West Nile virus (WNV), first recognized in North America in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. Despite the welldescribed epidemiological patterns of WNV in North America, the basis for the emergence of WNVassociated avian pathology, particularly in the American crow (AMCR) sentinel species, and the large scale of the North American epidemic and epiornitic is uncertain. We report here that the introduction of a T249P amino acid substitution in the NS3 helicase (found in North American WNV) in a low-virulence strain was sufficient to generate a phenotype highly virulent to AMCRs. Furthermore, comparative sequence analyses of full-length WNV genomes demonstrated that the same site (NS3-249) was subject to adaptive evolution. These phenotypic and evolutionary results provide compelling evidence for the positive selection of a mutation encoding increased viremia potential and virulence in the AMCR sentinel bird species.

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV-or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca 2+ )-calmodulin-protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca 2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1␣ (PP1␣) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1␣ releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca 2+ signaling, PP2B and PP1␣ alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription.[Keywords: P-TEFb; CDK activation; transcriptional elongation; Ca 2+ signal transduction; protein phosphatases] Supplemental material is available at http://www.genesdev.org. Received November 21, 2007; revised version accepted March 31, 2008. In eukaryotes, the transcription of protein-coding genes is performed by RNA polymerase (Pol) II in a cyclic process consisting of several tightly regulated stages (Sims et al. 2004). During the elongation stage, the C-terminal domain of the largest subunit of Pol II is phosphorylated by the positive transcription elongation factor b (P-TEFb) (Peterlin and Price 2006;Zhou and Yik 2006). This modification is crucial for Pol II to change from abortive to productive elongation and produce full-length RNA transcripts. Consisting of Cdk9 and cyclin T1 (or the minor forms T2 and K), P-TEFb is considered a general transcription factor required for the expression of a vast array of protein-coding genes (Chao and Price 2001;Shim et al. 2002). Not only is P-TEFb critical for cellular gene transcription, it is also a specific host cofactor for the HIV-1 Tat protein. Tat recruits P-TEFb to the TAR RNA element located at the 5Ј end of nascent viral transcripts, allowing P-TEFb to phosphorylate stalled Pol II and enhance HIV-1 elongation (Peterlin and Price 2006).However, not every P-TEFb in the nucleus is in a transcriptionally active state. A major reservoir of P-TEFb (∼50% of total P-TEFb in HeLa cells) actually exists in an inactive complex termed 7SK snRNP that also contains the 7SK snRNA (Nguyen et al. 2001;Yang et al. 2001) and three nuclear...

Complete genome sequencing of 22 West Nile virus isolates suggested 2 independent introductions into Mexico. A previously identified mouse-attenuated glycosylation variant was introduced into southern Mexico through the southeastern United States, while a common US genotype appears to have been introduced incrementally into northern Mexico through the southwestern United States.

A single helicase amino acid substitution, NS3-T249P, has been shown to increase viremia magnitude/mortality in American crows (AMCRs) following West Nile virus (WNV) infection. Lineage/intra-lineage geographic variants exhibit consistent amino acid polymorphisms at this locus; however, the majority of WNV isolates associated with recent outbreaks reported worldwide have a proline at the NS3-249 residue. In order to evaluate the impact of NS3-249 variants on avian and mammalian virulence, multiple amino acid substitutions were engineered into a WNV infectious cDNA (NY99; NS3-249P) and the resulting viruses inoculated into AMCRs, house sparrows (HOSPs) and mice. Differential viremia profiles were observed between mutant viruses in the two bird species; however, the NS3-249P virus produced the highest mean peak viral loads in both avian models. In contrast, this avian modulating virulence determinant had no effect on LD50 or the neurovirulence phenotype in the murine model. Recombinant helicase proteins demonstrated variable helicase and ATPase activities; however, differences did not correlate with avian or murine viremia phenotypes. These in vitro and in vivo data indicate that avian-specific phenotypes are modulated by critical viral-host protein interactions involving the NS3-249 residue that directly influence transmission efficiency and therefore the magnitude of WNV epizootics in nature.

Abstract. A West Nile virus (WNV) isolate from Mexico (TM171-03) and BIRD1153, a unique genotype from Texas, have exhibited reduced murine neuroinvasive phenotypes. To determine if murine neuroinvasive capacity equates to avian virulence potential, American crow ( Corvus brachyrhynchos ) and house sparrows ( Passer domesticus ) were experimentally inoculated with representative murine neuroinvasive/non-neuroinvasive strains. In both avian species, a plaque variant from Mexico that was E-glycosylation competent produced higher viremias than an E-glycosylation-incompetent variant, indicating the potential importance of E-glycosylation for avian replication. The murine non-neuroinvasive BIRD1153 strain was significantly attenuated in American crows but not house sparrows when compared with the murine neuroinvasive Texas strain. Despite the loss of murine neuroinvasive properties of nonglycosylated variants from Mexico, our data indicate avian replication potential of these strains and that unique WNV virulence characteristics exist between murine and avian models. The implications of reduced avian replication of variants from Mexico for restricted WNV transmission in Latin America is discussed.

The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.

The N-linked glycosylation motif at amino acid position 154–156 of the envelope (E) protein of West Nile virus (WNV) is linked to enhanced murine neuroinvasiveness, avian pathogenicity and vector competence. Naturally occurring isolates with altered E protein glycosylation patterns have been observed in WNV isolates; however, the specific effects of these polymorphisms on avian host pathogenesis and vector competence have not been investigated before. In the present study, amino acid polymorphisms, NYT, NYP, NYF, SYP, SYS, KYS and deletion (A’DEL), were reverse engineered into a parental WNV (NYS) cDNA infectious clone to generate WNV glycosylation mutant viruses. These WNV glycosylation mutant viruses were characterized for in vitro growth, pH-sensitivity, temperature-sensitivity and host competence in American crows (AMCR), house sparrows (HOSP) and Culex quinquefasciatus . The NYS and NYT glycosylated viruses showed higher viral replication, and lower pH and temperature sensitivity than NYP, NYF, SYP, SYS, KYS and A’DEL viruses in vitro . Interestingly, in vivo results demonstrated asymmetric effects in avian and mosquito competence that were independent of the E-protein glycosylation status. In AMCRs and HOSPs, all viruses showed comparable viremias with the exception of NYP and KYS viruses that showed attenuated phenotypes. Only NYP showed reduced vector competence in both Cx . quinquefasciatus and Cx . tarsalis . Glycosylated NYT exhibited similar avian virulence properties as NYS, but resulted in higher mosquito oral infectivity than glycosylated NYS and nonglycosylated, NYP, NYF, SYP and KYS mutants. These data demonstrated that amino acid polymorphisms at E154/156 dictate differential avian host and vector competence phenotypes independent of E-protein glycosylation status.

No abstract