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Rubella Virus Replication and Links to

Teratogenicity
Jia-Yee Lee and D. Scott Bowden
Clin. Microbiol. Rev. 2000, 13(4):571. DOI:
10.1128/CMR.13.4.571-587.2000.

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... when the clinical manifestations of the disease were described by two German the cataracts were often associated with other deformities........... In the of rubella was reported by two independent groups (111.......................573 EPIDEMIOLOGY ........................... who further reported other de- Several decades passed without significant research into ru...574 Attachment and Entry................... another German physician........ rectly demonstrated the presence of RV by resistance to chal- oratory............................................................. de Bergan in 1752 and Orlow in 1758 (155)....................................................00⫹0 Copyright © 2000........... All Rights Reserved.........577 Morphogenesis ..................................................... Locked Bag 815........................................................org/ on May 9.......................................................................................................................................................................................................584 REFERENCES ................................................................................ 68 mothers gave a had become known as rötheln (155).......... documented the illness as a distinct entity...................... There was considerable congenital abnormalities and rubella infection early in the conjecture about the relationship of rubella to measles and to mother’s pregnancy.................. but it was not until the bella........................... the illness became persistence finally led to the discovery of the link between the popularly known as German measles......... Veale........................... E-mail: jia-yee....................................................... No....... Processing............577 Role of Cellular Proteins in RV Replication........ He physicians...... The disease was renamed history of rubella infection early in pregnancy............................................asm................ Thus...............org....................... 13...................................584 INTRODUCTION spring and summer of 1940................574 Structural Proteins: Translation.......................583 TERATOGENICITY: CONCLUDING REMARKS......................572 CLINICAL FEATURES .................. Fax: 61-3-9342 2666............................................ George tice................................ the idea rubella (from the Latin for reddish things) in 1866 by Henry that viruses could be teratogenic agents was introduced..................................... Victorian Infectious Diseases Reference Laboratory............................................................... it was considered to be a derivative of measles...................573 Downloaded from http://cmr..................................... 2013 by guest Capsid Protein.............. The notion that rubella was England Journal of Medicine (155) that the true significance of only a mild illness of children was dispelled in 1941..............................................au..... (111) infected African green monkey kidney cells with throat washings from patients with rubella and indi- * Corresponding author.................. p......................... Rubella Virus Replication and Links to Teratogenicity JIA-YEE LEE* AND D.. an Australian ophthalmic surgeon..............................................574 Replication .................................. SCOTT BOWDEN Research and Molecular Development Division.............................................................580 Mitochondrial Changes...................................... Victoria 3053...............572 Pathology of CRS................577 Replication Complexes ........................ and with colleagues he managed to collate a total of 78 de Maton..................... fects associated with maternal rubella..................................................................................................................................... 4 0893-8512/00/$04.................................................... by his Australian colleagues.......................... American Society for Microbiology............................ Australia............576 Nonstructural Proteins: Translation and Processing ..................................................................................573 VIRUS STRUCTURE .......CLINICAL MICROBIOLOGY REVIEWS................ which cases of children with cataracts...........................583 ACKNOWLEDGMENTS ..574 Genome Organization .......... North Melbourne............... Norman Gregg..................................................................................................................................................................................573 VIRUS LIFE CYCLE....................... 571–587 Vol.................................................................. lenge with an echovirus..........................582 Apoptosis................lee@mh cytopathic effects in human amnion cells infected with RV ............................................................................................................................................. and Assembly ................................................................................. His because of the strong German influence...........................................................................572 VACCINES............................ a British Army surgeon................................... who found the original term Gregg’s report initially drew little attention.......................................... when Gregg’s earlier observations was accepted.........................................580 Cytoskeletal Changes... and babies in an attempt to assign a cause to the outbreak................................................................. Victoria 3051............................. whose spread was probably enhanced by Rubella virus (RV) is the causative agent of the disease wartime mobilization...............................................................................................571 CLASSIFICATION ...........572 Rubella and Its Complications ................................. the isolation in cell culture of the etiological agent the devastating teratogenic effects of the virus (47)............................................ while Weller and Neva (154) detected Phone: 61-3-9342 2604..... Australia had experienced an epidemic of rubella............ Carlton South......................................... Mailing address: Research and Molecular Development Division........... It has now been shown that 571 ....... It was supported “harsh and foreign to our ears” (31)...... Gregg observed an unusually large num- commonly known as German measles.................................................................... 2000.............................................. and of rubella appears to date back to the 1700s.......................... Australia INTRODUCTION .............................................. Gregg had 13 such cases in his own prac- scarlet fever until in 1814............................. 154)..................... At the obtained careful histories from the mothers of the affected time.................... from blood and urine specimens.................578 LINKS TO TERATOGENICITY ............ despite documentation of the viral etiology of the disease comprehensive review by Wesselhoeft published in the New by Hiro and Tasaka in 1938 (31)...................................................... Oct........................ Victorian Infectious Diseases Reference Lab.............. of these................................................................. reported In 1962................ Parkman et al...... The earliest description ber of cases of congenital cataracts in newborn children..............................573 E1 and E2 Glycoproteins .

Vascular aerosol presentation to the nasopharyngeal mucosa. which is been reported as a late complication of childhood rubella (1. As with RV-induced be the first sites of virus replication. other eye structures such as the iris and the retina can be affected. from the Latin “toga. the name “togavirus” is derived reported after 12 weeks gestation. 159). No cases of rubella reinfection causing CRS have been within the family Togaviridae. swelling of the mito- nopathy. The genus Alphavirus is the only other Pathology of CRS genus within this family and comprises at least 26 members. MICROBIOL. Lymphadenopathy chondria. 153). The major public health concern posed by rubella is its Genome Single-stranded. being common in the structures of the eyes. with transient joint in. E1. with maternal infection early in pregnancy lead- 3 ⫻ 106 to 4 ⫻ 106 Da in size ing to the congenital rubella syndrome (CRS) in infants. loreticular structures. 159). Other clinical features include car- proteins. bosis of small vessels and necrosis of surrounding tissue. Loss of cytoplasmic ground substance. RI representing 100% of infected fetuses developing congenital defects. and general malaise. with noninflammatory necrosis phaviruses. Virus aspect Description of property 159). brain. and dilation of the endoplasmic reticulum (ER) were can be characteristic. examination of RV-infected fetuses to regional lymph nodes (26. and the virus then spreads deafness in CRS infants. and ocular conditions such as cataracts and glaucoma. complexes in vascular endothelial cells from many organs (75). RV and the alphaviruses possess similar character. lens development was found to be retarded (143. encapsidated with multiple copies of the capsid proteins 34. The earlier in gestation the maternal infection Two ORFs. vertebrates and Microscopic analyses of aborted infected fetuses revealed cel- arthropods. The Methyl7guanosine capped at the 5⬘ terminus and time at which infection occurs during gestation can influence polyadenylated at the 3⬘ terminus the outcome. More serious complications including thrombocytopenic Virion Spherical particle measuring 50–70 nm in diameter purpura and postinfectious encephalopathy or encephalomy- Virion envelope composed of host-derived lipid bilayer elitis are very occasionally associated with postnatally acquired embedded with spikes made up of the E2. and rubella (34. mental retardation. the risk of developing any defects is low (44). the prototype. with Sindbis virus. infections are asymptomatic. subgenomic RNA. As with primary rubella infection. Insulin-dependent diabetes mellitus occurs commonly as a late sequela of CRS. infection can be induced by destruction of the myocardium is common (143. General properties of togaviruses quent. cell tory aerosols. after 17 weeks gesta- replication complexes are sites of RV replication tion. REV. 2013 by guest partial dsRNA. Interestingly. RV can be grown in a wide variety of cell culture systems. The contribution of RV replication Rubella can cause complications. are the only known natural hosts for RV.org/ on May 9. and defects such as deafness may not be initially detected (32. and Semliki Forest virus RV generally establishes a chronic nonlytic infection in the (SFV) being the best-characterized members. The rash first appears on the face reported the presence of nuclear bodies and tubuloreticular and then spreads over the trunk and later over the extremities. which can persist after the rash has resolved. Interestingly. Maternal and the 3⬘ ORF encodes structural proteins infection during the first 8 weeks after the last menstrual pe- riod results in nearly all fetuses becoming infected and up to Replication The genomic RNA. and RF representing fully dsRNA are produced in RV-infected cells risk of fetal infection and the severity of congenital abnormal- Virus-modified endosomes/lysosomes termed ities decreases after the first trimester. some of the features observed in cells displaying these tubu- cipital nodes. 153). cytoplasmic vacuoles. lular damage in multiple sites. with deafness mRNA for the synthesis of ns and structural being the most common. The clinical symptoms of revealed cellular damage to the epithelium of the cochlear duct RV infections acquired postnatally are usually mild. The upper necrotic lesions within the walls of the cerebral blood vessels respiratory tract and nasopharyngeal lymphoid tissue appear to may contribute to ischemic brain damage. 159). 153). involving the posterior cervical and oc.asm. are recognized hosts for al. sore throat. 44). not been elucidated. a reference to the virus envelope (99). respectively diac disease. In volunteer studies. ears of aborted RV-infected fetuses (143. such as mosquitoes. positive-polarity RNA teratogenicity. tion. revealed pyknotic nuclei. TABLE 1.” meaning cloak or shroud. Other symptoms typically include low-grade fever. the 5⬘-proximal ORF encodes ns proteins. The first clinical manifestation of To date there has only been one report of ultrastructural rubella is usually the appearance of a macropapular rash some examination of cells from RV-infected fetuses (75). The clinical The genomic and subgenomic RNA serves as the manifestations of CRS are numerous and varied. heart. although this does appear to be a rare phenomenon (131).572 LEE AND BOWDEN CLIN. Examination of The general characteristics of togaviruses are summarized in RV-induced cataractous eye lenses from first-trimester fetuses Table 1. The study 16 to 20 days after exposure. and many and/or stria vascularis (143. the gestational age at the CLASSIFICATION time of reinfection influences the chance of fetal abnormali- RV is classified as the only member of the genus Rubivirus ties. the more severe is the damage to the fetus. 153). . and cases of CRS have been reported following maternal reinfec- this has been the cornerstone of vaccine development. these symptoms are more prevalent and severe in RV-infected women than in RV-infected men (142. and inclusion bodies in primary lens cells. 159). Necrosis is also detected in the endothelial cells Rubella and Its Complications within the blood vessels lining the heart and can cause throm- The virus is transmitted from person to person via respira. lymphade. occurs. The Downloaded from http://cmr. and/or virus-induced host factors to such cellular changes has volvement such as arthritis and arthralgia being the most fre. and istics in terms of replication strategy and genomic organization. While humans fetus and has the potential to infect any organ (153. A rare and usually fatal neurodegenerative sometimes E3 glycoproteins disorder termed progressive rubella panencephalitis has also Icosahedral core comprising the viral genome. While the lens is the predominant site of CLINICAL FEATURES necrosis.

A number of molecular mass of 58 kDa. at least 20. indicating that they were green monkey kidney cells. domain.VOL. many rubella vaccination policies have been subse. two different strate. which have been a result. the positively notification rates in males. 2013 by guest the mainstay of vaccination programs in most developed coun. 110. In developed countries before vaccine develop. the interaction of the capsid protein to eradicate measles. In 1969. HPV-77. The including the United Kingdom. vent of childhood vaccination in the United States. Both strategies resulted in a substan. allowing the virus to circulate to susceptible individuals Capsid Protein when opportunity arose. The virion contains an electron- United States. possible to look at the genetic makeup of the virus itself and The RV E1 glycoprotein migrates as discrete band with a perform molecular epidemiological studies. As an indication of the effectiveness The capsid protein is a nonglycosylated. 13. mainte. It remains to be determined EPIDEMIOLOGY whether other domains of the protein are involved in nucleo- capsid formation. 66. Europe. Parkman et al. The E1 ment.to 9-year-old and E2 proteins each contain a putative transmembrane (TM) group. no apparent signature mutations or became the first rubella vaccine licensed for use in the United other changes could be identified to distinguish CRS strains States. 159). from initial rubella isolates collected in the 1960s through to demic of rubella. Soon afterward. Geographical isolates were de- rived from three continents and included wild-type. With the advent of vaccine availability. charged 7-residue sequence. it is now also domain (58). made. while in Australia and many European countries. The virion envelope proteins. rubella infection was still prevalent among males. 108). there were much higher region which acts as a signal sequence for E1. 146). rubella epidemics occurred every 6 to 9 years in the United States and at shorter intervals of 3 to 5 years in Europe. Overall. 98). other live attenuated RV vaccines be. (112) developed the first live atten. a 28- the development of the trivalent measles-mumps-rubella vac. following passaging of antigenically very similar. As part of a global effort genome (88). the putative TM domain is followed by a positively shift in disease incidence to young adults. In countries practic. where the two uated vaccine. The full impact of the teratogenic properties of RV infection and vaccine strains. infection was most common in the 5. With the ad. As clusters of proline and arginine residues. the spikes are composed of the E2 and E1 glycoproteins tial decrease in the number of cases of rubella and CRS. and Ja- bella epidemic added great impetus to the development of a pan. not evident at the deduced amino acid level. corresponding to the early school years. universal vaccination of all preschool children lucent spherical core composed of multiple copies of the RV was used. the latter strategy. With to form the viral nucleocapsids (20. it was widely atively charged phospholipid head groups of the lipid bilayer. 19. from other strains. The virus strains analyzed were derived was demonstrated from 1962 to 1965 following a global epi. and one of these. The consequences of the ru. became Downloaded from http://cmr. 108. 159). envelope E1 gene and performed phylogenetic analyses (for a Amino acid sequence analysis of the E1 protein has since . see reference 35). The peak incidence stretch (nt 347 to 375) interacts with the capsid protein. the TM domain is followed by a 13-residue cytoplasmic the advent of molecular techniques such as PCR. respectively (34). including the United Kingdom (110. However. capsid protein and a single copy of the viral RNA genome. as expected. In the prevaccine era. amino-acid domain containing a large number of basic resi- cine. RRACRRR.asm. On the RV genome. and genotype II represented only 3 Asian isolates from suitable vaccine. the RA27/3 strain. a 29-nucleotide (nt) Rubella has a worldwide distribution. With For E1. 144). In the imately 60 nm in diameter. the United States consistently disulfide-linked homodimer with a reported molecular mass of reported fewer cases of CRS than the United Kingdom did 33 to 38 kDa (18. while the E2 glycoprotein migrates groups have examined the nucleotide sequence of the RV as broad heterogeneous band of 42 to 47 kDa (18. 92.to 6-nm-long spikes which project from the virion sur- and susceptible women. E1 and E2. 2000 RUBELLA VIRUS REPLICATION AND TERATOGENICITY 573 VACCINES compilation. The isolates included strains isolated HPV-77 in duck embryo fibroblasts at Merck. which This figure would have been even more overwhelming if not differ from each other by 8 to 10% at the nucleotide level. are type 1 mem- nance of rubella requires continuous access to a susceptible brane glycoproteins observed as spikes in the form of E1-E2 population. of basic residues because other basic regions within the protein were found to bind poorly. with the viral RNA may not be solely dependent on the density bella vaccine schedules has enhanced rubella immunity (110). face. laboratory. RV could be divided into two genotypes.org/ on May 9. a combined infant and adolescent immunization strategy dues appears to be directly involved in binding to the RNA has been adopted by many countries. phosphorylated. (159). HPV-77 DE-5 from patients with CRS. of the immunization policies. although the disease is present throughout the year the genome (88). heterodimers on the virion surface (18.000 infants the 1990s. The mature RV virion is a round or ovoid particle approx- gies were used to avert further rubella epidemics. came available. 108. With (34. by passaging RV 77 times in African genotypes differed by only 1 to 3%. postulated to be involved in binding to the RV genomic RNA quently expanded to be inclusive rather than selective. charged 7-residue region is believed to interact with the neg- From studies with monoclonal antibodies. In the United States. agreed that there is essentially only one serotype of RV. al- occurs during spring months in countries with a temperate though it is not clear whether this is sufficient for packaging of climate. allowing some evolutionary comparisons to be suffered permanent damage as a result of in utero infection. a selective vaccination policy RV core is surrounded by a host-derived lipid bilayer contain- was used concentrating on school-age girls (10 to 15 years old) ing 5. and a 20-residue ing the selective vaccination policy. the 63 isolates derived from North America. The diversity seen at the nucleotide level was In 1966. In particular. there was a For E2. 34). for the large number of therapeutic abortions carried out after Genotype I was a large intercontinental group containing 60 of maternal rubella infection (25). VIRUS STRUCTURE tries (26. China and India. E1 and E2 Glycoproteins Since humans are the only known reservoir for RV. implementation of measles-mumps-ru. The capsid protein contains despite having a significantly higher population base (110). which is 22 and 39 residues in length.

