The research of the Krug laboratory focuses on how animal viruses exploit host cell functions for their replication. Among animal viruses with RNA genomes (other than retroviruses), influenza virus has the most extensive dependence on host cell functions in the nucleus. The investigations of the molecular mechanisms that mediate the interactions between cellular and influenza viral functions is providing novel information about the regulation of both cellular and viral gene expression.
As shown by this laboratory, influenza virus is unique in that it cannibalizes cellular capped RNAs in the nucleus to produce the primers needed for the initiation of viral mRNA synthesis. An endonuclease that is intrinsic to the viral polymerase cleaves host cell RNA polymerase II transcripts to produce capped (m7GpppNm-containing) RNA fragments 10-13 nucleotides long that serve as primers to initiate viral mRNA synthesis (Figure 1).
One of the current research projects in the Krug laboratory focuses on the second distinctive biochemical property of the influenza virus polymerase, namely, the activation of its catalytic activities by specific viral genome RNA sequences. This viral polymerase lacks the catalytic activity for producing capped RNA primers unless the 5' and 3' terminal sequences of the viral genome RNA sequentially bind to specific amino acid sequences in one of the subunits of the polymerase, the PB1 subunit (Figure 2). Consequently, viral genome RNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions of the polymerase.
We have identified the two RNA-binding sites on the polymerase and also the two new functional sites, i.e., for cap-binding and endonucleolytic cleavage, that are activated by these two RNA sequences. Presumably, the two genome RNA sequences that bind to the PB1 subunit induce conformational changes in both the polymerase subunits that are needed for the acquisition of catalytic activity. We are currently determining precisely how this RNA binding induces the protein subunits of the polymerase to refold to form the active sites that are needed for the production of capped RNA primers and for the ensuing synthesis of viral mRNA.
The major focus of the Krug laboratory is the regulation of the processing and nuclear export of cellular and viral mRNAs in virus-infected cells, and the structure and function of viral proteins that regulate this mRNA processing. This research centers on the NS1 protein encoded by influenza A viruses (NS1A protein), which is an unique regulator of several cellular post-transcriptional activities in both the nucleus and cytoplasm. To carry out one of its functions, the viral NS1A protein interacts with two essential proteins of the cellular system for the 3' end processing of cellular pre-mRNAs: the 30kD subunit of cleavage and polyadenylation specificity factor (CPSF); and poly(A)-binding protein II (PABII) (Figure 3). One of the domains of the NS1A protein, the effector domain, which is located in its carboxy half, mediates the interaction with 30kD CPSF and PABII. This interaction inhibits the functions of CPSF and PABII, causing the inhibition of 3' end processing of cellular pre-mRNAs and hence the nuclear export of cellular mRNAs. Because the 3' poly (A) ends of viral mRNAs are produced by another mechanism, i.e., reiterative copying of a short stretch of uridines in the viral genome RNA by the viral polymerase, the nuclear export of viral mRNAs is not inhibited in influenza virus-infected cells.
We are continuing to dissect the complex interactions of the NS1A protein with the cellular 3' end processing machinery. The NS1A protein also binds the viral double-stranded RNA molecules that are produced during infection, thereby blocking the activation of at least one cellular protein (PKR kinase) that would otherwise shut down the synthesis of viral proteins. This binding is mediated by the RNA-binding/dimerization domain, which is located at the amino terminus of the NS1A protein. The dimeric RNA-binding domain, which has several RNA targets including double-stranded RNA, exhibits a novel six-helical bundle fold (Figure 4). The dimer structure is essential for RNA-binding. A specific arginine side chain in helix 2 of each monomer is the only amino-acid side chain that is absolutely required only for RNA-binding and not for dimerization, indicating that this side chain probably interacts directly with the RNA target.
Based on this evidence, we have proposed that helix 2 and helix 2', which are antiparallel and next to each other in the dimer conformation, constitute the interaction face between the NS1A RNA-binding domain and its RNA target. To determine whether this is the case, we are determining the three-dimensional structure of the complex of the NS1A protein with its specific RNA targets. Another function of the NS1A protein is the inhibition of the splicing of pre-mRNAs in the nucleus. We are determining the mechanism(s) by which this protein interacts with components of the splicing system to cause splicing inhibition. We have identified one mechanism that is mediated by the NS1A RNA-binding domain: the specific binding of U6 snRNA, one of the snRNAs that is required for splicing. However, other experiments suggest that there is at least one other mechanism of splicing inhibition and that this mechanism is mediated by the effector domain of the NS1A protein. Other key issues about splicing inhibition need to be resolved: (1) because the NS1A protein-mediated inhibition of the 3' end processing of cellular pre-mRNAs is sufficient to block the production of mature cellular mRNAs that reach the cytoplasm of infected cells, it is not known what specific role that the NS1 protein-mediated inhibition of pre-mRNA splicing plays in infected cells; and (2) because two viral mRNAs continue to be spliced by the cellular splicing system in infected cells, the selective mechanism that spares the splicing of viral mRNAs needs to be identified.
We anticipate that our investigations into the mechanisms by which the NS1A protein regulates cellular functions will continue to provide novel information about the regulation of both cellular and influenza viral mRNA processing. In addition, the research projects in this laboratory are expected to provide new approaches for the development of influenza virus antivirals. Influenza virus causes widespread human disease. In a typical year, influenza virus afflicts 10-20% of the U.S. population, causing up to 40,000 deaths. During pandemics, a much greater loss of life occurs. The 1918 pandemic was the worst. At least 20 million people died worldwide, many of whom were young adults in the prime of life. The reason for the high pathogenicity of this particular virus strain has not yet been ascertained . The potential for the re-emergence of a highly pathogenic influenza virus strain is exemplified by the sudden appearance of a lethal virus in Hong Kong in 1957, which killed six out of the eighteen infected individuals.
Currently the primary means of controlling influenza virus infection is vaccination. The vaccine, which is comprised of killed virus, has to be reformulated each year because the hemagglutinin, the viral protein that attaches to the cell and that elicits the primary neutralizing immune response, usually undergoes changes that render the previous year's vaccine ineffective against the new strain. Each year the new vaccine takes about six months to produce, which is clearly too long to control a fast-moving epidemic. In contrast to vaccines, antivirals can be rapidly employed to combat an epidemic, and the efficacy of antiviral therapy against influenza virus has been greatly enhanced because the virus can now be detected at an early time of infection using rapid diagnostic tests. A few antiviral drugs are currently being used to combat influenza virus epidemics, but these drugs have several problems that limit their effectiveness. Better antiviral drugs are needed.
