Teryl K. Frey

Molecular biology of rubella virus.

Publisher Summary This chapter summarizes the present medical significance of rubella virus. Rubella virus infection is systemic in nature and the accompanying symptoms are generally benign, the most pronounced being a mild rash of short duration. The most common complication of rubella virus infection is transient joint involvement such as polyarthralgia and arthritis. The primary health impact of rubella virus is that it is a teratogenic agent. The vaccination strategy is aimed at elimination of rubella and includes both universal vaccination of infants at 15 months of age with the trivalent measles, mumps, rubella (MMR) vaccine and specific targeting with the rubella vaccine of seronegative women planning pregnancy and seronegative adults who could come in contact with women of childbearing age, although it is recommended that any individual over the age of 12 months without evidence of natural infection or vaccination be vaccinated. Medically, the current challenge posed by rubella virus is to achieve complete vaccination coverage to prevent resurgences.

Sequence of the genome RNA of rubella virus: evidence for genetic rearrangement during togavirus evolution.

Abstract The nucleotide sequence of the rubella virus (RUB) genomic RNA was determined. The RUB genomic RNA is 9757 nucleotides in length [excluding the poly(A) tail] and has a G/C content of 69.5%, the highest of any RNA virus sequenced to date. The RUB genomic RNA contains two long open reading frames (ORFs), a 5′-proximal ORF of 6656 nucleotides and a 3′-proximal ORF of 3189 nucleotides which encodes the structural proteins. Thus, the genomic organization of RUB is similar to that of alphaviruses, the other genus of the Togavirus family, and the 5′-proximal ORF of RUB therefore putatively codes for the nonstructural proteins. Sequences homologous to three regions of nucleotide sequence highly conserved among alphaviruses (a stem-and-loop structure at the 5′ end of the genome, a 51-nucleotide conserved sequence near the 5′ end of the genome, and a 20-nucleotide conserved sequence at the subgenomic RNA start site) were found in the RUB genomic RNA. Amino acid sequence comparisons between the nonstructural ORF of RUB and alphaviruses revealed only one short (122 amino acids) region of significant homology, indicating that these viruses are only distantly related. This region of homology is located at the NH2 terminus of nsP3 in the alphavirus genome. The RUB nonstructural protein ORF contains two global amino acid motifs conserved in a large number of positive-polarity RNA viruses, a motif indicative of helicase activity and a motif indicative of replicase activity. The order of the helicase motif and the nsP3 homology region in the RUB genome is reversed with respect to the alphavirus genome indicating that a genetic rearrangement has occurred during the evolution of these viruses.

Viral calciomics: interplays between Ca2+ and virus.

Abstract Ca2+ is one of the most universal and versatile signaling molecules and is involved in almost every aspect of cellular processes. Viruses are adept at utilizing the universal Ca2+ signal to create a tailored cellular environment that meets their own demands. This review summarizes most of the known mechanisms by which viruses perturb Ca2+ homeostasis and utilize Ca2+ and cellular Ca2+-binding proteins to their benefit in their replication cycles. Ca2+ plays important roles in virion structure formation, virus entry, viral gene expression, posttranslational processing of viral proteins and virion maturation and release. As part of the review, we introduce an algorithm to identify linear “EF-hand” Ca2+-binding motifs which resulted in the prediction of a total of 93 previously unrecognized Ca2+-binding motifs in virus proteins. Many of these proteins are nonstructural proteins, a class of proteins among which Ca2+ interactions had not been formerly appreciated. The presence of linear Ca2+-binding motifs in viral proteins enlarges the spectrum of Ca2+–virus interplay and expands the total scenario of viral calciomics.

Global distribution of rubella virus genotypes.

Phylogenetic analysis of a collection of 103 E1 gene sequences from rubella viruses isolated from 17 countries from 1961 to 2000 confirmed the existence of at least two genotypes. Rubella genotype I (RGI) isolates, predominant in Europe, Japan, and the Western Hemisphere, segregated into discrete subgenotypes; intercontinental subgenotypes present in the 1960s and 1970s were replaced by geographically restricted subgenotypes after ~1980. Recently, active subgenotypes include one in the United States and Latin America, one in China, and a third that apparently originated in Asia and spread to Europe and North America, starting in 1997, indicating the recent emergence of an intercontinental subgenotype. A virus that potentially arose as a recombinant between two RGI subgenotypes was discovered. Rubella genotype II (RGII) showed greater genetic diversity than did RGI and may actually consist of multiple genotypes. RGII viruses were limited to Asia and Europe; RGI viruses were also present in most of the countries where RGII viruses were isolated.