46). viral replicative intermediates (RI) of 21S. RV virions could be clones of E1 and E2 have shown that all the N-linked glyco. 161).345 nt encodes the viral ns proteins. with N-linked sugars representing adjacent to or in coated pits (Fig. both the RV 40S RNA and Attachment and Entry 24S RNA were detected at the end of the viral latent period. unlabeled viri- coli have indicated that glycosylation may be required for cor. However. 78). Similarly. Although the host portion of cells infected by RV at any one time is cell type cell receptor has not been identified. This is in contrast to the mechanisms of al- intracellular forms of E2 (39 kDa). The E2 protein of the M33 and HPV.762 nt and contains alphaviruses (73. Studies using RV-infected cells and full-length cDNA predominantly via the endocytic route. 162). The RV Sugiura (70) showed that exposure of the RV E1 and E2 5⬘-proximal ORF of 6. the the endosome. 134. 1). Using monoclonal antibodies.org/ on May 9. In addi- erodimer formation (164). REV. It has immunogenic epitopes (140). E2 does contain partial hemagglutination and neu- a messenger for the nonstructural (ns) proteins and as a tem- tralizing epitopes and may also carry strain-specific epitopes plate for the synthesis of a 40S negative-polarity RNA strand. the RV capsid pro- shown that removal of any of the N-linked sites results in tein undergoes a structural change from having hydrophilic to Downloaded from http://cmr. preembedding immunogold-labeling The role of N-linked glycosylation on the antigenicity and studies using antibodies to RV revealed that during the viral immunogenicity of E1 has been investigated by several groups. This hypothesis all strains so far sequenced (127). The minus strand in turns acts as a template for the transcrip- tion of both the 40S RNA and the 24S RNA (34).8 ⫻ 103 kDa) and a attachment of the virus to the cell.2 ⫻ 103 kDa) that corresponds to the revealed that a 28-residue internal hydrophobic domain of E1 3⬘ one-third of the genomic RNA are present in infected cells is responsible for the fusogenic activity of RV (164). while the 3⬘ ORF of 3. mutagenesis studies have been shown that between pH 5. this region is involved in the binding to E2 for het- terminus and a polyadenylate tail at the 3⬘ terminus. glycoproteins to pH 6. In addition to N-linked sugars. The the cytoplasm.4. gold-labeled RV virions were seen attached to Studies in which recombinant E1 was expressed in Escherichia the plasma membrane adjacent to coated pits. Pulse-chase to the endosomal membrane but also to trigger uncoating of labeling of RV-infected cells has revealed the presence of the capsid protein. representing The function of E2 has been more difficult to determine.0 and 5. During RV and neutralization (21.0 or less induced a conformational p150 and p90. 52. 134. while the and penetration studies by thin-section electron microscopy E2 protein of the Therien and RA27/3 strains possesses three (TSEM) indicate that at physiological pH of 7. capsid (C). with viral structural proteins appearing 4 h later (52). 52.asm. A to be the main surface protein. 2013 by guest slower glycan processing and lower stability. During viral replication. tion. For E2. 162). (28. which migrate more rapidly phavirus uncoating. E1 appears infection. 109. by the binding of the capsid proteins to ribosomes (135). MICROBIOL. Moreover. RV can establish infections able to infect every cell at any specific time. Nascent 40S VIRUS LIFE CYCLE RNA is packaged with the RV capsid protein to form nucleo- capsids. allowing the release of viral genomic RNA into RV E2 protein contains O-linked carbohydrates (89). the 40S RV genomic RNA serves as However. some of which are associated with hemagglutination the long viral latent period of 8 to 12 h (18. rect folding of E1 for the expression of important antigenic and The uncoating event for RV is also not well defined. 160. it seems that the low-pH environment of presence of these O-linked sugars most probably contributes to the endosome serves not only to induce virion envelope fusion the heterogeneous nature of the virion form of E2. 67. respectively (60. have been exposed (66. However. 139. 162). Thus. monolayer.574 LEE AND BOWDEN CLIN. In terms of viral kinetics. irrespective of the in a variety of cell lines (34). which is reflected in epitopes. ons were observed in endosome-like vacuoles (Fig. lysosomotropic agents (116). observed attached to cell surface projections which were either sylation sites are utilized. produce high titers of virus. Preliminary viral attachment 77 strains possesses four N-linked glycosylation sites. 166). these events were ob- approximately 6 kDa and 15 to 20 kDa of the molecular mass served as early as 3 min after the addition of virus to the cell of the mature E1 and E2. it appears that membrane dependent (52. the number of was further supported by more recent studies which demon- N-linked glycosylation sites of the E2 protein appears to vary strated the inhibition of viral replication following the use of depending on the strain. The route of RV entry into the host cell is not well under- stood. 2). There is some evidence to suggest that RV enters cells Genome Organization via the endocytic pathway. with domains involved in the single-stranded 40S RV genomic RNA (3. revealed that it contains three N-linked glycosylation sites for viral envelope to the endosomal membrane. latent period. 152. capsid protein presumably allows uncoating to occur within ation sites removed (127). Both contain a methyl7guanosine cap at the 5⬘ tion. 34).). 127). Therefore.189 nt encodes the change within the glycoproteins that favored the fusion of the structural proteins. For SFV. More recent studies have 24S subgenomic RNA (1. E2. and viral replicative is disulfide-linked to E1 in the mature virion and is poorly forms (RF) of 19 to 20S. 134). RV enters (127). This conformational change in the of the defect increasing with the number of N-linked glycosyl. 152). ment (94). similar to that reported for the The full-length RV genome measures 9. the viral nucleocapsid is uncoated than the virion form of E2 (42 to 47 kDa) (18. as infection proceeds. indicating that the host cell re- titer of the input virus (20. and E1 (27. Peak Molecular characterization of RV has been largely per- virus production occurs during the period from 36 to 48 h formed in Vero and BHK-21 cells because of their ability to postinfection (p. Thus. 140. the pro- ceptor is likely to be a ubiquitous molecule. In addi- (68. E2 partial double-stranded RNA (dsRNA). Early biochemical studies by Katow and two long open reading frames (ORFs) (27) (Fig. with the severity hydrophobic properties (95). perhaps due to the absence of an One-step multiplication studies have shown that RV is un- interferon system (97). it has been Replication shown that the E1 protein contains at least six nonoverlapping RV is characterized by slow replication. 108). the phospholipids and glycolipids may be involved in viral attach- entire culture eventually becomes infected. representing full dsRNA. In contrast. accessible to characterization by monoclonal antibodies (151). the . the antigenic sites of E2 are less detected in RV-infected cells (134. four distinct viral RNA species can be detected. The functions of the RV E1 and E2 glycoproteins have been studied extensively.i.5. 3).

