Ellen G. Strauss

Structure of Dengue Virus: Implications for Flavivirus Organization, Maturation, and Fusion

The first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal beta barrels of domain II of the glycoprotein E are inserted into the cellular membrane.

Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis.

Proteolytic processing of the Sindbis virus non‐structural polyproteins (P123 and P1234) and synthesis of minus‐ and plus‐strand RNAs are highly regulated during virus infection. Although their precise roles have not been defined, these polyproteins, processing intermediates or mature cleavage products (nsP1‐4) are believed to be essential components of viral replication and transcription complexes. In this study, we have shown that nsP4 can function as the polymerase for both minus‐ and plus‐strand RNA synthesis. Mutations inactivating the nsP2 proteinase, resulting in uncleaved P123, led to enhanced accumulation of minus‐strand RNAs and reduced accumulation of genomic and subgenomic plus‐strand RNAs. In contrast, no RNA synthesis was observed with a mutation which increased the efficiency of P123 processing. Inclusion of this mutation in a P123 polyprotein with cleavage sites 1/2 and 2/3 blocked allowed synthesis of both minus‐ and plus‐strand RNAs. We conclude that nsP4 and uncleaved P123 normally function as the minus‐strand replication complex, and propose that processing of P123 switches the template preference of the complex to minus‐strands, resulting in efficient synthesis of plus‐strand genomic and subgenomic RNAs and shut‐off of minus‐strand RNA synthesis.

Evolutionary Relationships and Systematics of the Alphaviruses

ABSTRACT Partial E1 envelope glycoprotein gene sequences and complete structural polyprotein sequences were used to compare divergence and construct phylogenetic trees for the genus Alphavirus. Tree topologies indicated that the mosquito-borne alphaviruses could have arisen in either the Old or the New World, with at least two transoceanic introductions to account for their current distribution. The time frame for alphavirus diversification could not be estimated because maximum-likelihood analyses indicated that the nucleotide substitution rate varies considerably across sites within the genome. While most trees showed evolutionary relationships consistent with current antigenic complexes and species, several changes to the current classification are proposed. The recently identified fish alphaviruses salmon pancreas disease virus and sleeping disease virus appear to be variants or subtypes of a new alphavirus species. Southern elephant seal virus is also a new alphavirus distantly related to all of the others analyzed. Tonate virus and Venezuelan equine encephalitis virus strain 78V3531 also appear to be distinct alphavirus species based on genetic, antigenic, and ecological criteria. Trocara virus, isolated from mosquitoes in Brazil and Peru, also represents a new species and probably a new alphavirus complex.

Structure and Replication of the Alphavirus Genome

In the last few years, our knowledge of the molecular biology of viruses has been greatly expanded by the technology of nucleic acid sequencing. Determination of the complete sequence of virus genomes coupled with mapping of the virus-encoded proteins on those genomes has resulted in a wealth of information about the structure of the genome, the nature of the encoded proteins, the translation strategy used by the virus, and the nature of proteolytic processing or other modification events involved in maturation of viral proteins. Comparison of the nucleic acid and deduced amino acid sequences of related viruses can reveal conserved domains, suggesting that these regions play key roles in either virus replication or morphology. Recombinant DNA technology makes it possible to design experiments to test the function of such domains directly; in particular, manipulation of viral genomes may lead to a more directed approach to vaccine production than the empirical strategies used heretofore. In a number of cases, the single base changes (and resulting amino acid substitutions) responsible for the temperature-sensitive phenotype of certain mutants have been determined. Nucleic acid sequencing is also being used to locate immunological epitopes as well as protein domains involved in virulence and specific tissue tropisms.

Fine Mapping of a cis-Acting Sequence Element in Yellow Fever Virus RNA That Is Required for RNA Replication and Cyclization

ABSTRACT We present fine mapping of a cis-acting nucleotide sequence found in the 5′ region of yellow fever virus genomic RNA that is required for RNA replication. There is evidence that this sequence interacts with a complementary sequence in the 3′ region of the genome to cyclize the RNA. Replicons were constructed that had various deletions in the 5′ region encoding the capsid protein and were tested for their ability to replicate. We found that a sequence of 18 nucleotides (residues 146 to 163 of the yellow fever virus genome, which encode amino acids 9 to 14 of the capsid protein) is essential for replication of the yellow fever virus replicon and that a slightly longer sequence of 21 nucleotides (residues 146 to 166, encoding amino acids 9 to 15) is required for full replication. This region is larger than the core sequence of 8 nucleotides conserved among all mosquito-borne flaviviruses and contains instead the entire sequence previously proposed to be involved in cyclization of yellow fever virus RNA.

Identification of the active site residues in the nsP2 proteinase of Sindbis virus.

