- Volume 96, Issue 11, 2015

Human immunodeficiency virus type 1 (HIV-1) splicing has to be strictly controlled to ensure the balanced production of the unspliced and all differently spliced viral RNAs. Splicing at the major 5′ splice site (5′ss) that is used for the synthesis of all spliced RNAs is modulated by the local RNA structure and binding of regulatory SR proteins. Here, we demonstrate that the suboptimal sequence complementarity between this 5′ss and U1 small nuclear RNA (snRNA) also contributes to prevent excessive splicing. Analysis of a large set of HIV-1 sequences revealed that all three regulatory features of the 5′ss region (RNA structure, SR protein binding and sequence complementarity with U1 snRNA) are highly conserved amongst virus isolates, which supports their importance. Combined mutations that destabilize the local RNA structure, remove binding sites for inhibitory SR proteins and optimize the U1 snRNA complementarity resulted in almost complete splicing and accordingly reduced virus replication.

Hordeivirus movement protein encoded by the first gene of the triple gene block (TGB1 protein, TGBp1) interacts in vivo with viral genomic and subgenomic RNAs to form ribonucleoprotein (RNP) particles that are considered to be a form of viral genome (non-virion transport form) capable of cell-to-cell and long-distance transport in infected plants. The structures of these RNPs have not been elucidated. The poa semilatent virus (PSLV) TGBp1 contains a structured C-terminal NTPase/helicase domain and an N-terminal extension region consisting of two domains – a completely intrinsically disordered extreme N-terminal domain and an internal domain (ID) with structure resembling a partially disordered molten globule. Here, we characterized the structures assembled in vitro by the full-length PSLV TGBp1 alone or in the presence of viral RNA. The PSLV TGBp1 was capable of multimerization and self-assembly into extended high-molecular-mass complexes. These complexes disassembled to apparent monomers upon incubation with ATP. Upon incubation with viral RNA, the PSLV TGBp1 in vitro formed RNP structures that appeared as filamentous particles resembling virions of helical filamentous plant viruses in morphology and dimensions. By comparing the biophysical characteristics of PSLV TGBp1 and its domains in the presence and absence of RNA, we show that the ID plays the main structural role in the self-interactions and RNA interactions of TGBp1 leading to the assembly of virus-like RNP particles.

Recently, four new viruses belonging to an unassigned family within the order Picornavirales were identified in excrements of healthy carp (fisavirus) and pigs (posavirus 1, 2 and 3). We report the detection and characterization of a fifth virus present in human faeces. The virus, named human stool-associated RNA virus (husavirus), contains a single ORF encoding a putative 2993 AA polyprotein, with a Hel-Pro-Pol replication block, typical for the Picornavirales. Phylogenetic analysis revealed that the closest relative to husavirus is posavirus 1, and together they cluster with fisavirus, posavirus 2 and 3 and a roundworm (Ascaris suum) derived virus. Husavirus was detected in eight human stool samples collected in 1984 (n = 2), 1985 (n = 4), 1995 (n = 1) and 2014 (n = 1). From three strains of husavirus from 1984 and 1985 the full genome sequence was determined, showing less than 5 % intraspecies variation in the nucleotide composition. The host of this virus remains to be determined.

The presence of disease-associated prions in tissues and bodily fluids of chronic wasting disease (CWD)-infected cervids has received much investigation, yet little is known about mother-to-offspring transmission of CWD. Our previous work demonstrated that mother-to-offspring transmission is efficient in an experimental setting. To address the question of relevance in a naturally exposed free-ranging population, we assessed maternal and fetal tissues derived from 19 elk dam–calf pairs collected from free-ranging Rocky Mountain elk from north-central Colorado, a known CWD endemic region. Conventional immunohistochemistry identified three of 19 CWD-positive dams, whereas a more sensitive assay [serial protein misfolding cyclic amplification (sPMCA)] detected CWD prion seeding activity (PrPCWD) in 15 of 19 dams. PrPCWD distribution in tissues was widespread, and included the central nervous system (CNS), lymphoreticular system, and reproductive, secretory, excretory and adipose tissues. Interestingly, five of 15 sPMCA-positive dams showed no evidence of PrPCWD in either CNS or lymphoreticular system, sites typically assessed in diagnosing CWD. Analysis of fetal tissues harvested from the 15 sPMCA-positive dams revealed PrPCWD in 80 % of fetuses (12 of 15), regardless of gestational stage. These findings demonstrated that PrPCWD is more abundant in peripheral tissues of CWD-exposed elk than current diagnostic methods suggest, and that transmission of prions from mother to offspring may contribute to the efficient transmission of CWD in naturally exposed cervid populations.