Anaerocolumna sedimenticola sp. nov., isolated from fresh water sediment

Strain CBA3638^T was isolated from the Geum River sediment, Republic of Korea. The cells of strain CBA3638^T were Gram-stain-positive, strictly anaerobic, rod-shaped, and 0.5–1.0 μm wide, and 4.0–4.5 μm long. Optimal growth occurred at 37 °C, pH 7.0, and 1.0% (w/v) NaCl. Based on the 16S rRNA gene sequence, the phylogenetic analysis showed that strain CBA3638^T belongs to the genus Anaerocolumna in the family Lachnospiraceae, and is most closely related to Anaerocolumna cellulosilytica (94.6–95.0%). The DDH value with A. cellulosilytica SN021^T showed 15.0% relatedness. The genome of strain CBA3638^T consisted of one circular chromosome that is 5,500,435 bp long with a 36.7 mol% G?+?C content. The genome contained seven 16S-5S-23S rRNA operons and one antibiotic resistance-related transporter gene (mefA). Quinones were not detected. The predominant cellular fatty acids were C_16:0 and C_14:0 and the polar lipids were diphosphatidylglycerol, phosphatidylcholine, and uncharacterised polar lipids. Based on the polyphasic taxonomic analysis, we propose strain CBA3638^T as a novel species in the genus Anaerocolumna, with the name Anaerocolumna sedimenticola sp. nov. The type strain is CBA3638^T (=?KACC 21652^T?=?DSM 110663^T).

     

A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression

During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.     

Construction and Manipulation of a New Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Clone

Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (～5 × 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.     

Anticapsid immunity level, not viral persistence level, correlates with the progression of Theiler's virus-induced demyelinating disease in viral P1-transgenic mice

Intracranial infection of Theiler's murine encephalomyelitis virus (TMEV) induces demyelination and a neurological disease in susceptible SJL/J (SJL) mice that resembles multiple sclerosis. While the virus is cleared from the central nervous system (CNS) of resistant C57BL/6 (B6) mice, it persists in SJL mice. To investigate the role of viral persistence and its accompanying immune responses in the development of demyelinating disease, transgenic mice expressing the P1 region of the TMEV genome (P1-Tg) were employed. Interestingly, P1-Tg mice with the B6 background showed severe reductions in both CD4(+) and CD8(+) T-cell responses to capsid epitopes, while P1-Tg mice with the SJL background displayed transient reductions following viral infection. Reduced antiviral immune responses in P1-Tg mice led to >100- to 1,000-fold increases in viral persistence at 120 days postinfection in the CNS of mice with both backgrounds. Despite the increased CNS TMEV levels in these P1-Tg mice, B6 P1-Tg mice developed neither neuropathological symptoms nor demyelinating lesions, and SJL P1-Tg mice developed significantly less severe TMEV-induced demyelinating disease. These results strongly suggest that viral persistence alone is not sufficient to induce disease and that the level of T-cell immunity to viral capsid epitopes is critical for the development of demyelinating disease in SJL mice.     

Epitope-Specific CD8+ T Cells Play a Differential Pathogenic Role in the Development of a Viral Disease Model for Multiple Sclerosis

Theiler''s virus-induced demyelinating disease has been extensively investigated as a model for persistent viral infection and multiple sclerosis (MS). However, the role of CD8⁺ T cells in the development of disease remains unclear. To assess the role of virus-specific CD8⁺ T cells in the pathogenesis of demyelinating disease, a single amino acid substitution was introduced into the predominant viral epitope (VP3 from residues 159 to 166 [VP3_159-166]) and/or a subdominant viral epitope (VP3_173-181) of susceptible SJL/J mice by site-directed mutagenesis. The resulting variant viruses (N160V, P179A, and N160V/P179A) failed to induce CD8⁺ T cell responses to the respective epitopes. Surprisingly, mice infected with N160V or N160V/P179A virus, which lacks CD8⁺ T cells against VP3_159-166, did not develop demyelinating disease, in contrast to wild-type virus or P179A virus lacking VP3_173-181-specific CD8⁺ T cells. Our findings clearly show that the presence of VP3_159-166-specific CD8⁺ T cells, rather than viral persistence itself, is strongly correlated with disease development. VP3_173-181-specific CD8⁺ T cells in the central nervous system (CNS) of these virus-infected mice expressed higher levels of transforming growth factor β, forkhead box P3, interleukin-22 (IL-22), and IL-17 mRNA but caused minimal cytotoxicity compared to that caused by VP3_159-166-specific CD8⁺ T cells. VP3_159-166-specific CD8⁺ T cells exhibited high functional avidity for gamma interferon production, whereas VP3_173-181-specific CD8⁺ T cells showed low avidity. To our knowledge, this is the first report indicating that the induction of the IL-17-producing CD8⁺ T cell type is largely epitope specific and that this specificity apparently plays a differential role in the pathogenicity of virus-induced demyelinating disease. These results strongly advocate for the careful consideration of CD8⁺ T cell-mediated intervention of virus-induced inflammatory diseases.     

The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma(1), a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies(2,3). A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells(4). We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA(2) and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry(5,6). Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.