The RV genome has an assembled particle. Vero cells were inoc- ulated with RV at a multiplicity of infection of 50 and incubated at 37°C.9% at the nucleotide is unlikely that the virion within the endosome-like vacuole represented a newly and amino acid levels.6 to 98. (A) An RV virion (solid arrow). 2. Therien (150). The complete nucleotide sequence of RV has been labeling EM using polyclonal antibodies to RV as described previously (84). 13. Bar. 100 nm. Since the cells were harvested during the viral latent period. Nucleotide and deduced amino acid sequencing extracellularly. 2013 by guest FIG. respectively. FIG.org/ on May 9.5%. plasma membrane. Amino acid sequence analysis of the RV 5⬘ ORF has re- vealed global amino acid motifs indicative of RNA-dependent RNA polymerase (replicase). Detection of an RV virion within an endosome-like vacuole.asm. An unlabeled RV virion (long solid arrow) is seen in an endosome- analysis reveals a high degree of homology between strains. RV- gene order for the RV 40S RNA is 5⬘-p150-p90-C-E2-E1-3⬘ infected Vero cells were harvested at 4 h p. Note the virion (short solid arrow) attached near a coated pit (open arrow). 121. (B) A virion (solid arrow) can be seen located within a coated vesicle (open arrow). 3). . 148). comprising an electron-lucent core surrounded by a host-derived lipid envelope. like vacuole (v).i. 138. it ranging from 97. the cell monolayer was harvested and processed for TSEM as described by Lee (81). PM. the highest of any known RNA virus to date.2 to 99% and 97. 30. 15. and 45 min after the addition of virus. methyltransferase. 5. can be seen attached to a cell surface projection (SP) adjacent to a coated pit (open arrow). 10 nm) were found associated with virions located RA27/3 (121). determined for three strains. and Downloaded from http://cmr. At 3. and this has undoubtedly contrib- uted to some discrepancies in the reported RV genomic se- quences (27. 2000 RUBELLA VIRUS REPLICATION AND TERATOGENICITY 575 extraordinarily high G⫹C content of 69. Attachment and entry of RV into Vero cells. 100 nm. M33 (165).VOL. helicase. and processed for immunogold- (Fig. Bars. and Gold particles (diameter. 1.

E2. cleavage in the ER. The assembly pathway of RV has not been fully elucidated. A polyprotein precursor. 34). and E1. is found within p150 and been suggest that both AUG codons are used indiscriminately is also present in the alphavirus nsP3 protein and the hepatitis (24. cleavage of RV capsid protein contrast. helicase (H). (RLP). The RNA transcripts the insertion of the proteins into the ER (54. A unique feature of the RV capsid protein is tifs relative to each other has led to the hypothesis that genetic the retention of the E2 signal peptide on the carboxy terminus rearrangement has occurred during togavirus evolution (34). The RV genome comprises two long nonover- lapping ORFs. Unlike the alphaviruses. in the order NH2-C-E2-E1-COOH from the following translation of these proteins. 2013 by guest FIG. peptide of 20 amino acids is retained on the carboxy terminus nome. a short region of un. This inversion of the mo.asm. The p110 protein is translocated into the ER by two E virus 5⬘ ORF product (27. significant progress has been made in under- which the cDNA clone was derived. REV. 137). Similarly. as evidenced by the production of virus that was phe. two possible AUG initia. 107). The amino acid motifs for separate signal peptides. and Assembly ding to their wild-type counterpart. In addition. notypically and genetically similar to the parental virus from Nevertheless. In from the polyprotein precursor. Of particular significance is further to the understanding of RV replication and pathogenesis. 109). Following proteolytic 24S subgenomic mRNA (106. 126). 24. 59). a full-length infectious clone derived from 54. the RV gene order for the 5⬘ ORF where an autoprotease cleaves the alphavirus capsid protein is 5⬘-methyltransferase-X-protease-helicase-replicase-3⬘ (34). the development of cDNA constructs that express RV struc- tural proteins that assemble into rubella virus-like particles Structural Proteins: Translation. These RLPs have similar morphology and sites of bud- Processing. 92). proteinase and methyltransferase are located in the carboxy located at the amino termini of E2 and E1. 23 and 20 amino acids in length. Within terminus of p150. is translated from the 5⬘ ORF of the RV genomic RNA and undergoes cis cleavage to produce two ns proteins. with the 5⬘ ORF coding for the ns proteins and the 3⬘ ORF coding for the structural proteins. the ER (24. is translated from the subgenomic RNA and undergoes several posttranslational modifications to ultimately produce the mature capsid (C). p110. ated (150). 54. p150 and p90. terminus of p90 (27). while the motifs for the replicase (GDD) and the ER. tion codons are present within the RV 24S RNA. and they serve as a conve- nient tool in dissecting the RV assembly pathway (58. For The RV structural proteins are translated as a polyprotein RV. 137). MICROBIOL. Downloaded from http://cmr. Schematic representation of the translation and processing strategy of the RV ns and structural proteins. of the capsid protein (10. 36. p100. The RV structural proteins are synthesized from a 24S subgenomic RNA transcribed from the 3⬘ ORF. 59. The respective signal peptides of E2 and E1 direct the M33 strain was also produced (165). It is anticipated that the standing RV assembly with the use of cDNA constructs con- genetic manipulation of such RV infectious clones will contribute taining the RV structural genes.576 LEE AND BOWDEN CLIN. the gene order for SIN virus 5⬘ ORF is 5⬘-methyltrans. Recently. Thus. 36). and replicase (R) are indicated on the 5⬘ ORF. the first cDNA copy of the RV genomic RNA was gener- of the E2 protein after cleavage from E1 by host signalase (36. the E1 signal Following the complete sequencing of the Therien strain ge. p200. generated from both the full-length RV clones were found to be infectious. is mediated by a cellular signalase found within the lumen of ferase-helicase-protease-X-replicase-3⬘. and it has known function. the RV capsid protein is cleaved from E2 and E2 is the helicase (GxGKT and DExx) are located in the carboxy cleaved from E1 (10. respectively. papain-like cysteine protease (P). X motif. The locations of the putative amino acid motifs for methyltransferase (M). termed the X motif. the E1 and E2 proteins form disulfide- . 3. 92.org/ on May 9. the assembly of structural proteins begins immediately precursor. proteinase activities (34). A polyprotein precursor. 92.

152). Collectively. it has plexes (77). the RV protease transmembrane domains of the protein. catalytic residues of Cys-1151 and His-1272. 100. in the absence of E2. 75. protein of Bowden and Westaway (18) in RV-infected cells linked homodimers (11. 165). Moreover. The p150 protein that masks the ER retention signal. A synthetic RNA modeled defined. Thus. It would be of interest to determine whether calreti- erodimers have yet to be elucidated. the are several sequences at the 5⬘ and 3⬘ termini of the RV data suggest that the E1 TM and cytoplasmic domains play a genome that can potentially form stable stem-loop structures critical role in the very late stages of virus budding. The 87-kDa rough ER (RER). it has been proposed cell proteins that obscured the detection of the ns proteins that this interaction may induce RV teratogenesis (3). It has been suggested that E1 and E2 dimerization facil. 150. p200. Pulse-chase Morphogenesis studies during hypertonic salt treatment of RV-infected cells Numerous investigators have employed TSEM to study RV demonstrated that the ns200 protein was cleaved to ns150 (18). functions to retain was proposed to be a Main protease with similarities to cellular unassembled E1 subunits and immature E2-E1 dimers in the proteases (45). implying that it interacts with RV RNA cent observations that RV virions and RLPs can be secreted (30. In contrast. 136). re- cellular protein. 119. 87.asm.VOL. Transfection studies have since confirmed that ER until folding and heterodimer formation are complete the RV protease is indeed a Main protease because it can (55). E1-E2 heterodimer formation is most probably represents the 90 kDa protein observed in trans- necessary for the transport of E1 from the ER to the Golgi fection studies (33. Nonstructural Proteins: Translation and Processing In addition to host proteins interacting with the RV genome. there cDNA has revealed similar findings (165). into two arrested in the post-ER. A better-defined mutagenesis study on replication. Earlier studies pos- a host protein that is involved in the modulation of genes and tulated that the interaction of the E1 cytoplasmic domain and that binds specifically to regions located at the 3⬘ end of the the RV capsid protein triggered virus budding (58). The E2-E1 heterodimers are retained in acid region encompassing the GxGKT and DExx motifs of the the Golgi complex by a retention signal of 18 amino acids putative helicase (50). 166). 102. glycoproteins to the Golgi complex (126). 93. 102. 2000 RUBELLA VIRUS REPLICATION AND TERATOGENICITY 577 linked heterodimers while the capsid proteins form disulfide. Although the 3⬘ untrans- recent studies have revealed that this interaction is not the lated region contains cis-acting elements necessary for RV driving force for virus budding (41). Because the RB protein is cells.org/ on May 9. fragments is mediated by a protease residing in p150 with 61). thereby allowing the trans. One such protein has been identified as calreticulin. Recent studies using replicase have not been shown. 22. complex and the cell surface. The mechanisms involving replication. Recently. spanning both the cytoplasmic and comparative amino acid sequence analysis. BHK- More recent work employing cDNA constructs containing the 21. and 27 kDa in RV-infected cells (18). RV maturation is a budding process in which the viral cleaved to produce two products. suggesting that cellular or host proteins may also the E1 TM and cytoplasmic domain within a RV infectious be involved in viral RNA replication (103). NTPase involved in E1-E2 interaction. leading to the formation of the activity has been demonstrated to be associated with the amino heterodimer (164). E1 is The cleavage of the polyprotein precursor. have raised questions whether the assembly and budding The Ro/SS-A antigen has also been suggested to interact with events are similar for both compartments. the TM Studies using RV-infected cells showed that the addition of and cytoplasmic domains of E1 were shown not to be required actinomycin D (an inhibitor of DNA-dependent RNA poly- for this process but were necessary for the secretion of RLPs merase) in the early stages of RV infection inhibited virus into the medium (41). the 5⬘(⫹) stem-loop structure. pre-Golgi complex compartment (57. The Golgi apparatus. cytoplasmic vacuoles. 3) core acquires an envelope membrane after passing through (33. it was shown that this RNA structure is into the apical and basolateral surfaces of polarized cells (40) involved in translation rather that in viral replication (124). The task was further complicated by the presence of host involved in the regulation of cell growth. morphogenesis in a variety of cell lines including RK13. was recently shown to be localized to RV replication com- port of the heterodimers to the Golgi complex. La. An ER retention signal cleavage site is found within Gly-1300–Gly-1301 (23). The only other functional activity demonstrated been shown that an internal hydrophobic domain in E1 is within the RV 5⬘ ORF is that of the RV helicase (50). of 200. However. ORF-encoded protein is NH2-p150-p90-COOH. of 150 and 90 kDa (Fig. 13. Early stud- ies reported the detection of several ns proteins. Based on on E1 of 22 amino acids. but it is not known whether this interaction is specific (102). It has been proposed that maintenance of the E2 from the 3⬘ stem-loop structure has been shown to interact signal sequence as part of the capsid protein after cleavage specifically with three phosphorylated cytoplasmic proteins allows the capsid protein to be transported along with the (100. 93. the role of calreticulin binding to this region is the interaction between the nucleocapsid and E2-E1 het- unclear. the gene product order for the RV 5⬘ modified host cell membranes (98). and plasma mem- . RV RNA (4. In the process. RLPs have indicated that the TM and cytoplasmic domain of E2 are required for the targeting of the heterodimers to the Role of Cellular Proteins in RV Replication sites of budding such as the Golgi complex. Early TSEM studies on RV-infected cells RV 5⬘ ORF of the Therien and M33 strains demonstrated that reported predominantly on the morphology and maturation of the 200-kDa protein is an ns polyprotein precursor that is RV. culin associates with RV replication complexes where active While studies with RLPs have provided important insights viral RNA synthesis occurs (22). 119). E1 undergoes a conformational change function in cis and trans cleavage (87. Recently. and Vero cells. representing possible sites for interactions between RV RNA The role of the capsid protein in RV assembly is less well and host proteins (101. because RV does not inhibit host protein synthesis. 120). 2013 by guest located in the TM domain of E2 (62). the protease itates the proper folding of E1 (10. 165). the cellular retinoblastoma (RB) protein interacts with a The characterization of RV ns proteins has largely been known RB-binding motif located within the carboxy-terminal hampered by the limited production of ns proteins in infected half of the RV p90 protein (3). 119). The enzymatic functions of the putative Downloaded from http://cmr. Moreover. 56). the precise mechanisms for budding where the virions cis elements of RNA could be cross-linked in vitro to the acquire the host membrane are far from clear. into RV assembly with respect to virus protein-protein inter- Other studies have demonstrated that the 5⬘(⫹) stem-loop actions.

