Lewis Markoff

5′- and 3′-noncoding regions in flavivirus RNA

Publisher Summary The flavivirus genome is a capped, positive-sense RNA approximately 10.5 kb in length. It contains a single long open reading frame (ORF), flanked by a 5´ noncoding regions (NCR), which is about 100 nucleotides in length, and a 3´ NCR ranging in size from about 400 to 800 nucleotides in length. The conserved structural and nucleotide sequence elements of these NCRs and their function in RNA replication and translation are the subjects of this chapter. The 5´ and 3´ NCRs play a role in the initiation of negative-strand synthesis on virus RNA released from entering virions, switching from negative-strand synthesis to synthesis of progeny plus strand RNA at late times after infection, and possibly in the initiation of translation and in the packaging of virus plus strand RNA into particles. The presence of conserved and nonconserved complementary nucleotide sequences near the 5´ and 3´ termini of flavivirus genomes suggests that ‘‘panhandle’’ or circular RNA structures are formed transiently by hydrogen bonding at some stage during RNA replication.

A Live, Attenuated Dengue Virus Type 1 Vaccine Candidate with a 30-Nucleotide Deletion in the 3′ Untranslated Region Is Highly Attenuated and Immunogenic in Monkeys

ABSTRACT The Δ30 deletion mutation, which was originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172 to 143 from the 3′ untranslated region (3′ UTR), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (DEN1). The resulting virus, rDEN1Δ30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4Δ30 vaccine candidate. rDEN1Δ30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3′ UTR, and both vaccines were highly protective against challenge with wt DEN1. Both rDEN1Δ30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1Δ30 nor rDEN1mutF showed restricted replication following intrathoracic inoculation in the mosquito Toxorhynchites splendens. The ability of the Δ30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the Δ30 mutation into wt DEN viruses belonging to each of the four serotypes.

The topology of bulges in the long stem of the flavivirus 3' stem-loop is a major determinant of RNA replication competence

ABSTRACT All flavivirus genomes contain a 3′terminal stem-loop secondary structure (3′SL) formed by the most downstream ∼100 nucleotides (nt) of the viral RNA. The 3′SL is required for virus replication and has been shown to bind both virus-coded and cellular proteins. Results of the present study using an infectious DNA for WN virus strain 956 initially demonstrated that the dengue virus serotype 2 (DEN2) 3′SL nucleotide sequence could not substitute for that of the WN 3′SL to support WN genome replication. To determine what WN virus-specific 3′SL nucleotide sequences were required for WN virus replication, WN virus 3′SL nucleotide sequences were selectively deleted and replaced by analogous segments of the DEN2 3′SL nucleotide sequence such that the overall 3′SL secondary structure was not disrupted. Top and bottom portions of the WN virus 3′SL were defined according to previous studies (J. L. Blackwell and M. A. Brinton, J. Virol. 71:6433-6444, 1997; L. Zeng, L., B. Falgout, and L. Markoff, J. Virol. 72:7510-7522, 1998). A bulge in the top portion of the long stem of the WN 3′SL was essential for replication of mutant WN RNAs, and replication-defective RNAs failed to produce negative strands in transfected cells. Introduction of a second bulge into the bottom portion of the long stem of the wild-type WN 3′SL markedly enhanced the replication competence of WN virus in mosquito cells but had no effect on replication in mammalian cells. This second bulge was identified as a host cell-specific enhancer of flavivirus replication. Results suggested that bulges and their topological location within the long stem of the 3′SL are primary determinants of replication competence for flavivirus genomes.

Points to consider in the development of a surrogate for efficacy of novel Japanese encephalitis virus vaccines

Although an effective killed virus vaccine to prevent illness due to Japanese encephalitis virus (JEV) infection exists, many authorities recognize that a safe, effective live JEV vaccine is desirable in order to reduce the cost and the number of doses of vaccine required per immunization. A large-scale clinical efficacy trail for such a vaccine would be both unethical and impractical. Therefore, a surrogate for the efficacy of JE vaccines should be established. Detection of virus-neutralizing antibodies in sera of vaccinees could constitute such a surrogate for efficacy. Field studies of vaccinees in endemic areas and studies done in mice already exist to support this concept. Also, titers of virus-neutralizing antibodies are already accepted as a surrogate for the efficacy of yellow fever virus vaccines and for the efficacy of other viral vaccines as well. In developing a correlation between N antibody titers and protection from JEV infection, standard procedures must be validated and adopted for both measuring N antibodies and for testing in animals. A novel live virus vaccine could be tested in the mouse and/or the monkey model of JEV infection to establish a correlation between virus-neutralizing antibodies elicited by the vaccines and protection from encephalitis. In addition, sera of subjects receiving the novel live JEV vaccine in early clinical trials could be passively transferred to mice or monkeys in order to establish the protective immunogenicity of the vaccine in humans. A monkey model for JEV infection was recently established by scientists at WRAIR in the US. From this group, pools of JEV of known infectivity for Rhesus macaques may be obtained for testing of immunity elicited by live JE vaccine virus.

Cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences

We investigated the requirements of the carboxyterminal sequence for surface expression of the influenza viral hemagglutinin (HA). Deletions in the cloned hemagglutinin gene were introduced at locations upstream from and spanning into the region that codes for the hydrophobic carboxyl terminus. Primate cells infected with recombinants of the deleted HA gene and an SV40 vector were negative for surface immunofluorescence and failed to adsorb erythrocytes. Polypeptide analysis showed that the mutant hemagglutinins lacking the normal hydrophobic carboxy-terminal sequences were secreted into the medium. These data provide evidence that these sequences of the influenza hemagglutinin are responsible for accumulation at the cell surface. During infection with each deletion mutant, a truncated HA polypeptide was found intracellularly. Both intracellular and extracellular HAs were glycosylated, since a third species representing the unglycosylated mutant hemagglutinin was detected in the presence of tunicamycin. Interestingly, the secreted and intracellular mutant HA polypeptides differ from the surface HA in their sensitivity to endoglycosidase H, indicating that an alteration of glycosylation has occurred.

Derivation and Characterization of a Dengue Type 1 Host Range-Restricted Mutant Virus That Is Attenuated and Highly Immunogenic in Monkeys

ABSTRACT We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3′ stem-loop structure (3′-SL) that is conserved among all flavivirus species. The 3′-SL constitutes the downstream terminal ∼95 nucleotides of the 3′ noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3′-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.

Amino acid sequence changes in antigenic variants of type A influenza virus N2 neuraminidase

Abstract Pronase-released neuraminidase heads from six strains of influenza type A virus (of the H2N2 and H3N2 subtypes) isolated between 1957 and 1975 were examined for changes in amino acid sequence. In 469 residues 19 changes which occurred during this period were located. Two regions of the neuraminidase molecule were not examined, residues 188–210 which were insoluble, and residues 1–73 (or 76) which formed part of the stalk of the neuraminidase and were removed during Pronase digestion. Neuraminidase heads from seven variants of A/Tokyo/3/67 virus, selected with different monoclonal antibodies to the neuraminidase were also examined for sequence changes. In four of these, a single sequence change at position 344 of arginine to isoleucine was found, in another variant an asparagine residue at position 221 changed to histidine and in another the lysine at position 368 changed to glutamic acid. This last change also occurred in the field strains isolated in 1972 and 1975. In the seventh monoclonal variant the change could not be found and may be in the insoluble region. Some of these sequence changes were clustered into two highly variable regions in the neuraminidase comprising residues 344–347 and 367–370, suggesting that these regions may be involved in antigenic sites on the neuraminidase molecule. An antigenic map of N2 neuraminidase [R. G. Webster, V. S. Hinshaw, and W. G. Laver, Virology 117, 93–104 (1982)] suggested three or four nonoverlapping antigenic areas and the variants with changes at residues 344 and 368 were grouped in one of these antigenic regions. Sequence changes in the region of 344–347 were also associated with changes in the stability of the neuraminidase. The four monoclonal variants with a sequence change of Arg (344) to Ile possessed neuraminidase molecules which were destroyed by Pronase at 37° (but not at 20°) whereas wild-type neuraminidase was stable during digestion at 37°.

Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5′- and 3′-terminal regions (TR) with the conserved sequence elements. Our results show that both 5′- and 3′-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3′-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5′-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5′- nor 3′-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5′-cap inhibits the (–)-strand RNA synthesis from the 3′ fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that “cyclization” of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis.

Sequence of the influenza A/Udorn/72 (H3N2) virus neuraminidase gene as determined from cloned full-length DNA

A complete nucleotide sequence was determined from cloned full-length DNA of the influenza A/Udorn/72 (H3N2) neuraminidase gene. The neuraminidase DNA has a total length of 1466 bases, excluding G-C linker residues. The cloned segment retained the common sequences known to be present at the 3′ and 5′ termini of virion RNA. Also present was a nonviral oligonucleotide nine bases long at the 5′(+) end of the DNA which represents cellular sequences acquired during influenza mRNA synthesis. The sense strand of the DNA contained a single open-reading frame coding for 469 amino acids. A possible polyadenylation site was noted downstream from the termination codon. No mammalian gene splice sequences and no alternate open-reading frame were present. The deduced amino acid sequence displayed the features of other neuraminidase genes, including the conservation of a sequence of 12 amino acids at the NH2 terminus and the presence of a hydrophobic region from amino acid positions 7 to 34. The data were consistent with the hypothesis that this hydrophobic region of the surface glycoprotein represents the site for membrane insertion. When compared to the complete nucleotide and amino acid sequences of the influenza A/PR/8/34 (H1N1) neuraminidase, the N2 sequence revealed conservation of the positions of cysteine residues and potential glycosylation sites. Regions of homology were detected between N1 and N2 amino acid sequences, if cysteine residues of the two polypeptides were aligned. Nucleotide sequence homology between N1 and N2 was evident to a lesser degree. The data suggest that the N2 gene sequence differs from that of the N1 gene by numerous point mutations, as well as by insertion/deletion, and that the antigenic “shift” in the human neuraminidase antigenic subtype in 1957 is most likely to have occurred by replacement of the human N1 gene with one from the animal reservoir of influenza A viruses through gene reassortment.

Requirements for West Nile Virus Minus- and Plus-Strand Subgenomic RNA Synthesis in vitro by the viral RNA- dependent RNA Polymerase Expressed in E. coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5′- and 3′-terminal regions (TR) with the conserved sequence elements. Our results show that both 5′- and 3′-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3′-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5′-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5′- nor 3′-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5′-cap inhibits the (–)-strand RNA synthesis from the 3′ fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that “cyclization” of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis.