Antimicrobial sulfonamides clear latent Kaposi sarcoma herpesvirus infection and impair MDM2-p53 complex formation.

Kaposi sarcoma herpesvirus (KSHV), also known as human herpesvirus 8, is the causative agent of Kaposi sarcoma; this malignant angiosarcoma is usually treated with conventional antitumor agents that can control disease evolution, but do not clear the latent KSHV episome that binds to cellular DNA. Some commercial antibacterial sulfonamides were tested for the ability to suppress latent KSHV. Quantitative PCR (qPCR) and cytofluorometry assays were used for detecting both viral DNA and the latency factor LANA (latency-associated nuclear antigen) in BC3 cells, respectively. The capacity of sulfonamides to impair MDM2-p53 complex formation was detected by an enzyme-linked immunosorbent assay method. The analysis of variance was performed according to one-way analysis of variance with Fisher as a post hoc test. Here we show that sulfonamide antibiotics are able to suppress the KSHV latent state in permanently infected BC3 lymphoma cells and interfere with the formation of the MDM2-p53 complex that KSHV seemingly needs to support latency and to trigger tumor cell transformation. These findings detected a new molecular target for the activity of sulfonamides and offer a new potential perspective for treating KSHV-induced lymphoproliferative diseases.

The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Augments Viral Lytic Replication.

UNLABELLED: The K1 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV life cycle, we constructed a set of recombinant viruses that contained either wild-type (WT) K1, a deleted K1 ORF (KSHVΔK1), stop codons within the K1 ORF (KSHV-K15×STOP), or a revertant K1 virus (KSHV-K1REV). We report that the recombinant viruses KSHVΔK1 and KSHV-K15×STOP displayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1REV upon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHVΔK1 and KSHV-K15×STOP also yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV- and KSHV-K1REV-infected cells. Upon reactivation from latency, WT KSHV- and KSHV-K1REV-infected cells displayed activated Akt kinase, as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV life cycle. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies, and KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the life cycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to the WT virus and also yielded smaller numbers of infectious progeny. We report that K1 plays an important role in the life cycle of KSHV.

Interleukin 1 receptor-associated kinase 1 (IRAK1) mutation is a common, essential driver for Kaposi sarcoma herpesvirus lymphoma.

Primary effusion lymphoma (PEL) is an AIDS-defining cancer. All PELs carry Kaposi sarcoma-associated herpesvirus (KSHV). X chromosome-targeted sequencing of PEL identified 34 common missense mutations in 100% of cases. This included a Phe196Ser change in the interleukin 1 receptor-associated kinase 1 (IRAK1). The mutation was verified in primary PEL exudates. IRAK1 is the binding partner of MyD88, which is mutated in a fraction of Waldenström macroglobulinemia. Together, these two mediate toll-like receptor (TLR) signaling. IRAK1 was constitutively phosphorylated in PEL and required for survival, implicating IRAK1 and TLR signaling as a driver pathway in PEL and as a new drug development target.

Hsp90 inhibitors are efficacious against Kaposi Sarcoma by enhancing the degradation of the essential viral gene LANA, of the viral co-receptor EphA2 as well as other client proteins.

Heat-shock protein 90 (Hsp90) inhibitors exhibit activity against human cancers. We evaluated a series of new, oral bioavailable, chemically diverse Hsp90 inhibitors (PU-H71, AUY922, BIIB021, NVP-BEP800) against Kaposi sarcoma (KS). All Hsp90 inhibitors exhibited nanomolar EC(50) in culture and AUY922 reduced tumor burden in a xenograft model of KS. KS is associated with KS-associated herpesvirus (KSHV). We identified the viral latency associated nuclear antigen (LANA) as a novel client protein of Hsp90 and demonstrate that the Hsp90 inhibitors diminish the level of LANA through proteasomal degradation. These Hsp90 inhibitors also downregulated EphA2 and ephrin-B2 protein levels. LANA is essential for viral maintenance and EphA2 has recently been shown to facilitate KSHV infection; which in turn feeds latent persistence. Further, both molecules are required for KS tumor formation and both were downregulated in response to Hsp90 inhibitors. This provides a rationale for clinical testing of Hsp90 inhibitors in KSHV-associated cancers and in the eradication of latent KSHV reservoirs.

