Identification of a Gammaherpesvirus Selective Chemokine Binding Protein That Inhibits Chemokine Action

Whether a particular organ showed signal (red plus signs) or not (black minus signs) at the given time is shown below the images. HE stain. MCODE analyses to identify functional protein clusters within the networks [62] identified either unique clusters or clusters in which the composition of proteins represented varied according to infection (Figs. Better understanding of the host signaling pathways that contribute to the establishment and maintenance of a latent infection is critical for the development of therapeutics against infection and virus-associated malignancies. Because the miRNA mutations in MHV68.Zt6 were inserted in the genomic region proximal to the protein-coding genes M1, M2, and M3, we verified the expression of these genes using qRT-PCR (Fig. However, viral transcripts for M9, M3, gB, M8, Rta, and K3 were readily detected in peritoneal cells isolated from infected IFN-γR−/− mice. The x axis shows the numbers of cells per reaction; the y axis shows the percentages of 12 reactions positive for CPE.

KSHV K1 and RRV R1 have also been shown to oligomerize through disulfide bonding of their extracellular domains (24, 71, 76, 77). (E) B220 is detected in residual B-cell follicles (×10). Y129, but not Y120, of M2 is required for differentiation of infected B cells to plasma cells. (B) Densitometric analyses were performed on resulting immunoblots to compare levels of p53 between CHX-treated or vehicle-treated samples relative to that for the loading control β-actin, which is not affected by CHX treatment. 4B). Regulation of gammaherpesvirus infection by granzymes A and B.Our examination of the role of granzymes in regulating γHV68 infection focused initially on granzymes A and B because they are the most extensively characterized granzymes and the only two for which specific knockout mice are available. It is striking that γHV68 maintains an IFN-γ-responsive lytic switch gene given that IFN-γ is a key regulator of chronic infection in vivo.

The data are shown as a color matrix with columns representing time points p.i. The transfected cells were harvested at the indicated time points and analyzed for viral DNA replication (C), viral gene expressions (D), and virion production (E) as described in the legend to Fig. 11, 371-379. U. To verify that CHX was effective under these conditions, a similar experiment was carried out by using twice the amount of CHX and similar results were obtained (data not show). NIH 3T3 cells were transiently transfected with a GST or GST-M2 expression vector. Caspase-11 controls a noncanonical inflammasome pathway that is activated by LPS from Gram-negative bacteria (25, 38).

Blots were sequentially probed with rabbit polyclonal anti-v-cyclin antiserum (top panel), chicken polyclonal anti-ORF59 (middle panel), and mouse anti-β-actin (bottom panel). (C) Expression kinetics of the ORF49 protein during de novo infection of MHV-68. Here, we demonstrate that PML can exert a repressive effect on establishment of the latent state and reactivation of the MHV68 strain of gammaherpesvirus (summarized in Fig. Cells were prepared and stained to detect IL-6, IL-12, TNF-α, IFN-γ and IL-10 as described above. Trends Immunol. 4A). Cell debris was removed by centrifugation at maximum speed.

The digested DNAs were analyzed by Southern blotting with a digoxigenin-labeled probe hybridizing to vector pST76K-SR. β-Actin antibody was used as a loading control. In conclusion, we show that T cells recognizing the M3 protein of MHV-68 can afford partial protection from the acute phase of infection, but protection from the early latent phase of infection was not observed with consistency. Blots were hybridized overnight with digoxigenin (DIG)-labeled probes and developed using an enhanced chemiluminescence system (Boehringer Mannheim) according to the instructions of the manufacturer. Briefly, splenocytes harvested at days 16, 42, and 182 postinfection and stored at −80°C in cMEM supplemented with 10% dimethyl sulfoxide were thawed, counted, and resuspended in isotonic buffer. J. MHV68 naturally infects rodents and infection of laboratory strains of mice has been extensively studied as a small animal model of gammaherpesvirus pathogenesis.

This further illustrates that early establishment of MHV68 latency is dependent on the participation of germinal-center reactions to gain entry into the memory B-cell compartment. The initial screening identified M2 as a ZAP-responsive element (28). After removing the inoculum, cells were incubated for 5 days at 37°C with fresh medium containing methylcellulose. This pronounced T cell expansion and activation is a hallmark of MHV68 infection in many inbred mouse strains and is observed in peripheral lymphoid organs, as well as the blood, reaching peak levels after the virus has established latency [3], [4]. D. Our laboratory is particularly interested in the interactions between PML NBs and herpesviruses. Acute infections are not associated with a detectable viremia, consistent with a predominance of direct, cell-to-cell virus transfer.

Immunofluorescence and cellular fractionation analyses comparing wild-type (WT) to mLANA-null MHV68 infections demonstrated mLANA-dependent recruitment of Hsc70 to nuclei of productively infected cells. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation.