Biology subjects

Haller, S. L.

Publications and source records attributed to Haller, S. L..

5 recordsLinked to original sources

bioRxiv2024

Molecular basis for the host range function of the poxvirus PKR inhibitor E3

The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix G) determined sensitivity to K3. Expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix-G was resistant to wild type VACV infection, and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA. SignificanceThe molecular mechanisms that govern the host range of viruses are incompletely understood. A small number of poxvirus genes have been identified that influence the host range of poxviruses. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. While there is a substantial body of work demonstrating host-specific interactions with K3, the current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters dsRNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete, and that dsRNA sequestration is not the primary mechanism for E3 activity.

microbiology↗

bioRxiv2020

Cascade evolution in poxviruses: retrotransposon-mediated host gene capture, complementation and recombination

There is ample phylogenetic evidence that many critical virus functions, like immune evasion, evolved by the acquisition of genes from their hosts through horizontal gene transfer (HGT). However, the lack of an experimental system has prevented a mechanistic understanding of this process. We developed a model to elucidate the mechanisms of HGT into vaccinia virus, the prototypic poxvirus. All identified gene capture events showed signatures of long interspersed nuclear element-1 (LINE-1)-mediated retrotransposition, including spliced-out introns, polyadenylated tails and target site duplications. In one case, the acquired gene integrated together with a polyadenylated host U2 small nuclear RNA. Integrations occurred across the genome, in some cases knocking out essential viral genes. These essential gene knockouts were rescued through a process of complementation by the parent virus followed by non-homologous recombination during serial passaging to generate a single competent virus. This work links multiple evolutionary mechanisms into one adaptive cascade and identifies host retrotransposons as major drivers for virus evolution.

microbiology↗

bioRxiv2020

Emergence of Zika virus: Role of founder effects, drift and mutational reversions in spread to the Americas

Summary ParagraphMosquito-borne viruses have recently spread globally, with major impacts on human health. Zika virus (ZIKV) emerged from obscurity in 2013 to spread from Asia to the South Pacific and the Americas, where millions of people were infected. For the first time, severe clinical manifestations, including Guillain Barre syndrome and defects to the fetuses of pregnant women, were detected. Phylogenetic studies have shown that ZIKV evolved in Africa and later spread to Asia, and that the Asian lineage is responsible for the recent epidemics. However, the reasons for the sudden emergence of ZIKV remain incompletely understood. Accumulating evidence on other arboviruses like chikungunya and West Nile suggest the likelihood that viral mutations could be determinants of change in ZIKV transmission efficiency responsible for efficient spread. Using evolutionary analyses, we determined that four mutations, which occurred just before ZIKV introduction to the Americas, represent direct reversions of previous mutations that accompanied spread many decades ago from ZIKVs native Africa to Asia and early circulation there. Experimental infections of mosquitoes, human cells, and mice using ZIKV strains with and without these mutations demonstrated that the original mutations reduced fitness for urban transmission, while the reversions restored fitness, increasing epidemic risk. Overall, our findings include the three newly identified, transmission-adaptive ZIKV mutations, and demonstration that these and one identified previously restored fitness for epidemic transmission soon before introduction into the America. The initial mutations may have followed founder effects and/or drift when the virus was introduced into Asia, or could be related to changes on host or vector utilization within Asia.

microbiology↗

bioRxiv2020

Orthopoxvirus K3 orthologs show virus- and host-specific inhibition of the antiviral protein kinase PKR

The antiviral protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2 is highly conserved, some K3 orthologs were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2 interactions, by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined. Authors summaryMost virus families are composed of large numbers of virus species. However, in general, only a few prototypic viruses are experimentally studied in-depth, and it is often assumed that the obtained results are representative of other viruses in the same family. In order to test this assumption, we compared the sensitivity of the antiviral protein kinase PKR from various mammals to inhibition by multiple orthologs of K3, a PKR inhibitor expressed by several closely related orthopoxviruses. We found strong differences in PKR inhibition by the K3 orthologs, demonstrating that sensitivity to a specific inhibitor was not indicative of broad sensitivity to orthologs of these inhibitors from closely related viruses. We also show that PKR from even closely related species displayed markedly different sensitivities to these poxvirus inhibitors. Furthermore, we identified amino acid residues in these K3 orthologs that are critical for enhanced or decreased PKR inhibition and found that distinct amino acid combinations affected PKRs from various species differently. Our study shows that even closely related inhibitors of an antiviral protein can vary dramatically in their inhibitory potential, and cautions that results from host-virus interaction studies of a prototypic virus genus member cannot necessarily be extrapolated to other viruses in the same genus without experimental verification.

microbiology↗

bioRxiv2018

Rapid, seamless generation of recombinant poxviruses using host-range and visual selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since this first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACVs easily manipulated genetics and broad host range as an outstanding platform to generate recombinant therapeutics. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host-range selection method coupled with visual identification. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. This method permits rapid, seamless generation of rVACV in a variety of cell types.

microbiology↗