Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs

Virus-vector relationship in the Citrus leprosis pathosystem

Citrus leprosis has been one of the most destructive diseases of citrus in the Americas. In the last decade important progress has been achieved such as the complete genome sequencing of its main causal agent, Citrus leprosis virus C (CiLV-C), belonging to a new genus Cilevirus. It is transmitted by Brevipalpus yothersi Baker (Acari: Tenuipalpidae), and is characterized by the localized symptoms it induces on the leaves, fruits and stems. It occurs in the American continents from Mexico to Argentina. The virus was until recently considered restricted to Citrus spp. However, it was found naturally infecting other plants species as Swinglea glutinosa Merrill and Commelina benghalensis L., and has been experimentally transmitted by B. yothersi to a large number of plant species. Despite these advances little is known about the virus-vector relationship that is a key to understanding the epidemiology of the disease. Some components of the CiLV-C/B. yothersi relationship were determined using the common bean (Phaseolus vulgaris L. cv. ‘IAC Una’) as a test plant. They included: (a) the virus acquisition access period was 4 h; (b) the virus inoculation access period was 2 h; (c) the latent period between acquisition and inoculation was 7 h; (d) the period of retention of the virus by a single viruliferous mite was at least 12 days; (d) the percentage of viruliferous individuals from mite colonies on infected tissues ranged from 25 to 60%. The experiments confirmed previous data that all developmental stages of B. yothersi (larva, protonymph and deutonymph, adult female and male) were able to transmit CiLV-C and that transovarial transmission of the virus did not occur. CiLV-C can be acquired from lesions on leaves, fruits and stems by B. yothersi. Based on the distribution of lesions produced by single viruliferous B. yothersi on bean leaves, it is concluded that they tend to feed in restricted areas, usually near the veins. The short latent and transmission periods during the larval stage suggest that the CiLV-C/B. yothersi relationship is of the persistent circulative type.Fundación de apoyo a la investigación estatal/[2014/08458-9]/Fapesp/BrasilConsejo Nacional de desarrollo científico y tecnológico/[47.2425/2013-7]/CNPq/BrasilFundación de apoyo a la investigación estatal/[2008/57477-5]/Fapesp/BrasilFundación de apoyo a la investigación estatal/[2013/25713-0]/Fapesp/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM