Genetic Adaptation to Untranslated Region-Mediated Enterovirus Growth Deficits by Mutations in the Nonstructural Proteins 3AB and 3CD

Abstract

Both untranslated regions (UTRs) of plus-strand RNA virus genomes jointly control translation and replication of viral genomes. In the case of the Enterovirus genus of the Picornaviridae family, the 5UTR consists of a cloverleaf-like terminus preceding the internal ribosomal entry site (IRES) and the 3 terminus is composed of a structured 3UTR and poly ( Enterovirus (EV) plus-strand RNA genome translation and replication are controlled by cis-acting sequence elements mapping to the open reading frame and both 5Ј and 3Ј untranslated regions (UTRs) as well as nonstructural viral gene products. Their ϳ7,400-nucleotide (nt) genomes feature complex structured 5Ј and 3ЈUTRs of ϳ750-and ϳ80-to 110-nt length, respectively, flanking the open reading frame for a single polyprotein (24). The EV 5ЈUTR consists of a ϳ90-nt "cloverleaf" structure serving as a cis-acting replication signal (4) preceding a ϳ450-nt internal ribosomal entry site (IRES) involved in cap-independent translation initiation (23, 39). The 3ЈUTRs of EVs are structurally highly conserved, except coxsackie B viruses (CBVs), whose 3ЈUTRs contain a stem-loop domain (SLD) added to the conventional EV arrangement (42) (see Fig. 1A).The 5Ј cloverleaf and 3ЈUTR are recognized by the same viral proteins with RNA-binding properties and have been implicated in genome replication functions. Viral proteins 3AB (the precursor of 3A, a small membrane-anchoring and RNAbinding protein and 3B, the genome-linked protein VPg) and 3CD (the precursor of the 3C proteinase and the 3D RNAdependent RNA polymerase) interact with these terminal RNA structures (3,15,21,37). Both the cloverleaf and the 3ЈUTR are required for efficient minus-strand synthesis (3,5,27,28,43,50), although viable EVs with 3ЈUTR deletions have been derived (52).In addition to these cis-acting genetic elements and viral RNA-binding proteins, host factors may play a role in the control of EV genome replication and translation. In addition to 3CD and 3AB, the cloverleaf attracts the poly(rC)-binding protein 2 (PCBP2) (15,35). PCBP2 has been proposed to be involved in a switch from translation to replication at plusstrand RNA templates (16). Also, an interaction of PCBP2 with the poly(A)-binding protein has been speculated to mediate template circularization by linking the cloverleaf and poly(A) (22). The complexity of EV translation and RNA synthesis has obstructed unraveling the mechanistic details of their regulation. Several multifunctional viral proteins interact with cisacting signals on both ends on a potentially circular template. Often, the functions of precursor polypeptides are disparate from those of processed viral proteins. For example, although they differ only by the removal of the carboxy-terminal 22 amino acids of 3B, 3AB is believed to costimulate RNA replication by supporting the function of 3CD and 3D, while 3A does not (32,36). cis-acting signals recognized by viral RNAbinding proteins simultaneously contribute to template stability, translation, and/or RNA replication. Moreover, the vir...

Human Enterovirus 109: a Novel Interspecies Recombinant Enterovirus Isolated from a Case of Acute Pediatric Respiratory Illness in Nicaragua

“…Chimeric polioviruses possessing heterologous 5Ј UTRs from coxsackievirus or rhinovirus have exhibited growth deficiencies in cell culture and attenuated neuropathogenesis in poliomyelitis mouse models though they share ϳ70% nucleotide identity across the 5Ј UTRs (13,18). Other investigators have observed complementary mutations in 3A/3AB and 3C/3CD that alleviate cell-type-specific growth defects in enteroviruses with incompatible UTRs; although the mutations were confined to the 3A and 3C regions, homogenous mutations were not identified at defined sites (9). Future site-directed mutagenesis studies using EV109 could elucidate the selected coding region mutations needed to maintain UTR-viral protein interactions within the context of a natural recombination event.…”

Attenuation of Neurovirulence, Biodistribution, and Shedding of a Poliovirus:Rhinovirus Chimera after Intrathalamic Inoculation in Macaca fascicularis

“…Prior to the growth curve experiments, the cultures were rinsed with DMEM (Invitrogen). The experimental setup was a standard one-step growth curve (13). Briefly, cells in a control well were treated with trypsin and counted using a hemocytometer to determine the amount of virus needed to achieve a multiplicity of infection (MOI) of 10.…”

Adaptation of an ICAM-1-Tropic Enterovirus to the Mouse Respiratory Tract

“…Sequencing of viral genomes followed earlier published procedures (17 (Table 1) were used to generate overlapping PCR products covering Ͼ99% of the viral genome (we were unable to determine the sequence of the ϳ30 5Ј-most and 3Ј-most nucleotides of the viral RNA). A variable combination of primers 6 to 39 (Table 1) was used for sequencing of the RT-PCR products.…”

“…Virus obtained after the final passage was used for plaque purification by standard methods. One-step growth curves were established as previously described (17). To analyze hICAM-1 signaling upon CAV21 binding, HeLa H1 cells were serum starved for 48 h and infected at an MOI of 10.…”

“…ICAM-La-4-adapted virus was cloned with PCR fragments encompassing parts of VP2/VP3 (nt 767 to 2210) and VP1/2A/2C (nt 3162 to 5069) as described above. Mutant viruses were derived after transfection of HeLa H1 cells with infectious in vitro transcript RNA as described before (17).…”