A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

BACKGROUND: Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression

Weathering as controlled by shock processes

Shock processes affect all bodies in the solar system and the effect on rocky targets is profound on both the scale of hundreds of km and on the microscopic scale, as evidenced by mineral textures [1, 2]. We examine the micro-environments of weathering in L6 chondrites, both in terms of textural and chemical alteration; and we relate them to shock created features. Weathering, when mediated by fluid, is sped up or slowed down by the presence of fractures [3]. The most profound difference seen in more shocked samples is the formation of compositionally heterogeneous, polycrystalline sulphides (similar to those found in ALH 84001 by [4]). The shocked sulphides contain micro-fractures, which allow enhanced retention of fluid throughout the crystal. The sulphides also have significant compositional heterogeneity which makes the minerals more vulnerable to weathering [4]. This in turn modifies the altering fluid to a more acidic form. The acid then attacks surrounding silicates. The effect of the acidic fluid is seen through the trace elements of the affected olivine and pyroxene, and a decrease in Mn, Fe and Mg can be seen relative to Si adjacent to weathering sulphides. The estimated pressures needed to create polycrystalline sulphides is 35-60 GPa [5], well within the limits of martian crater formation [6], and therefore is likely to play a part in fluid based crater alteration processes. [1] Schwenzer, S. P. et al. (2012) Planetary and Space Science, 70: 84-95. [2] Rubin, A. E. (2003) Geochimica et Cosmochimica Acta, 67: 2695-2709. [3] Barber, D. J. & Scott, E. R. D. (2006) Meteoritics and Planetary Science, 41: 643-662. [4] Kwong, Y-T.J, (1993) MEND/NEDEM report 1.32.1. [5] Bennett, M. E., McSween, H. (1996) Meteoritics and Planetary Science, 31: 255-264. [6] Abramov, O. et al. (2012) Icarus, 218: 906-916

Relationships Between Shock and Fluid Processes

High shock pressure in rocks occurs across the solar system, with impacts into planetary bodies having been frequent features of the past and still occurring today, e.g., as seen in meteorite falls on Earth. The shock pressures that rocks undergo create unique physical characteristics in the targets and in the impactors. Creation of shock melt pockets, destruction of original porosity and restructuring of minerals can occur. The change of physical properties in the rocks affects later processes like fluid alteration. This study looks at shock created features in L6 chondrites and relates the features to fluid driven alteration processes in Antarctica. Several shock features in samples in this study have been found to be directly altering weathering patterns. The shock feature with the most pronounced effect on alteration processes is the creation of polymineralic melt veins, which are more weatherable and so make pathways for alteration fluids. Also seen is a reconstruction of sulphides to create polycrystalline structures with many planes of weakness, more easily exploited by fluids, which then have greater access to the minerals to alter the sulphides. A mixture of metals and sulphides is created in some of the samples where a “fizz” mixture has been formed. The addition of planes of weakness, differential behaviour in rapidly changing temperatures and differences in weatherability have created concentrated weathering centres which affect the system around. These processes should be common in other solar system bodies with similar targets and shock features, if water was present on those bodies

Mineralogical controls on cold desert weathering

Textural and chemical alteration of silicates is studied in L6 chondrites with relation to local mineral reactions and distance from the rim of the samples. Alteration is seen to be complex and more related to adjacent mineralogy than rim distance

The 40 bp internal repeat of MHV-68 is involved in latency amplification by regulating the expression of latency-associated genes.

<p>A) RT-PCR analysis of the expression of K3 after infection of fibroblasts (NIH3T3) and B cells (Ag8). As control, the expression of the murine ribosomal protein L8 gene, which was amplified in parallel, was determined. Lanes 1: Parental virus; Lanes 2: Delta 40 bp mutant; Lanes 3: Delta 100 bp mutant; M: marker. The sizes of the PCR products are indicated on the right. B) Quantitative RT-PCR analysis of the expression of K3 after infection of fibroblasts (NIH3T3) and B cells (Ag8). The data are presented as relative copy number of K3 to L8. C) Determination of the viral genomic load by PCR using primers specific for ORF 50 of MHV-68. As control, the murine ribosomal protein L8 gene was amplified in parallel. Lanes 1: Parental virus; Lanes 2: Delta 40 bp mutant; Lanes 3: Delta 100 bp mutant; M: marker. The sizes of the PCR products are indicated on the right. D) Quantitative PCR analysis of the genomic load after infection of fibroblasts (NIH3T3) and B cells (Ag8). The data are presented as relative copy number of ORF50 to L8. E) RT-PCR analysis of the expression of K3, ORF72 and ORF73 after infection of fibroblasts (NIH3T3) and B cells (Ag8). As control, the expression of the murine ribosomal protein L8 gene, which was amplified in parallel, was determined. Lanes 1: Parental virus; Lanes 2: Delta 40 bp mutant; Lanes 3: 40 bp revertant; M: marker. The sizes of the PCR products are indicated on the right. The data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000733#pone-0000733-g007" target="_blank">figure 7</a> are from representative experiments which were repeated 3 times with similar results.</p

The absence of CD8⁺T cells partially reverses the phenotype of the Delta 40 bp mutant.

<p>A) Splenomegaly. C57BL/6 or CD8^−/− mice (5 mice per group) were i.n. infected with 5×10⁴ PFU. At day 17 after infection, spleens were harvested and the number of spleen cells was determined. Data shown are means±SD. The asterisk indicates a statistically significant difference (p = 0.01; Student's t-test). B) <i>Ex vivo</i> reactivation. C57BL/6 mice or CD8^−/− mice were i.n. infected with 5×10⁴ PFU. The extent of <i>ex vivo</i> reactivation was determined 17 days after infection. Splenocytes pooled from 5 mice per group were used.</p

Generation of MHV-68 mutants.

<p>A) Schematic presentation of viral mutants. B) Scheme of the expected fragments after digestion of viral DNA with the restriction enzyme <i>EcoR</i>I. Digestion of DNA from both parental virus and the 40 bp revertant with <i>EcoR</i>I results in a 5.2 kb wildtype fragment. Deletion of the 40 bp repeat results in the loss of the 5.2 kb fragment and in the generation of a new 2.8 kb fragment. Partial loss of repeat units results in a shift of the 5.2 kb fragment to 4.6 kb and 4.2 kb fragments, respectively. “P” indicates the probe used for Southern blot analysis, corresponding to nucleotides 25889-26711. C) Structural analysis of reconstituted virus genomes by ethidium bromide-stained agarose gel analysis of viral DNA digested with <i>EcoR</i>I. Lane 1: Parental virus; Lane 2: 40 bp revertant; Lane 3: Delta 40 bp mutant; Lane 4: 40 bp moderate mutant; Lane 5: 40 bp low mutant; Lane 6: M6STOP mutant. D) Southern blot analysis of the gel shown in panel C using probe “P” indicated in panel B. The expected fragments are indicated by dots (panel C) or by arrows (panel D). Marker (M) sizes (in kilobase pairs) are indicated on the left.</p