Ribosomal protein S6 interacts with the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA.

Distinct p53, p53:LANA, and LANA complexes in Kaposi's Sarcoma--associated Herpesvirus Lymphomas.

The role of p53 in primary effusion lymphoma (PEL) is complicated. The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) binds p53. Despite this interaction, we had found that p53 was functional in PEL, i.e., able to induce apoptosis in response to DNA damage (C. E. Petre, S. H. Sin, and D. P. Dittmer, J. Virol. 81:1912-1922, 2007), and that hdm2 was overexpressed. To further elucidate the relationship between LANA, p53, and hdm2, we purified LANA complexes from PEL by column chromatography. This confirmed that LANA bound p53. However, the LANA:p53 complexes were a minority compared to hdm2:p53 and p53:p53 complexes. The half-life of p53 was not extended, which is in contrast to the half-life of simian virus 40 T antigen-transformed cells. p53:p53, LANA:p53, and LANA:LANA complexes coexisted in PEL, and each protein was able to bind to its cognate DNA element. These data suggest that under normal conditions, p53 is inactive in PEL, thus allowing for exponential growth, but that this inactivation is driven by the relative stoichiometries of LANA, hdm2, and p53. If p53 is activated by DNA damage or nutlin-3a, the complex falls apart easily, and p53 exercises its role as guardian of the genome.

Pathogenicity of a new China variety of Metarhizium anisopliae (M. Anisopliae var. Dcjhyium) to subterranean termite Odontotermes formosanus.

The pathogenicity of a new China variety of Metarhizium anisopliae (M. anisopliae var. dcjhyium) against the subterranean termite Odontotermes formosanus and the effect of the fungal fermented solution on hemolymph intracellular calcium were studied in laboratory. Conidia from the M. anisopliae var. dcjhyium were highly virulent for O. formosanus causing approximately 100% mortality 3 days post-inoculation in the concentration of 3x10(8) conidia/ml. The conidial clumps with conidial chains distributed on the cadavers of termite. When the termite was treated with 3x10(5) conidia/ml for 2 days, two constitutive proteins (91 and 105kDa) disappeared and a new specific protein (40kDa) appeared in the hemolymph of survivors relative to the controls. Hemolymph cells treated by the fungal fermented solution had a significantly higher level of intracellular calcium than controls 30min after treatment (x1.7). When the termite O. formosanus was infected by the entomopathogenic fungus M. anisopliae var. dcjhyium, hyphae invaded the integument and body cavity of the termite; well-developed muscles and fat tissue in the thorax of termite were decomposed and absorbed by hyphae, and formed the net structure; Hyphae seriously destroyed hemolymph, various tissues, pipelines and produced large number of conidia in the body of termite.

Characterization of the RNA-binding regions in protein p36 of Heliothis armigera cypovirus 14.

Some proteins of cypovirus (CPV) bind to RNA, probably contributing to the replication of viral genome. However, little is known about whether any protein from Heliothis armigera cypovirus (HaCPV) could bind to RNA. In this study, we cloned the ORF of segment 9 (S9) of HaCPV, serotype 14, into pMAL-c2X for the generation and purification of maltose binding protein (MBP) fused protein p36 (MBP-p36). The analysis of the RNA-binding properties of MBP-p36 revealed that p36, but not MBP alone, bound to ssRNA of CPV. Furthermore, the ssRNA-binding activities of p36 were significantly inhibited or completely eliminated by protein denaturants or unsuitable concentrations of NaCl. Importantly, the formation of ssRNA/p36 was only competitively inhibited by a heavy dose of competitive non-viral ssRNA or dsRNA, but not by ssDNA and dsDNA, suggesting that p36 bound to both ssRNA and dsRNA, but not DNA. Moreover, the characterization of different mutants of p36 revealed that the regions 1-26aa, 154-170aa, and 229-238aa, but not region 291-320aa, may be crucial for the ssRNA-binding ability of p36. Conceivably, the sensitivity of p36 to denaturants and the synergetic effect of different regions suggest that the RNA-binding ability of p36 may be conformation-dependent. Thus, our findings provide new insights into understanding the genomic function of HaCPV-14.

