Plant Dicer-like (DCL) and Argonaute (AGO) are the key enzymes involved in anti-virus post-transcriptional gene silencing (AV-PTGS). Here we show that AV-PTGS exhibited nucleotide preference by calculating a relative AV-PTGS efficiency on processing viral RNA substrates. In comparison with genome sequences of dicot-infecting Turnip mosaic virus (TuMV) and monocot-infecting Cocksfoot streak virus (CSV), viral-derived small interfering RNAs (vsiRNAs) displayed positive correlations between AV-PTGS efficiency and G+C content (GC%). Further investigations on nucleotide contents revealed that the vsiRNA populations had G-biases. This finding was further supported by our analyses of previously reported vsiRNA populations in diverse plant-virus associations, and AGO associated Arabidopsis endogenous siRNA populations, indicating that plant AGOs operated with G-preference. We further propose a hypothesis that AV-PTGS imposes selection pressure(s) on the evolution of plant viruses. This hypothesis was supported when potyvirus genomes were analysed for evidence of GC elimination, suggesting that plant virus evolution to have low GC% genomes would have a unique function, which is to reduce the host AV-PTGS attack during infections.
Arabidopsis ; enzymology ; genetics ; virology ; Base Composition ; Dactylis ; enzymology ; genetics ; virology ; Genes, Plant ; Genes, Viral ; Models, Genetic ; Mustard Plant ; enzymology ; genetics ; virology ; Plant Diseases ; genetics ; virology ; Plant Proteins ; metabolism ; Plant Viruses ; genetics ; pathogenicity ; Plants ; enzymology ; genetics ; virology ; Potyvirus ; genetics ; pathogenicity ; RNA Interference ; RNA, Plant ; genetics ; RNA, Small Interfering ; chemistry ; genetics ; metabolism ; RNA, Viral ; chemistry ; genetics ; metabolism ; RNA-Induced Silencing Complex ; metabolism ; Ribonuclease III ; metabolism ; Selection, Genetic ; Substrate Specificity

In this study, Leishmania RNA virus 1-4 (LRV1-4) particles purified from host Leishmania guyanensis promastigotes were examined for capsid endoribonuclease. Temperature optimum for the endoribonulease activity was found to be at 37degrees C to 42degrees C and the activity was specifically inhibited by the aminoglycoside antibiotics, neomycin, kanamycin, and hygromycin and by 100 mM levels of NaCl or KCl. To determine the catalytic domain of the capsid endoribonuclease activity, three point-mutation at cysteine residues at C47S (P1), C128/ 133S (P2), and C194R (P3) were prepared and each gene was constructed into baculoviruses and expressed in Sf9 insect cells. LRV1-4 capsid N- terminus (N2 and N3) and C-terminus (C1 and C2) deletion mutants (Cadd et al., 1994) were also examined by in vitro RNA cleavage assay. The results showed that the capsid mutants; C1, C2, N3, P1, and P2 were capable of forming proper virus-like particles (VLPs) and they all possessed the specific endoribonuclease activity. However, two assembly-defective capsid mutants, N2 (N- terminus 24-amino acids deletion) and P3 mutants, did not retain the specific endoribonuclease activity. Taken together, the results suggest that at least 24 amino acids from the N-terminal region and C194 residue in LRV1-4 capsid protein are functionally important for LRV1-4 viral assembly and the capsid endoribonuclease activity may be dependent upon the properly assembled LRV1-4 virus particles.
Amino Acid Substitution ; Animals ; Anti-Bacterial Agents/pharmacology ; Baculoviridae ; Capsid/*enzymology ; Cell Line ; Cysteine/genetics ; Endoribonucleases/antagonists & inhibitors/chemistry/genetics/isolation & purification/*metabolism ; Enzyme Activation/drug effects ; Heat ; Insects ; Leishmania guyanensis/*virology ; RNA/chemistry ; RNA Viruses/*enzymology/genetics ; Recombinant Proteins/antagonists & inhibitors/genetics/isolation & purification/metabolism ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S. ; Substrate Specificity/genetics ; Transduction, Genetic

BACKGROUNDRespiratory syncytial virus (RSV) is a common pathogen in the lower respiratory tract of infants and children. Recent studies have shown that in adults, especially in the elderly population who have relatively weak immunity, lower respiratory tract infection is not uncommon. RSV was detected in 22% hospitalized patients with acute exacerbation of chronic obstructive pulmonary disease (COPD), and the detection rate was only next to that of parvovirus and influenza virus respectively. Further studies revealed that lung infection of RSV could lead to inflammatory destruction and structural remodeling. This study was undertaken to examine the effect of infection with RSV on matrix metalloproteinase (MMPs) in mice, and to explore the role of RSV in the pathogenesis of pulmonary diseases.

METHODSTwenty BALB/c mice were divided randomly into an RSV infection group and a blank control group. The mice in the RSV infection group were given 100 microl liquid containing 10(6) PFU of RSV by intranasal instillation. Three days after the infection, the bronchoalveolar lavage fluid (BALF) was harvested and RT-PCR and Western blotting were used to detect MMP-9 and the expression of tissue inhibitors of matrix metalloproteinase (TIMP)-1 mRNA in lung tissues. Gelatin zymography was employed to detect the activities of MMP-9 and MMP-2 in BALF. Immunohistochemistry was used to determine the expressions of E-cadherin (E-cd) and proliferating cell nuclear antigen (PCNA) in the lung tissues.

RESULTSIn the BALF of the mice infected with RSV, the activities of MMP-9 and MMP-2 were significantly increased (t = 6.08, P < 0.01 and t = 5.68, P < 0.01). The levels of mRNA and proteins of MMP-9 in the lung tissues of the mice infected with RSV were significantly elevated, and the mRNA and protein levels were significantly higher than those of the blank controls. Though the ratio of MMP-9/TIMP-1 mRNA in the lung tissues of the infected mice was not significantly different from that of the normal controls, the ratio of the MMP-9/TIMP-1 protein in the RSV infection group was significantly higher than that in the control group. Moreover, the number of cells with positive E-cd expression was decreased and the number of cells positive for PCNA was increased, with an enhanced expression.

CONCLUSIONSIn mice, infection with RSV can significantly increase the activities of MMP-9 and MMP-2, and conspicuously elevate the mRNA transcription of MMP-9. RSV infection can increase the activity of gelatinase while up-regulating its inhibitory factors but increase its protein ratio of MMP-9/TIMP-1 in lung tissues, thereby facilitating fibrosis after structural destruction of the airway. The resultant status might be an important factor causing chronic reconstruction of the airway.

Animals ; Cadherins ; analysis ; HeLa Cells ; Humans ; Lung ; enzymology ; virology ; Matrix Metalloproteinase 2 ; analysis ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; analysis ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Proliferating Cell Nuclear Antigen ; analysis ; RNA, Messenger ; analysis ; Respiratory Syncytial Virus Infections ; enzymology ; Respiratory Syncytial Viruses ; isolation & purification