ciated with replication complexes (Fig. Downloaded from http://cmr. Consequently. and biogenesis to alphavirus cytopathic vacuoles (77). that facilitate the accumulation The presence of RV replication complexes in infected cells and assembly of the proteins in the respective organelles. 53. 83.578 LEE AND BOWDEN CLIN. RER was found to associate only with play an important role in viral entry via the endocytic pathway. the capsid protein is likely to re- RV research has focused mainly on the characterization of RV main attached to the RER membrane (54. the side of the replication complex vacuole where the vesicles the use of these organelles as sites of viral replication appears were located. It is now recognized that the results of these is expelled from the vesicles of the replication complex (Fig. these complexes (91). respectively. This is not surprising. less. (83) first described RV replication complexes as oles of the complexes (83). but it is most likely that these virus-induced structures tion reveals that the vesicles contain irregular internal struc. Thus. these morphological studies re. the RER sur. they do not origin. RV replication 83). rounded the whole replication complex (83). 4). Furthermore. This is in contrast to the alphaviruses. coincident with the membrane is the only site of virus budding (159). structures are associated with viral RNA synthesis (84). A model for the biogenesis of . the cytoplasmic side of the replication complex and the adja- annulate lamellae have been found in other virus-infected cent RER membrane. aber. protein hydrophobic. precise site of viral replication appears to be the vesicles within Moreover. marker for viral RI and RF. coinciding with peak presence of an ER and a Golgi retention signal on the E1 and production of the virus (83). protein confirmed the identity of the particles (85). RV replication complexes appear to serve as sites for nucleocapsid Replication Complexes assembly. thereby indicating the vealed that RV replication complexes are similar in morphol. replication the vacuole via a membranous neck. In addition. inter. 37. signal peptidase involved in cleaving E2 from the capsid and tant findings on the morphology and maturation of RV. brane have been identified as sites of RV maturation (64.i. membrane whorls were frequently observed within the vacu- Lee et al. and E1 proteins occurs in the RER with the host Although early TSEM studies have produced several impor. 85. 84. was first postulated by Bowden et al. but as the infection progressed. 49). 48. since the processing of the capsid. it appears that these com- RV virions have not been detected in the vacuoles of the plexes are derived from existing lysosomes during RV infection replication complex where the vesicles were found (83). It must be noted that RV In terms of the biogenesis of RV replication complexes. Cytoplasmic inclusions have been the replication complex. type 1 (CPV-1) (2. another group has reported the colocaliza- replication.i. E2. a nisms are involved at the different maturation sites. indicating that the occurrence of these structures is prob. in the past two decades. where the plasma complexes were detected as early as 8 h p..org/ on May 9. E2 glycoproteins. thus. 4). 65. interest in RV morphogenesis has continued. 113). Close examina. to be unique to the togaviruses. association of capsid proteins with RV replication complexes ogy. Furthermore. 4). 58). Recently. Antibodies to endosomes were not used in these RV constitute immature or aberrant virion forms. 57. 5) studies complement existing molecular information on RV (84). dsRNA was localized within the vacuole of with these findings exist. close association of the RER with the vacuole complex (Fig. Interestingly. replication complexes were so named in reference to their these structures were initially proposed to be virus-modified functional role while their alphavirus counterparts were lysosomes because degenerating material such as myelin-like termed CPV-1 according to their morphology. 91). Although lysosomes in tures which are distinct from the spherical electron-lucent RV-infected cells have been recruited for virus replication. More recent studies employing membrane-bound cytoplasmic vacuoles lined internally with confocal microscopy and immunogold-labeling EM with anti- vesicles measuring approximately 60 nm in diameter (Fig. rather than stimulating the production of new lysosomes for A distinctive feature of RV replication complexes is the conversion into replication complexes. end of the viral latent period.. 2013 by guest descriptions of virus-like particles in these studies did not cor. immunofluorescence assay using antibodies to dsRNA. bodies specific to lysosome proteins confirmed that the com- These vesicles contained thread-like inclusions and were found plexes were indeed virus-modified lysosomes similar to their either free in the vacuole or attached to the inner membrane of alphavirus counterparts (91). RV replication it appears unlikely that these structures were associated with complex-associated core particles were often detected between RV replication as has been previously reported. RV core particles were demonstrated to be asso- relate with the characteristic morphology of RV virions. For the alphaviruses. (17). The localization of RV p150 not known. 149). tion of RV capsid proteins with p150. it is cores observed in mature RV virions (83). 98. Neverthe. since acid phosphatase activity was detected in surrounded by a single membrane (9. likely that lysosomal functions continue within replication rant forms of RV virions were reported as multicore structures complexes. function. Importantly. However. While it is well recognized that endosomes and lysosomes During RV infection. indicating that these virus-induced reported in RV-infected RK13 cells (65) but were never de. Hence. MICROBIOL. studies. these particles were frequently found at cells. who observed dis- Mechanisms responsible for triggering virion assembly are crete cytoplasmic fluorescent foci in RV-infected cells in an poorly defined. The matu. Annulate lamellae were observed in LLC-MK2 and within these complexes further confirms their role in viral RK13 cells infected with RV (74. and peak numbers of these ration of RV at cytoplasmic sites is presumably due to the structures were reported at 24 h p. but measurements and replication (77). In addition. 38. Given the replication at the molecular and biochemical level. Although these vesicles complexes were found to be both endosomal and lysosomal in have similar dimensions to those of RV virions. The tected in a similar cell line studied by other investigators (51). E2 from E1. 4) (85). the significance of these cytoplasmic inclusions is the replication complex (84). When later studies employing Early morphological studies revealed several cellular immunogold-labeling EM and anti-dsRNA were performed on changes associated with RV infection. close proximity of the RER to the replication complex (Fig. and it is not known whether similar mecha. leading to it is conceivable that the assembled capsid protein then asso- the discovery of “replication complexes” in RV-infected cells ciates with newly synthesized genomic RNA as the viral RNA (81. REV.asm. the base of the vesicles lining the vacuole complex. are also virus-modified endosomes. Immuno- ably due to a nonspecific cellular response to viral infection gold-labeling studies using monoclonal antibodies to the capsid (71). discrepancies RV-infected cells. The attachment of the E2 signal peptide at the est in RV morphogenesis waned with the advent of molecular carboxy terminus of the capsid protein tends to render the biology applications.

200 nm. 4. 6) (81). The virion is then transported into the cell induce conformational changes within the E1 and capsid pro- . The findings from RV studies (Fig. Electron-dense zones (arrowheads) are frequently observed between the outer membranes of adjacent mitochondria. 13. In this model. Cellular changes in RV-infected cells.3) to surface projection. 2000 RUBELLA VIRUS REPLICATION AND TERATOGENICITY 579 Downloaded from http://cmr.VOL. A typical replication complex is observed with the characteristic vesicles (double-headed arrows) and the close association of the RER (open arrow). RV replication complexes is proposed based on the collective when the coated pit invaginates to form a coated vesicle. Note the clustering of mitochondria near the replication complex.asm. Core particles (small solid arrows) can also be detected in association with the outer membrane of mitochondria. 2013 by guest FIG.org/ on May 9. RV core particles (long solid arrows) can be seen at the cytoplasmic side of the vesicles of the replication complex. the RV virus is subsequently delivered through a series of endosomes virion binds preferentially to a cell surface projection and then with progressively acidic compartments until it reaches an en- translocates into an electron-dense coated pit adjacent to the dosome where the environment is sufficiently acidic (pH 5. Bar.