Phylogenetic analysis of Heliothis armigera cytoplasmic polyhedrosis virus type 14 and a series of dwarf segments found in the genome.

Full-length nucleotide sequences for the genome segments (S1-S6) of Heliothis armigera cytoplasmic polyhedrosis virus type 14 (HaCPV-14) have been characterized. Each segment consists of a single open reading frame with conserved motifs AGAA and AGCU at the 5' and 3' ends, respectively. Comparison of the proteins of HaCPV-14 with those of other members of the family Reoviridae suggests that S1 encodes an RNA-dependent RNA polymerase (RdRp), whilst S2 encodes a major capsid protein of the virus. Phylogenetic analysis of RdRps from 16 viruses in the family Reoviridae reveals that the genera Cypovirus and Oryzavirus may have originated from a common insect virus ancestor. A series of viable dwarf segments originating from S5 of HaCPV-14 has been identified. Analysis of the predicted secondary structures for these dwarf segments suggests that the signals essential for replication and packaging are located within the terminal sequences of these segments.

Characterization of a newly discovered China variety of Metarhizium anisopliae (M. anisopliae var. dcjhyium) for virulence to termites, isoenzyme, and phylogenic analysis.

The efficacy of a new virulent Metarhizium anisopliae variety (M. anisopliae var. dcjhyium, DQ288247) obtained from Odontotermes formosanus in China was evaluated against the subterranean termite, O. formosanus, in the laboratory. The new variety was compared with four other virulent M. anisopliae isolates and was found to be highly infectious and virulent against termites. M. anisopliae var. dcjhyium could cause approximately 100% mortality of termites 3 days post-inoculation in the concentration of 3x10(8) conidia/ml. There were also differences in relative hyhal growth and isoenzymes. M. anisopliae var. dcjhyium showed a different isoenzyme band pattern from the four isolates of M. anisopliae (AB027337, AB099510, AB099941 and AF280631). The phylogenetic tree of the 18S rDNA sequences revealed the taxonomic and evolutionary position of M. anisopliae var. dcjhyium. M. anisopliae var. dcjhyium and four isolates of M. anisopliae formed a monophyletic group, supported by a 99% bootstrap value. M. anisopliae var. dcjhyium formed a distinct variety, which had a special characterization of unique bands of isoenzyme, high virulence and low repellency against termites when compared with four other isolates of M. anisopliae.

Identification of transmembrane domain of a membrane associated protein NS5 of Dendrolimus punctatus cytoplasmic polyhedrosis virus.

We examined the intracellular localization of NS5 protein of Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV) by expressing NS5-GFP fusion protein and proteins from deletion mutants of NS5 in baculovirus recombinant infected insect Spodoptera frugiperda (Sf-9) cells. It was found that the NS5 protein was present at the plasma membrane of the cells, and that the N-terminal portion of the protein played a key role in the localization. A transmembrane region was identified to be present in the N-terminal portion of the protein, and the detailed transmembrane domain (SQIHMVWVKSGLVFF, 57-71aa) of N-terminal portion of NS5 was further determined, which was accorded with the predicted results, these findings suggested that NS5 might have an important function in viral life cycle.

Sequence analysis of coat protein gene of Wuhan nodavirus isolated from insect.

