MCMV infected filtering macrophages, which supported virus replication poorly. When these macrophages were depleted, MCMV infected susceptible fibroblasts and spread faster. The capacity of filtering macrophages to limit MCMV spread argued that their infection is an important bottleneck in host colonization and might be a good vaccine target. Human cytomegalovirus (HCMV) chronically infects more than 90% of the world's population. Latent virus is detectable in circulating monocytes; there also may be persistent stromal infection, and infectious virus is shed long term in secretions such as saliva (1). While most infections are asymptomatic, immune control can require substantial resources (2); immunocompromised patients suffer multiorgan disease, and HCMV is a leading cause of congenital disability. Therefore, better control would improve human health. So far, vaccines have failed to prevent infection or reduce long-term viral loads (3). One problem is that early infection events, which are key vaccine targets, remain ill defined. Virus behavior is cell type dependent, so an important unknown is which cell types HCMV infects first when colonizing new hosts.Sporadic, asymptomatic HCMV transmission makes early infection hard to study (1). It is important to recognize, there...

Herpesviruses have coevolved with their hosts over hundreds of millions of years and exploit fundamental features of their biology. Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells, and it has been hypothesized that systemic dissemination arises from infected stem cells in bone marrow. However, poor CMV transfer by stem cell transplantation argues against this being the main reservoir. To identify alternative pathways for CMV spread, we tracked murine CMV (MCMV) colonization after mucosal entry. We show that following intranasal MCMV infection, lung CD11c+ dendritic cells (DC) migrated sequentially to lymph nodes (LN), blood, and then salivary glands. Replication-deficient virus followed the same route, and thus, DC infected peripherally traversed LN to enter the blood. Given that DC are thought to die locally following their arrival and integration into LN, recirculation into blood represents a new pathway. We examined host and viral factors that facilitated this LN traverse. We show that MCMV-infected DC exited LN by a distinct route to lymphocytes, entering high endothelial venules and bypassing the efferent lymph. LN exit required CD44 and the viral M33 chemokine receptor, without which infected DC accumulated in LN and systemic spread was greatly reduced. Taken together, our studies provide the first demonstration of virus-driven DC recirculation. As viruses follow host-defined pathways, high endothelial venules may normally allow DC to pass from LN back into blood.

Viruses transmit via the environmental and social interactions of their hosts. Herpesviruses have colonized mammals since their earliest origins, suggesting that they exploit ancient, common pathways. Cytomegaloviruses (CMVs) are assumed to enter new hosts orally, but no site has been identified. We show by live imaging that murine CMV (MCMV) infects nasally rather than orally, both after experimental virus uptake and during natural transmission. Replication-deficient virions revealed the primary target as olfactory neurons. Local, nasal replication by wild-type MCMV was not extensive, but there was rapid systemic spread, associated with macrophage infection. A long-term, transmissible infection was then maintained in the salivary glands. The viral m131/m129 chemokine homolog, which influences tropism, promoted salivary gland colonization after nasal entry but was not required for entry per se. The capacity of MCMV to transmit via olfaction, together with previous demonstrations of experimental olfactory infection by murid herpesvirus 4 (MuHV-4) and herpes simplex virus 1 (HSV-1), suggest that this is a common, conserved route of mammalian herpesvirus entry.

Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells. Murine CMV (MCMV) spreads from the lungs via infected CD11c+cells, consistent with an important role for dendritic cells (DC). We show here that MCMV entering via the olfactory epithelium, a natural transmission portal, also spreads via infected DC. They reached lymph nodes, entered the blood via high endothelial venules, and then entered the salivary glands, driven by constitutive signaling of the viral M33 G protein-coupled receptor (GPCR). Intraperitoneal infection also delivered MCMV to the salivary glands via DC. However, it also seeded F4/80+infected macrophages to the blood; they did not enter the salivary glands or require M33 for extravasation. Instead, they seeded infection to a range of other sites, including brown adipose tissue (BAT). Peritoneal cells infectedex vivothen adoptively transferred showed similar cell type-dependent differences in distribution, with abundant F4/80+cells in BAT and CD11c+cells in the salivary glands. BAT colonization by CMV-infected cells was insensitive to pertussis toxin inhibition of the GPCR signaling through Gi/osubstrate, whereas salivary gland colonization was sensitive. Since salivary gland infection required both M33 and Gi/o-coupled signaling, whereas BAT infection required neither, these migrations were mechanistically distinct. MCMV spread from the lungs or nose depended on DC, controlled by M33. Infecting other monocyte populations resulted in unpredictable new infections.IMPORTANCECytomegaloviruses (CMVs) spread through the blood by infecting monocytes, and this can lead to disease. With murine CMV (MCMV) we can track infected myeloid cells and so understand how CMVs spread. Previous experiments have injected MCMV into the peritoneal cavity. MCMV normally enters mice via the olfactory epithelium. We show that olfactory infection spreads via dendritic cells, which MCMV directs to the salivary glands. Peritoneal infection similarly reached the salivary glands via dendritic cells. However, it also infected other monocyte types, and they spread infection to other tissues. Thus, infecting the “wrong” monocytes altered virus spread, with potential to cause disease. These results provide a basis for understanding how the monocyte types infected by human CMV might promote different infection outcomes.

Cytomegaloviruses (CMVs) persistently and systemically infect the myeloid cells of immunocompetent hosts. Persistence implies immune evasion, and CMVs evade CD8+ T cells by inhibiting MHC class I-restricted antigen presentation. Myeloid cells can also interact with CD4+ T cells via MHC class II (MHC II). Human CMV (HCMV) attacks the MHC II presentation pathway in vitro, but what role this evasion might play in host colonization is unknown. We show that Murine CMV (MCMV) down-regulates MHC II via M78, a multi-membrane spanning viral protein that captured MHC II from the cell surface and was necessary although not sufficient for its degradation in low pH endosomes. M78-deficient MCMV down-regulated MHC I but not MHC II. After intranasal inoculation, it showed a severe defect in salivary gland colonization that was associated with increased MHC II expression on infected cells, and was significantly rescued by CD4+ T cell loss. Therefore MCMV requires CD4+ T cell evasion by M78 to colonize the salivary glands, its main site of long-term shedding.

Cytomegaloviruses (CMVs) establish chronic, systemic infections. Peripheral infection spreads via lymph nodes, which are also a focus of host defence. Thus, this is a point at which systemic infection spread might be restricted. Subcapsular sinus macrophages (SSM) captured murine CMV (MCMV) from the afferent lymph and poorly supported its replication. Blocking the type I interferon (IFN-I) receptor (IFNAR) increased MCMV infection of SSM and of the fibroblastic reticular cells (FRC) lining the subcapsular sinus, and accelerated viral spread to the spleen. Little splenic virus derived from SSM, arguing that they mainly induce an anti-viral state in the otherwise susceptible FRC. NK cells also limited infection, killing infected FRC and causing tissue damage. They acted independently of IFN-I, as IFNAR blockade increased NK cell recruitment, and NK cell depletion increased infection in IFNAR-blocked mice. Thus SSM restricted MCMV infection primarily though IFN-I, with NK cells providing a second line of defence. The capacity of innate immunity to restrict MCMV escape from the subcapsular sinus suggested that enhancing its recruitment might improve infection control.

Native chromatin IP assays were used to define changes in core histone acetylation at the lysozyme locus during developmental maturation of chicken macrophages and stimulation to high-level expression by lipo-polysaccharide. In pluripotent precursors the lysozyme gene (Lys) is inactive and there is no acetylation of core histones at the gene, its promoter or at the upstream cis-control elements. In myeloblasts, where there is a very low level of Lys expression, H4 acetylation appears at the cis-control elements but not at the Lys gene or its promoter: neither H3 nor H2B become significantly acetylated in myeloblasts. In mature macrophages, Lys expression increases 5-fold: H4, H2B and H2A.Z are all acetylated at the cis-control elements but H3 remains unacetylated except at the −2.4 S silencer. Stimulation with LPS increases Lys expression a further 10-fold: this is accompanied by a rise in H3 acetylation throughout the cis-control elements; H4 and H2B acetylation remain substantial but acetylation at the Lys gene and its promoter remains low. Acetylation is thus concentrated at the cis-control elements, not at the Lys gene or its immediate promoter. H4 acetylation precedes H3 acetylation during development and H3 acetylation is most directly linked to high-level Lys expression.