Time course of virus-specific macromolecular synthesis during rubella virus infection in vero cells

Virus specific macromolecular synthesis was studied in Vero cells infected with plaque-purified rubella virus under one-step multiplication conditions. Under these conditions, the rate of virus production was found to increase rapidly until 24 hr postinfection after which time the rate of virus production rose more slowly, reaching a peak level at 48 hr postinfection. This peak rate of virus production was maintained through 72 hr postinfection. A majority of the cells remained alive through 96 hr postinfection, although a 20 to 30% decrease in the number of living cells occurred between 24 and 48 hr postinfection, the time period at which cytopathic effect was first observed. The virus structural proteins were first detected intracellularly at 16 hr postinfection. The rate of synthesis of these proteins was already maximal at 16 hr postinfection and remained constant through 48 hr postinfection. By immunofluorescence, cells expressing virus proteins were first observed at 12 hr postinfection. At 24 hr postinfection, 35 to 50% of the cells in the infected culture were exhibiting immunofluorescence, at 36 hr postinfection, 65 to 90% of the cells were exhibiting immunofluorescence, and at 48 hr postinfection, all of the cells were exhibiting immunofluorescence. The virus genomic and subgenomic RNA species were first detectable by 12 hr postinfection. The rate of synthesis of both of these species peaked at 26 hr postinfection. Rubella virus infection was found to have no effect on total cell RNA synthesis. However, a modest inhibition of total cell protein synthesis which reached 40% by 48 hr postinfection was observed. When Northern analysis of RNA extracted from infected cells was performed, a negative-polarity, virus-specific RNA probe hybridized only to the virus genomic and subgenomic RNA species. A positive-polarity, virus-specific RNA probe hybridized predominantly to a negative-polarity RNA of genome length indicating that both the genomic and subgenomic RNAs are synthesized from a genome-length negative-polarity template. Defective interfering (DI) RNAs were not detected in infected cells through 96 hr postinfection or in cells onto which virus released through 96 hr postinfection was passaged. Thus, the generation of DI particles by rubella virus appears to play no role in the slow, noncytopathic replication of this virus or in the ability of rubella virus-infected cells to survive for extended periods of time.

Molecular Analysis of Rubella Virus Epidemiology across Three Continents, North America, Europe, and Asia, 1961–1997

E1 gene nucleotide sequences of 63 rubella virus isolates from North America, Europe, and Asia isolated between 1961 and 1997 were compared phylogenetically. Two genotypes were evident: Genotype I contained 60 viruses from North America, Europe, and Japan, and genotype II contained 3 viruses from China and India. The genotype I isolates prior to 1970 grouped into a single diffuse clade, indicating intercontinental circulation, while most post-1975 viruses segregated into geographic clades from each continent, indicating evolution in response to vaccination programs. The E1 amino acid sequences differed by no more than 3%; thus, no major antigenic variation was apparent. Among 4 viruses from congenital rubella syndrome that occurred following reinfection, only one amino acid substitution occurred in several important epitopes, indicating that antigenic drift is not important in this phenomenon. However, 2 viruses isolated from chronic arthritis exhibited changes in these epitopes. Isolates of the RA 27/3 vaccine strain were readily identifiable by nucleotide sequence.

Expression of the hepatitis E virus ORF1

Summary. Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose genome contains three open reading frames (ORFs). ORF1, the 5′ proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which share homology with the products of the corresponding ORF of members of the alphavirus-like superfamily of plus-strand RNA viruses. Among animal virus members of this superfamily (the alphavirus and rubivirus genera of the family Togaviridae), the product of this ORF is a nonstructural polyprotein (NSP) that is cleaved by a papain-like cysteine protease (PCP) within the NSP. To determine if the NSP of HEV is similarly processed, ORF1 was introduced into a plasmid vector which allowed for expression both in vitro using a coupled transcription/translation system and in vivo using a vaccinia virus-driven transient expression system. A recombinant vaccinia virus expressing ORF1 was also constructed. Both in vitro and in vivo expression under standard conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have been shown to activate other viral proteases, failed to change this expression pattern. However, in vivo following extended incubations (24--36 hours), two potential processing products of 107 kDa and 78 kDa were observed. N- and C-terminus-specific immunoprecipitation and deletion mutagenesis were used to determine that the order of these products within the NSP is NH

NEUROLOGICAL ASPECTS OF RUBELLA VIRUS INFECTION

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Complementation of a Deletion in the Rubella Virus P150 Nonstructural Protein by the Viral Capsid Protein

-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys483, predicted by computer alignment to be one member of the catalytic dyad of a PCP within the NSP, failed to abolish this cleavage. Additionally, sequence alignment across HEV strains revealed that the other member of the proposed catalytic dyad of this PCP, His590, was not conserved. Thus, the cleavage of the NSP observed following prolonged in vivo expression was not mediated by this protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary monkey hepatocytes were not successful and therefore this proteolytic cleavage could not be authenticated. Overall, the results of this study indicate that either the HEV NSP is not processed or that it is cleaved at one site by a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.

Development of a Rubella Virus Vaccine Expression Vector: Use of a Picornavirus Internal Ribosome Entry Site Increases Stability of Expression

Rubella virus is a single-stranded, plus-sense RNA virus belonging to the Togaviridae family. Rubella virus infection causes a benign disease known as rubella or German measles, however infection during early pregnancy can lead to severe birth defects known as congenital rubella syndrome (CRS). Sequelae of rubella virus infection include three distinct neurological syndromes: a postinfectious encephalitis following acute infection, a spectrum of neurological manifestations following congenital infection, and an extremely rare neurodegenerative disorder, progressive rubella panencephalitis (PRP), that can follow either congenital or postnatal infection. The pathogenesis of all three of these syndromes is incompletely understood. Virus invasion and replication in the brain has only been definitively demonstrated in CRS and appears to account for the majority of neurological lesions observed in this disease. Immune-mediated pathology is particularly evident in PRP and may be autoimmune in nature, possibly triggered by molecular mimicry between viral and host epitopes, considering the apparent lack of virus in the brain. The pathogenesis of rubella encephalitis following acute infection has not been determined.