Colleen B. Jonsson

A Global Perspective on Hantavirus Ecology, Epidemiology, and Disease

SUMMARY Hantaviruses are enzootic viruses that maintain persistent infections in their rodent hosts without apparent disease symptoms. The spillover of these viruses to humans can lead to one of two serious illnesses, hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome. In recent years, there has been an improved understanding of the epidemiology, pathogenesis, and natural history of these viruses following an increase in the number of outbreaks in the Americas. In this review, current concepts regarding the ecology of and disease associated with these serious human pathogens are presented. Priorities for future research suggest an integration of the ecology and evolution of these and other host-virus ecosystems through modeling and hypothesis-driven research with the risk of emergence, host switching/spillover, and disease transmission to humans.

Ribavirin Causes Error Catastrophe during Hantaan Virus Replication

ABSTRACT Except for ribavirin, no other antiviral drugs for treating hantaviral diseases have been identified. It is well established that ribavirin will inhibit the production of infectious Hantaan virus (HTNV); however, its mechanism of action is unknown. To characterize the inhibitory effect of ribavirin on HTNV, the levels of viral RNAs, proteins, and infectious particles were measured for 3 days posttreatment of HTNV-infected Vero E6 cells. HTNV-infected cells treated with ribavirin showed a slight reduction in the levels of cRNA, viral RNA, and mRNA populations on the first day postinfection. The amount of cRNA and viral RNA increased to that observed for untreated HTNV-infected cells on day 2, whereas mRNA levels were more greatly reduced on days 2 and 3. Despite the finding of S-segment mRNA, albeit low, three of the viral proteins—nucleocapsid (N) protein and glycoproteins G1 and G2—could not be detected by immunohistochemistry in ribavirin-treated cells. To test the hypothesis that these effects were caused by incorporation of ribavirin into nascent RNA and a resultant “error catastrophe” was occurring, we cloned and sequenced the S-segment cRNA/mRNA from ribavirin-treated or untreated cells from day 3. We found a high mutation frequency (9.5/1,000 nucleotides) in viral RNA synthesized in the presence of ribavirin. Hence, the transcripts produced in the presence of the drug were not functional. These results suggest that ribavirins mechanism of action lies in challenging the fidelity of the hantavirus polymerase, which causes error catastrophe.

Replication of Hantaviruses

Hantaviruses have a genome consisting of three segments of negative-sense, single-strand RNA (reviewed in Schmaljohn 1996a). The large (L), medium (M), and small (S) genomic segments, or vRNAs, code for the viral RNA-dependent RNA polymerase (RdRp), envelope glycoproteins (Gl and G2), and nucleoprotein (N), respectively. Nonstructural proteins have not been described for hantaviruses. One of the first molecular features found to distinguish hantaviruses from other viruses in the family Bunyaviridae is the sequence of their conserved, complementary terminal nucleotides on the L, M, and S segments (Schmaljohn and Dalrymple 1983). This characteristic, and the absence of serological cross-reactivity among other members of the family, were the bases for the proposal to establish the Hantavirus genus in the family Bunyaviridae in 1986.

The RNA Binding Domain of the Hantaan Virus N Protein Maps to a Central, Conserved Region

ABSTRACT The nucleocapsid (N) protein of hantaviruses encapsidates both viral genomic and antigenomic RNAs, although only the genomic viral RNA (vRNA) is packaged into virions. To define the domain within the Hantaan virus (HTNV) N protein that mediates these interactions, 14 N- and C-terminal deletion constructs were cloned into a bacterial expression vector, expressed, and purified to homogeneity. Each protein was examined for its ability to bind the HTNV S segment vRNA with filter binding and gel electrophoretic mobility shift assays. These studies mapped a minimal region within the HTNV N protein (amino acids 175 to 217) that bound vRNA. Sequence alignments made from several hantavirus N protein sequences showed that the region identified has a 58% identity and an 86% similarity among these amino acid sequences. Two peptides corresponding to amino acids 175 to 196 (N1) and 197 to 218 (N2) were synthesized. The RNA binding of each peptide was measured by filter binding and competition analysis. Three oligoribonucleotides were used to measure binding affinity and assess specificity. The N2 peptide contained the major RNA binding determinants, while the N1 peptide, when mixed with N2, contributed to the specificity of vRNA recognition.

Characterization of the Hantaan Nucleocapsid Protein-Ribonucleic Acid Interaction

The nucleocapsid (N) protein functions in hantavirus replication through its interactions with the viral genomic and antigenomic RNAs. To address the biological functions of the N protein, it was critical to first define this binding interaction. The dissociation constant, K d , for the interaction of the Hantaan virus (HTNV) N protein and its genomic S segment (vRNA) was measured under several solution conditions. Overall, increasing the NaCl and Mg2+ in these binding reactions had little impact on the K d . However, the HTNV N protein showed an enhanced specificity for HTNV vRNA as compared with the S segment open reading frame RNA or a nonviral RNA with increasing ionic strength and the presence of Mg2+. In contrast, the assembly of Sin Nombre virus N protein-HTNV vRNA complexes was inhibited by the presence of Mg2+ or an increase in the ionic strength. TheKd values for HTNV and Sin Nombre virus N proteins were nearly identical for the S segment open reading frame RNA, showing weak affinity over several binding reaction conditions. Our data suggest a model in which specific recognition of the HTNV vRNA by the HTNV N protein resides in the noncoding regions of the HTNV vRNA.

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment

ABSTRACT In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly, deriving the virion envelope from the endoplasmic reticulum (ER) or Golgi compartment. While it is generally accepted that Old World hantaviruses assemble and bud into the Golgi compartment, some studies with New World hantaviruses have raised the possibility of maturation at the plasma membrane as well. Overall, the steps leading to virion assembly remain largely undetermined for hantaviruses. Because hantaviruses do not have matrix proteins, the nucleocapsid protein (N) has been proposed to play a key role in assembly. Herein, we examine the intracellular trafficking and morphogenesis of the prototype Old World hantavirus, Hantaan virus (HTNV). Using confocal microscopy, we show that N colocalized with the ER-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells, not with the ER, Golgi compartment, or early endosomes. Brefeldin A, which effectively disperses the ER, the ERGIC, and Golgi membranes, redistributed N with the ERGIC, implicating membrane association; however, subcellular fractionation experiments showed the majority of N in particulate fractions. Confocal microscopy revealed that N was juxtaposed to and distributed along microtubules and, over time, became surrounded by vimentin cages. To probe cytoskeletal association further, we probed trafficking of N in cells treated with nocodazole and cytochalasin D, which depolymerize microtubules and actin, respectively. We show that nocodazole, but not cytochalasin D, affected the distribution of N and reduced levels of intracellular viral RNA. These results suggested the involvement of microtubules in trafficking of N, whose movement could occur via molecular motors such as dynein. Overexpression of dynamitin, which is associated with dynein-mediated transport, creates a dominant-negative phenotype blocking transport on microtubules. Overexpression of dynamitin reduced N accumulation in the perinuclear region, which further supports microtubule components in N trafficking. The combined results of these experiments support targeting of N to the ERGIC prior to its movement to the Golgi compartment and the requirement of an intact ERGIC for viral replication and, thus, the possibility of virus factories in this region.

cis-Acting Signals in Encapsidation of Hantaan Virus S-Segment Viral Genomic RNA by Its N Protein

ABSTRACT The nucleocapsid (N) protein encapsidates both viral genomic RNA (vRNA) and the antigenomic RNA (cRNA), but not viral mRNA. Previous work has shown that the N protein has preference for vRNA, and this suggested the possibility of a cis-acting signal that could be used to initiate encapsidation for the S segment. To map thecis-acting determinants, several deletion RNA derivatives and synthetic oligoribonucleotides were constructed from the S segment of the Hantaan virus (HTNV) vRNA. N protein-RNA interactions were examined by UV cross-linking studies, filter-binding assays, and gel electrophoresis mobility shift assays to define the ability of each to bind HTNV N protein. The 5′ end of the S-segment vRNA was observed to be necessary and sufficient for the binding reaction. Modeling of the 5′ end of the vRNA revealed a possible stem-loop structure (SL) with a large single-stranded loop. We suggest that a specific interaction occurs between the N protein and sequences within this region to initiate encapsidation of the vRNAs.

Structural Studies of Hantaan Virus

ABSTRACT Hantaan virus is the prototypic member of the Hantavirus genus within the family Bunyaviridae and is a causative agent of the potentially fatal hemorrhagic fever with renal syndrome. The Bunyaviridae are a family of negative-sense RNA viruses with three-part segmented genomes. Virions are enveloped and decorated with spikes derived from a pair of glycoproteins (Gn and Gc). Here, we present cryo-electron tomography and single-particle cryo-electron microscopy studies of Hantaan virus virions. We have determined the structure of the tetrameric Gn-Gc spike complex to a resolution of 2.5 nm and show that spikes are ordered in lattices on the virion surface. Large cytoplasmic extensions associated with each Gn-Gc spike also form a lattice on the inner surface of the viral membrane. Rod-shaped ribonucleoprotein complexes are arranged into nearly parallel pairs and triplets within virions. Our results differ from the T=12 icosahedral organization found for some bunyaviruses. However, a comparison of our results with the previous tomographic studies of the nonpathogenic Tula hantavirus indicates a common structural organization for hantaviruses.

Ebola Virus VP30 Is an RNA Binding Protein

ABSTRACT The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.