Abstract The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.

Replication Strategies of the Single Stranded RNA Viruses of Eukaryotes

Our knowledge of the molecular biology of virus replication has expanded dramatically in the last few years, especially with the advent of rapid techniques for obtaining the nucleotide sequence of viral genomes. Full or partial sequences of virus genomes are appearing monthly, and it seems appropriate at this time to review the subject of the strategies used for replication by RNA animal viruses in the hope of formulating a conceptual framework in which to organize the new sequence information. This chapter will be concerned with the single-stranded RNA viruses which replicate via RNA intermediates and will focus on the animal viruses, but selected plant viruses whose replication strategies are known will also be discussed. The primary topics will be RNA transcription (the production of virus-specific messages), RNA replication (synthesis of viral genomes), and mRNA translation (synthesis and processing of viral proteins).

Mutants of Sindbis Virus I. Isolation and Partial Characterization of 89 New Temperature-Sensitive Mutants

More than 100 new temperature-sensitive mutants of Sindbis virus have been isolated, following mutagenesis with nitrous acid, N-methyl-N’-nitro-N-nitrosoguanidine, Sazacytidine, and 5-fluorouridine. Thirty-six of these mutants synthesize at least 60% as much RNA at the nonpermissive temperature as does the parental strain and are designated RNA+; 23 mutants synthesize between 10 and 60% as much RNA as the parental strain at 40° and are designated RNA±; 30 mutants make less than 10% as much RNA at 40° and are called RNA-. The remaining mutants have not been tested for RNA incorporation. The thermal stability at 56° of most of the mutant particles has been examined. The majority of the RNA+ mutants is more sensitive to heating at 56° than the parental HR strain, and RNA+ mutations appear to reside primarily in genes coding for the structural proteins. Approximately 20% of either RNA± or RNA- mutants are thermosensitive, and these mutations thus appear to reside primarily in genes coding for the nonstructural proteins. Complementation assays have been performed with a number of these mutants and with those of Burge and Pfefferkorn (1966a, b). The existence of three complementation groups among the RNA+ mutants, which appear to encode the three major structural proteins, has been confirmed; no new complementation groups among RNA+ mutants have been identified. A total of four complementation groups has been identified among the RNA- mutants. Thus, Sindbis virus contains at least seven complementation groups.

Isolation and Characterization of the Hydrophobic COOH-terminal Domains of the Sindbis Virion Glycoproteins

Digestion of intact Sindbis virions with α-chymotrypsin produced a single membrane-associated peptide derived from each of the two virion glycoproteins (referred to as RE1 and RE2, or roots derived from El and E2, respectively). Amino acid composition data and NH_2-terminal sequence analysis established their location at the extreme COOR-terminal end of each glycoprotein. REI and RE2 are rich in hydrophobic amino acids and insoluble in aqueous solutions in the absence of detergents, and show differential solubility in organic solvent systems designed for the extraction of lipids. Essentially all of the covalently attached palmitic acid associated with El and E2 was found to be clustered in their hydrophobic. membrane-associated roots. Beginning six to seven residues from their NH2 termini, RE1 and RE2 contain uninterrupted sequences of hydrophobic amino acids similar in terms of amino acid composition and length to the transmembrane anchors found in other bitopic integral membrane proteins. By comparing the sequence and composition data obtained here with the sequences of E1 and E2 deduced from complementary DNA sequence analysis (Rice & Strauss, 1981) we can make several observations. First, following their uncharged, putative intramembrane segments (33 and 26 amino acids, respectively), El and E2 contain clusters of predominantly basic amino acids. By structural analogy to known transmembrane proteins, El probably spans the bilayer but contains only a few residues exposed on the inner face of the virion envelope. In contrast, E2 and PE2 (the precursor to E2), which have been shown to span the bilayer completely, contain an additional 33 COOR-terminal residues, which could be either exposed on the cytoplasmic face of the lipid bilayer or which could loop back into the membrane. This region at the extreme COOR-terminal end of E2, which is protected by the virion envelope from digestion by α-chymotrypsin, contains a second uncharged domain (23 amino acids in length) whose orientation is unknown, but which may be involved in the highly specific interaction of the transmembrane glycoproteins in the plasma membrane with the cytoplasmic nucleocapsid during budding.

Budding of alphaviruses

The icosahedral structures of alphaviruses and of the external shell of the viral nucleocapsid have been defined to very high resolutions, revealing details of the interactions between the glycoproteins to form trimeric spikes and the nucleocapsid. The structural studies complement biochemical and molecular genetic studies showing that a sequence-specific interaction between the cytoplasmic domains of the glycoproteins and the nucleocapsid drives budding.