Wuhan nodavirus (WhNV) particles are isometric, non-enveloped, and about 29 nm in diameter. In the previous study, we determine its physiochemical characterization and the nucleotide sequence of the larger genomic segment, RNA1 and identify it a nodavirus. WhNV RNA1 is 3,149 nt in length, encoding protein A, catalytic subunit of RNA-dependent RNA polymerase (RdRp). In this report, we complete the sequence determination of the smaller genomic segment, RNA2 of WhNV. WhNV RNA2 is determined to be 1,562 nt long, containing a 430-amino-acid open reading frame (ORF) encoding the coat protein of WhNV with a calculated molecular mass of 47,856 Da. The homology of the coat protein of WhNV and the homologous proteins of other nodaviruses either alphanodaviruses or betanodaviruses is very low. WhNV coat protein shares the highest identity (24%) with that of Lates calcarifer encephalitis virus (LCEV), a betanodavirus, and shares less than 16% identical amino acids with each of the alphanodaviruses. Furthermore, the prediction of WhNV capsid structure by 3D-PSSM shows that the capsid structure of WhNV resembles that of tomato bushy stunt virus (TBSV), a tombusvirus, which contains two domains, rather than the expected single-domain capsid protein of insect nodaviruses. The phylogenetic analysis indicates that WhNV is the most distantly related of both the alphanodaviruses and betanodaviruses, which provides significant new data for understanding the evolution of the nodavirus family.

Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1.

The non-structural (NS) proteins of parvoviruses are involved in essential steps of the viral life cycle. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to the protein NS1. Compared with the non-structural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been well characterized. Here, we describe the biochemical properties of NS1 of Periplaneta fuliginosa densovirus (PfDNV). We have expressed and purified NS1 using a baculovirus system and analyzed its enzymatic activity. The purified recombinant NS1 protein possesses ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg(2+) or Mn(2+) as a cofactor. The ATPase activity of NS1 can be efficiently stimulated by single-stranded DNA. The ATPase coupled helicase activity was detected on blunt-ended double-stranded oligonucleotide substrate. Using South-Western and Dot-spot assays, we identified a DNA fragment that is recognized specifically by the recombinant NS1 protein. The fragment consists of (CAC)(4) and is located on the hairpin region of the terminal palindrome. The domain for DNA binding was defined to the amino-terminal region (amino acids 1-250). In addition, we found that NS1 can form oligomeric complexes in vivo and in vitro. Mutagenesis analysis showed that ATP binding is necessary for oligomerization. Based on these results, it seems that PfDNV NS1, a multifunctional protein, plays an important role in viral DNA replication comparable to those of vertebrate parvovirus initiator proteins.

Identification and genome characterization of Heliothis armigera cypovirus types 5 and 14 and Heliothis assulta cypovirus type 14.

Genomic characterization of Heliothis armigera cypovirus (HaCPV) isolated from China showed that insects were co-infected with several cypoviruses (CPVs). One of the CPVs (HaCPV-5) could be separated from the others by changing the rearing conditions of the Heliothis armigera larvae. This finding was further confirmed by nucleotide sequencing analysis. Genomic sequences of segments S10-S7 from HaCPV-14, S10 and S7 from HaCPV-5, and S10 from Heliothis assulta CPV-14 were compared. Results from database searches showed that the nucleotide sequences and deduced amino acid sequences of the newly identified CPVs had high levels of identity with those of reported CPVs of the same type, but not with CPVs of different types. Putative amino acid sequences of HaCPV-5 S7 were similar to that of the protein from Rice ragged stunt virus (genus Oryzavirus, family Reoviridae), suggesting that CPVs and oryzaviruses are related more closely than other genera of the family Reoviridae. Conserved motifs were also identified at the ends of each RNA segment of the same virus type: type 14, 5'-AGAAUUU...CAGCU-3'; and type 5, 5'-AGUU...UUGC-3'. Our results are consistent with classification of CPV types based on the electrophoretic patterns of CPV double-stranded RNA.