Mutagenic analysis of the HIV restriction factor shiftless

The interferon-induced host cell protein shiftless (SFL) was reported to inhibit human immunodeficiency virus (HIV) infection by blocking the –1 programmed ribosomal frameshifting (–1PRF) required for expression of the Gag-Pol polyprotein. However, it is not clear how SFL inhibits –1PRF. To address this question, we focused on a 36 amino acids comprising region (termed required for antiviral activity (RAA)) that is essential for suppression of –1PRF and HIV infection and is missing from SFL short (SFLS), a splice variant of SFL with unknown function. Here, we confirm that SFL, but not SFLS, inhibits HIV –1PRF and show that inhibition is cell-type-independent. Mutagenic and biochemical analyses demonstrated that the RAA region is required for SFL self-interactions and confirmed that it is necessary for ribosome association and binding to the HIV RNA. Analysis of SFL mutants with six consecutive amino-acids-comprising deletions in the RAA region suggests effects on binding to the HIV RNA, complete inhibition of –1PRF, inhibition of Gag-Pol expression, and antiviral activity. In contrast, these amino acids did not affect SFL expression and were partially dispensable for SFL self-interactions and binding to the ribosome. Collectively, our results support the notion that SFL binds to the ribosome and the HIV RNA in order to block –1PRF and HIV infection, and suggest that the multimerization of SFL may be functionally important

Efeitos do tamanho da semente do algodoeiro Mocó (Gossypium hirsutum marie galante Hutch) da cultivar Veludo C - 71 sobre sua qualidade fisiológica.

Observou-se que a germinação e o vigor são mais baixos nas sementes de maior largura, isto é, sementes maiores, e que a qualidade fisiológica das sementes está mais estreitamente as sociada a densidade das sementes pertencentes ao lote do que à largura dessas sementes

HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyltRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation

Genetic Reconstruction of Protozoan rRNA Decoding Sites Provides a Rationale for Paromomycin Activity against Leishmania and Trypanosoma

Aminoglycoside antibiotics target the ribosomal decoding A-site and are active against a broad spectrum of bacteria. These compounds bind to a highly conserved stem-loop-stem structure in helix 44 of bacterial 16S rRNA. One particular aminoglycoside, paromomycin, also shows potent antiprotozoal activity and is used for the treatment of parasitic infections, e.g. by Leishmania spp. The precise drug target is, however, unclear; in particular whether aminoglycoside antibiotics target the cytosolic and/or the mitochondrial protozoan ribosome. To establish an experimental model for the study of protozoan decoding-site function, we constructed bacterial chimeric ribosomes where the central part of bacterial 16S rRNA helix 44 has been replaced by the corresponding Leishmania and Trypanosoma rRNA sequences. Relating the results from in-vitro ribosomal assays to that of in-vivo aminoglycoside activity against Trypanosoma brucei, as assessed in cell cultures and in a mouse model of infection, we conclude that aminoglycosides affect cytosolic translation while the mitochondrial ribosome of trypanosomes is not a target for aminoglycoside antibiotics

Efeito de várias disponibilidades hídricas, atuando como pré-condicionamento fisiológico durante a germinação das sementes de milho, no desenvolvimento das plântulas

The present paper considers the possible effects of physiological pre-treatment during the beginning of corn seeds germination in environments variable as to water availability (0 to -12 atm ). Periods of defficiency followed by 5 days without hydric limitations were compared with a control that remained equally, without reservations as to the disposability of water during 5 days. Results indicated that hydric pre-treatment of seeds can, if property adapted in its details, provide some advantages in the development of the seedlings derived from them. Chemical fungicide treatment of the seeds, submmited to the hydric pre-treatment may not be beneficial to the seedlings; as indicated by, in some isolated cases, the production of abnormalities in the embryonic structures.O presente trabalho buscou estimar os eventuais efeitos de pré-condicionamento fisiológico promovidos, durante o início da germinação das sementes de milho, por ambientes variáveis quanto à disponibilidade de água (0 a -12 atm). Para tanto, os períodos de deficiência eram sucedidos por prazos fixos de 5 dias sem limitações hídricas e comparados com uma testemunha que permaneceu, igualmente, sem restrições quanto à disponibilidade de água durante 5 dias. Os resultados encontrados indicaram que a técnica de pré-condicionamento hídrico das sementes pode, se devidamente adaptada em seus detalhes, propiciar algumas vantagens no desenvolvimento das plântulas delas oriundas. Adicionalmente, o tratamento químico fungicida das sementes, submetidas a pré-condicionamento hídrico, pode não trazer os benefícios esperados às plântulas; há casos isolados, ligados a produção de anormalidades nas estruturas embrionárias, em que o seu efeito é prejudicial