123). and a mech- RER migrates to the vicinity of the endosome. RER. and alanine synthesis during the same period replication complexes are not well understood. providing an architectural framework for the assembly of all the components of replication. 145). nat- tein. was reported to be present in RV lysosome. (6). The production of ns tant clues. For RV. While these viral content and metabolic changes in RV-infected cells. Although the presence of immunoglobulins such as immunoglobulin M (IgM). RV has been suggested based on early studies of viral lipid which has been lined internally with vesicles. Cellular damage seen during early ges- nucleocapsids for interaction with RV E2-E1 heterodimers have not been de- fined. the virus ns proteins are synthesized immediately from the A closer examination of some of the unusual features of RV newly released viral RNA and remain associated with the en. 105. rather than infectious viri. It has been postulated that the membranous structure of the replication complex acts as scaffolding. since no EM the RER. which notably. and/or host proteins contrib. MICROBIOL. Newly synthesized viral RNA is then the consequences of CRS are well documented. the outer membrane of a mitochondrion and one membrane of ficient to induce formation of these vesicles. but exactly rapidly encapsidated by RV capsid proteins. allowing the anism for the teratogenic effect of RV has been proposed (29. translation and processing of the viral structural proteins from 63. It is important to bear in mind that the life cycle of virions (7). that RV may bud from mitochondria. between the outer membranes of two adjacent mi- procedures were performed. In addition. tion in infected fetuses. resulting in the release of the genomic RNA into the ural killer cells. a phospholipid that is relatively specific to the inner replication complex.580 LEE AND BOWDEN CLIN. Previous work has shown two distinctive features of proteins. thereby expelling coincident with the period of viral adsorption and penetration the vesicles and other lysosomal by-products extracellularly. Concomitantly. tation of RV-infected fetuses is unlikely to involve the immune system. replicating viral RNA. the 24S subgenomic RNA to occur. glycolysis. 7) (82). It is unclear. 82. 96. since no fetal immune response can be detected at this early stage (153).org/ on May 9. As infection progresses. The vesicles of the replication complex are postulated to provide a protective environment for the mains to be elucidated.asm. confronting membranes . (8. Presumably. the vestigated the ability of RV to induce apoptosis. confirmed more than a decade later. mitochondrial abnormalities and disruption of the provides a protected environment for the transcription of the host cell cytoskeleton (17. 2013 by guest FIG. tochondria. which now represents a lipin. and IgA. Many of the steps leading to CRS and synthesis of nascent viral genomic RNA. 104. as well as pro- viding a large surface area for viral replication and hence a more rapid synthesis of progeny RNA (156). It is not known whether nsP1 alone was suf. and picornaviruses have utilized vesicles for viral replication. the explicit pathway leading to teratogenicity re- lication complex as precise sites of viral RNA synthesis. There were further sug- The decrease in the number of replication complexes late in gestions of mitochondrial involvement in RV infection when infection and the detection of vesicles containing thread-like the same group reported a decrease in the level of ATP in inclusions on the cell surface indicate that the virus-modified RV-infected BHK-21 cells within the first hour of infection lysosome has fused to the plasma membrane. LINKS TO TERATOGENICITY Downloaded from http://cmr. many single-stranded RNA viruses such as the picornaviruses and flaviviruses (14. when mitochondrial ons. T cells. 90. the endosome. was able to induce the formation of replication complexes changes were detected in RV-infected Vero cells by TSEM (114). replication and virus-host cell interactions may provide impor- dosome where uncoating has occurred. 5. continues in its life cycle and fuses to a mitochondrial membrane. The mechanisms involved in the translocation of the resulting of much speculation. Vesicles play a vital role in the replication of fronting membranes type 1 (CM-1). Cardio- events are occurring. IgG. Electron-dense zones associated with mitochon- found that the alphavirus nsP1 was localized to endosomes and dria were identified in three different configurations: between lysosomes (115). utes to the formation of vesicles within the endosome. These ultrastructural changes have been designated con- rus infection. and between two opposing membranes of the Vesicle formation is not a characteristic confined to togavi. 157). The vesicles induced by these viruses are postulated to represent replication complexes that are derived from or accumulate in the ER rather than in endosomes and lysosomes. the RER is found surrounding the entire vacuole. there appeared to be an increase in respira- The mechanisms involved in vesicle formation within RV tion. REV. which are synthesized from the how the virus causes this dramatic effect has been the subject adjacent RER. flaviviruses. and interferon can be detected by mid-gesta- cytoplasm (95). These findings led the investigators to speculate both the virus and the endosome/lysosome are intertwined. Schematic representation of the role of vesicles within the RV rep. confocal microscopy studies by the same group (Fig. The events from virus uncoating to early for. In addition. transfection studies have provided some important clues by A direct link between mitochondria and RV infection was showing that SFV genomic RNA. the double membrane vesicles may serve to protect the nascent single-stranded viral genomic RNA from degradation by cel- lular RNases (83). Later. how- ever. However. why the togaviruses. tribute to further fetal damage has yet to be determined (153). More recent work has in- genomic and possibly subgenomic RNAs. 156. the vesicles within the endosome increase in number as more Mitochondrial Changes ns proteins are synthesized either from newly synthesized RV genomic RNA or from the original viral genomic RNA. RV replication which may impact on normal host cell function. Late in A role for mitochondria in the budding and replication of infection. the extent to which they limit or con- mation of the replication complex are unknown. 85). 15.

the RER surrounds the entire vacuole. Furthermore. after fusion of the lysosomal vacuole membrane to the plasma membrane. The E1 and capsid proteins undergo conformational changes that result in the release of the viral genomic RNA into the cytoplasm. SFV-infected cells (82). The virion passes through a series of endosomes with progressively acidic pH until it arrives at an endosome where the environment is sufficiently acidic to trigger the uncoating process. The coated pit then pinches off to form a coated vesicle that contains the virion. the RER is associated with the side of the vacuole where the vesicles are located. As infection progresses. it was noted that during the course of RV infection. Step 3. and vesicles are induced to form within the endosome. the mitochondria appeared to become club shaped and were not present in the mock-infected preparations or in (82). The RV virion attaches to the cell surface and is translocated to the coated pit. the virus-modified endosome fuses to a lysosome as part of its life cycle. including the vesicles. 2013 by guest FIG.VOL. type 2 (CM-2). It remains to be determined whether mitochondrial func. Schematic representation of the biogenesis of RV replication complexes. Step 4.org/ on May 9. This leads to the formation of the replication complex. 6. Step 1. which is lined internally with vesicles.asm. the RER migrates to the vicinity of the virus-modified endosome. At this early stage of the infection. Concomitantly. Step 6. The replication complex continues in its life cycle as a virus-modified lysosome and eventually expels its lysosomal contents. Step 2. 13. Step 5. Structures similar to CM-2 have been . Release of the viral RNA triggers the transformation of the endosome. While these events are occurring. and confronting cisternae (CC). These findings were seen only in RV-infected cells infection. respectively tion is impaired as a result of such deformities during RV (82). 2000 RUBELLA VIRUS REPLICATION AND TERATOGENICITY 581 Downloaded from http://cmr.

it has been suggested that RV. Note an RV virion (long arrow) within a lumen of an ER.asm. amorphous clumps of fluorescent foci. Electron-dense zones (short arrows) are frequently observed between the outer membrane of an adjacent mitochondrion (m). The RV capsid pro- tein is a phosphorylated protein with a unique feature in pos. Instead of the filamentous actin cables observed in core particles localize to the mitochondria. 2013 by guest FIG. dition. have also been reported performed an ultrastructural examination of cells from RV. although in vitro and in vivo studies The effects of RV infection on the cytoskeletal components have revealed that it has an affinity for membranes (54. The infected human embryos and fetuses (75). 57). CM-1 appears unique to (82). In ad- of RV core particles with mitochondria (85). 56. detected in cells infected with Nodamura virus (42). For RV. to migrate in large numbers to viral assembly sites (132). Mitochondria in Vero cells infected with Af- studies do not correspond to those described by Kistler. This phenomenon does not appear to be confined to within these zones. have been found in a variety of cells including cells infected this was similarly observed with the SFV replication complexes with herpesviruses (43). repre- In addition to these observations. 100 nm. The composition of the electron-dense zone is not known.org/ on May 9. Mitochondrial changes in RV-infected cells. uninfected cells. REV. This association is proteins and mitochondrial chaperone proteins was observed perplexing. although analysis using RV-specific an. no such associa. cell metabolic and viral kinetic studies need to ing signal on the RV capsid protein. In light of these observations. quirement that induces mitochondria to migrate to their vicin- tibodies should show whether viral proteins are localized ity (82). The composition of the electron-dense zones within these togavirus replication complexes are sites of a high-energy re- structures is unknown. since there appears to be no mitochondrial target. were detected in RV- . Downloaded from http://cmr. Cytoskeletal Changes sessing the E2 signal peptide. characteristic of active respiration. A significant alteration in the or phosphorylation play a role in targeting the RV capsid arrangement of actin filaments following RV infection was proteins for assembly at mitochondria or whether assembled evident. It may be that the capsid be performed in parallel with ultrastructural studies to gain a protein has a specific affinity for mitochondrial membranes or better understanding of RV-induced mitochondrial changes. MICROBIOL. Bar. of cells have been investigated by immunofluorescence studies Further studies are necessary to see whether this signal peptide using antibodies to actin (17). The cellular changes observed in these the togaviruses. (132). The precise role of the peptide has not been determined. who rican swine fever virus. However. an affinity for cell membranes in general. 7. pronounced clustering of senting depolymerized actin filaments. a fourfold increase in the levels of mitochondrial stress tion has been reported for the alphaviruses.582 LEE AND BOWDEN CLIN. while CC mitochondria around RV replication complexes was reported. migration was accompanied by a dramatic change in mitochon- Another intriguing feature of RV infection is the association drial ultrastructure. a dsDNA virus.

and activation of cytokine receptors using a similar cell line. collapse of mitochondrial transmembrane pathway in RV-induced apoptosis. it can also cause cytopathic effects (CPE) in whether the noninflammatory necrosis observed in organs of cell lines such as Vero. respective TM domain. the E1 and E2 proteins. (29. fewer actin filaments were stained. 141).-Y. involved are not known. the caspases. However. 96. degradation of chromatin. Some of these viruses possess genes chromatin condensation within the nuclei of cells displaying that encode either homologues of the Bcl-2 family of proteins RV replication complexes and mitochondrial changes were or inhibitors of caspases. potential. leading to that cause CPE during infection. are typical fea. this effect was noted in both RV-infected Vero tween RV and the alphaviruses. Characteristic markers of apoptosis such as DNA fragmentation.asm. some of these viruses found. 13. E2. 63. It is now established that apo. ical features that can vary with tissue and cell type. and cellular frag. hepatitis B virus. The proteins within the Bcl-2 family best to be elucidated. persistence and teratogenicity (29. (63). 123). To date. and RK13. and an. detached from the monolayer as a consequence of CPE (29. nuclear chromatin condensation. The ability of RV to induce apoptosis varies consid. suggesting that the synthesis of some viral prod. adenoviruses. The RV teratogenic process most probably begins when erably with cell type and appears to be associated with cells placental infection occurs during maternal viremia. is due to apoptotic cell death ular tools in apoptosis are required. Biochemically. is generally char. in so doing. while Höfmann et al. However. which act directly or rectly that apoptosis during RV infection is mediated by indirectly to activate a family of proteases called caspases. A chronic or for RV-induced apoptosis during acute infection. 147). Little depolymerization was evident until 16 h transfection studies of cells expressing only RV structural pro- after infection. The inhibition of apoptosis in the RV- increases in the volume of literature on RV-induced apoptosis infected monolayer has clear advantages for the virus. Morpho. and in particular their reported similar findings. The cellular proteins and biochemical pathways involved in volved in the regulation of apoptosis. can induce apoptosis as shown in served to the microtubules. the presumed interplay between the ns proteins and sion of certain regulatory proteins that function to trigger or cellular proteins involved with the apoptotic pathway remains regulate apoptosis. Alternatively. It is becoming increasingly evident that apoptosis plays an Apoptosis in RV-infected cell cultures occurs asynchro- important role in the pathogenesis of many viruses (129. Megyeri et al. p53 activation. cally. The RV ns proteins may also determined by DNA fragmentation assays. which is in general noncytolytic. and capsid proteins may not uct may have been necessary to achieve this effect. intensely the trigger for virus-induced apoptosis may be different be- stained foci. although persistent infection ensues. 96. reported that cell death during RV such as fas/APO-1/CD 95. ther analyses using more sophisticated biochemical and molec- tachment from the monolayer.. a key player in in- ptosis is characterized by specific morphological and biochem. Lee. . estingly. Studies are antiapoptotic proteins such as bcl-2 and bcl-xL and pro. there has been no report on the role of apoptosis in While RV can establish persistent noncytocidal infection in RV teratogenicity. alpha interferon is present in 90% of RV-infected mentation into membrane apoptotic bodies. 96. latency. There are conflicting reports on the role of family of proteins can indirectly promote apoptosis through p53 in inducing apoptosis during RV infection. lomavirus. nously and is confined mainly to RV-infected cells that have Alphaviruses. 123). 123). 2013 by guest logically. Since RV replication requires the formation of replication Apoptosis complexes. 72). Recent studies by Kujala et al. no changes were ob. since no evidence of the apoptotic pathway. 91) and ns proteins (77) found within these structures play Apoptosis is a form of cell death that involves a genetically a role in the apoptotic process. the regulation of a variety of signal transduction stimuli. BHK-21. While this has not been shown for RV. The apparent lack of RV progressed. cytopathogenicity have been mapped to these proteins (122). It would be of interest to determine many cell lines. The versatility of the virus to control the cellular apoptotic infected. plasma membrane blebbing. it is possible that Downloaded from http://cmr. (96) demonstrated the involvement of the p53-dependent through ceramide. it is likely that viral components such as dsRNA (84. human papil. as gestation (21 to 29 weeks) (79). and it is likely that the Bcl-2 family of proteins are major effector proteins for apoptosis (76. 158).VOL. TERATOGENICITY: CONCLUDING REMARKS nexin V staining have been reported during acute infection with RV. Ultrastructural studies of Vero cell monolayers rus are some of the viruses known to induce or interfere with infected with RV confirm these findings. seen as cell rounding followed by de. The extent to which viral or host cellular proteins are machinery can result in a range of pathogenic outcomes such involved in controlling or limiting apoptosis in these infected as persistence. The recent large cells is not known. The Bcl-2 involved (29. unpublished have evolved proteins that block apoptosis and. triguing. play a role in RV-induced apoptosis. 2000 RUBELLA VIRUS REPLICATION AND TERATOGENICITY 583 infected cells. It is now clear that RV-infected fetuses is caused by RV-induced apoptosis. until almost all structural protein involvement with apoptosis indicates that the actin was found to be disaggregated into large. Epstein-Barr virus. changes within the cell. since determinants of acteristic of this form of cell death (133. such as nuclear chromatin the RV RI and RF RNAs may elicit similar responses. 39. inter- condensation. using chemically defined caspase inhibitors have shown indi- apoptotic proteins such as bax and bcl-xS. This asynchronous induction of apoptosis is in- prolong the life of the infected cell for virus dissemination (13. terferon-mediated host defense against viral infection (5. since have revealed some understanding of the mechanisms of RV persistence develops as a consequence. even at late stages of infection (J. another cytoskeletal component. Within the Bcl-2 family RV-induced apoptosis are also not well characterized. apoptotic events can be characterized by the overexpres. cDNA transfection studies (69). e. exemplify the diverse and complex biochemical pathways in. Fur- the RV-induced CPE. 63. particularly when the majority of the cells are RV- 86). As infection be required for this process (63). More specifi. the mechanisms during RV infection (77).g. and tissue tropism.org/ on May 9. Active replication is required dissemination of the virus throughout the fetus. 130. replication is mediated by a p53-independent pathway. tion of dsRNA-dependent protein kinase. Recent studies of vaccinia virus programmed series of events culminating in the destruction infection showed that dsRNA caused apoptosis via the activa- and disposal of unwanted cells. fetuses and is readily detectable in the sera of fetuses at mid- tures of apoptosis. and human immunodeficiency vi. the proto- and BHK-21 cells. For Sindbis virus. teins indicate that the RV E1. 96). 63. observations). (77) also type alphavirus. Interestingly.

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