Optimal lengths for DNAs encapsidated by Epstein-Barr virus.

We measured the efficiency of DNA packaging by Epstein-Barr virus (EBV) as a function of the length of the DNA being packaged. Plasmids that contain oriP (the origin of latent EBV DNA replication), oriLyt (the origin of lytic EBV DNA replication), the viral terminal repeats (necessary for cleavage and packaging by EBV), and various lengths of bacteriophage lambda DNA were introduced into EBV-positive cells. Upon induction of the resident EBV's lytic phase, introduced plasmids replicated as concatemers and were packaged. Plasmid-derived concatemers of DNA with certain lengths were found to predominate in isolated virion particles. We measured the distribution of lengths of plasmid concatemers found within cells supporting the lytic phase of the viral life cycle and found that this distribution differed from the distribution of lengths of concatemers found in mature virion particles. This finding indicates that the DNA packaged into mature virions represents a selected subset of those present in the cell during packaging. These observations together indicate that the length of DNA affects the efficiency with which that DNA is packaged by EBV. Finally, we measured the length of the packaged B95-8 viral DNA and found it to be approximately 165 kbp, or 10 kbp shorter than the originally predicted size for B95-8 based on its sequence. Together with the results of other studies, these findings indicate that the packaging of DNAs by EBV is dependent on two imprecisely recognized elements: the viral terminal repeats and the length of the DNA being packaged by the virus.     

Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines

Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.     

Nucleotide sequence relationships between vertebrate 5.8 S ribosomal RNAs.

Nucleotide sequences of the 5.8 S ribosomal RNAs from HeLa cells, Xenopus laevis and chick embryo fibroblasts were compared. Xenopus laevis 5.8 S RNA differs from that of HeLa cells in four internal positions and at the 3' end of the molecule. Chick 5.8 S RNA differs from that of HeLa cells in two positions. Six out of the seven interspecies differences are due to base substitutions. The other difference is due to the presence of an extra nucleotide, internally located, within the Xenopus 5.8 S sequence.     

Functional and structural interactions between measles virus hemagglutinin and CD46.

We analyzed the roles of the individual measles virus (MV) surface glycoproteins in mediating functional and structural interactions with human CD46, the primary MV receptor. On one cell population, recombinant vaccinia virus vectors were used to produce the MV hemagglutinin (H) and fusion (F) glycoproteins. As fusion partner cells, various cell types were examined, without or with human CD46 (endogenous or recombinant vaccinia virus encoded). Fusion between the two cell populations was monitored by a quantitative reporter gene activation assay and by syncytium formation. MV glycoproteins promoted fusion with primate cells but not with nonprimate cells; recombinant CD46 rendered nonprimate cells competent for MV glycoprotein-mediated fusion. Markedly different fusion specificity was observed for another morbillivirus, canine distemper virus (CDV): recombinant CDV glycoproteins promoted fusion with primate and nonprimate cells independently of CD46. Fusion by the recombinant MV and CDV glycoproteins required coexpression of H plus F in either homologous or heterologous combinations. To assess the role of H versus F in determining the CD46 dependence of MV fusion, we examined the fusion specificities of cells producing heterologous glycoprotein combinations. The specificity of HMV plus FCDV paralleled that observed for the homologous MV glycoproteins: fusion occurred with primate cells but not with nonprimate cells unless they produced recombinant CD46. By contrast, the specificity of HCDV plus FMV paralleled that for the homologous CDV glycoproteins: fusion occurred with either primate or nonprimate cells with no dependence on CD46. Thus, for both MV and CDV, fusion specificity was determined by H. In particular, the results demonstrate a functional interaction between HMV and CD46. Flow cytometry and antibody coprecipitation studies provided a structural correlate to this functional interaction: CD46 formed a molecular complex with HMV but not with FMV or with either CDV glycoprotein. These results highlight the critical role of the H glycoprotein in determining MV specificity for CD46-positive cells.     

Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus.

The organization of DNA within the HSV-1 capsid has been determined by cryoelectron microscopy and image reconstruction. Purified C-capsids, which are fully packaged, were compared with A-capsids, which are empty. Unlike A-capsids, C-capsids show fine striations and punctate arrays with a spacing of approximately 2.6 nm. The packaged DNA forms a uniformly dense ball, extending radially as far as the inner surface of the icosahedral (T = 16) capsid shell, whose structure is essentially identical in A-capsids and C-capsids. Thus we find no evidence for the inner T = 4 shell previously reported by Schrag et al. to be present in C-capsids. Encapsidated HSV-1 DNA closely resembles that previously visualized in bacteriophages T4 and lambda, thus supporting the idea of a close parallelism between the respective assembly pathways of a major family of animal viruses (the herpesviruses) and a major family of bacterial viruses.     

Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.

Packaging of retroviral RNA is attained through the specific recognition of a cis-acting encapsidation site (located near the 5' end of the viral RNA) by components of the Gag precursor protein. Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are two lentiviruses that lack apparent sequence similarity in their putative encapsidation regions. We used SIV vectors to determine whether HIV-1 particles can recognize the SIV encapsidation site and functionally propagate SIV nucleic acid. SIV nucleic acid was replicated by HIV-1 proteins. Thus, efficient lentivirus pseudotyping can take place at the RNA level. Direct examination of the RNA contents of virus particles indicated that encapsidation of this heterologous RNA is efficient. Characterization of deletion mutants in the untranslated leader region of SIV RNA indicates that only a very short region at the 5' end of the SIV RNA is needed for packaging. Comparison of this region with the corresponding region of HIV-1 reveals that both are marked by secondary structures that are likely to be similar. Thus, it is likely that a similar higher-order RNA structure is required for encapsidation.     

Heteroduplex study of the sequence relations between RD-114 and baboon viral RNAs.

The regions of sequence homology and nonhomology between the RNA genomes of RD-114 and baboon endogenous type C viruses have been mapped by an electron microscope heteroduplex study. Short complementary DNA (cDNA) copies (approximately 150 to 200 nucleotides in length) of RD-114 RNA were prepared by an endogenous synthesis; labels of polydeoxythymidylic acid [poly(dT)] were attached to the 3' ends of the cDNA molecules by a reaction catalyzed by deoxynucleotidyl terminal transferase. The cDNA-poly(dT) was hybridized to RD-114 RNA and to baboon viral RNA dimer (50 to 70S) units, and the position- of the poly(dT) labels were observed by electron microscopy. With RD-114, labels were distributed uniformly along the genome. With baboon virus RNA (monomer length, 9.5 kilobases [kb]), the regions of high homology with RD-114 cDNA were observed to lie in the intervals from 1.5 to 2.5 kb and from 3.7 to 5.5 kb from the 5' end. The relations of these heteroduplex maps to the known antigenic similarities and differences among the several viral proteins and to the genetic maps of the viruses are discussed.     

Development of antibody to measles virus polypeptides during complicated and uncomplicated measles virus infections.

Immune precipitation of 181 sera from 152 patients with natural measles was studied to determine the temporal course and frequency of antibody responses to nucleocapsid, fusion, hemagglutinin, and matrix proteins of measles virus. Large amounts of antibody to nucleocapsid protein developed in all patients by day one of the rash. Antibody to hemagglutinin and fusion proteins developed in all patients over the next 3 weeks, the former to high levels and the latter to low levels. Antibody to matrix protein developed to very low levels and was detectable in only 41% of the patients; this poor response to matrix protein was not correlated with the age of the patient or the acute neurological complications of measles.     

An examination of the electrostatic interactions between the N-terminal tail of the Brome Mosaic Virus coat protein and encapsidated RNAs

The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ～900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.     

Analysis of nucleotide sequence of wheat yellow mosaic virus genomic RNAs

Wheat yellow mosaic virus (WYMV) isolate HC was used for viral cDNA synthesis and sequencing. The results show that the viral RNA1 is 7629 nueleotides encoding a polyprotein with 2407 amino acids, from which seven putative proteins may be produced by an autolytie cleavage processing besides the viral coat protein. The RNA2 is 3639 nueleotides and codes for a polypretein of 903 amino acids, which may contain two putative non-structural proteins. Although WYMV shares a similarity in genetic organization to wheat spindle streak mosaic virus (WSSMV), the identities in their nucleotide sequences or deduced amino acid sequences are as low as 70% and 75 % respectively. Based on this result, it is confirmed that WYMV and WSSMV are different species within Bymovirus.     

Interactions between brome mosaic virus RNAs and cytoplasmic processing bodies

Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.     

In situ structural organization of encapsidated and unencapsidated Ectromelia virus DNA

The structural organization of Ectromelia virus DNA in infected mouse liver cells has been studied by using thin sections stained with the Feulgen-like osmium-ammine reaction. We found that in the cytoplasmic factories, free viral DNA was structured into completely extended filaments 2-3 nm thick. Viral DNA in immature virions, however, appeared to have a structural organization that superimposed that of eukaryotic chromatin. This was constituted by roundish subunits, with a diameter of 11-13 nm, composed of a DNA ring encircling an unstained inner core. The mature virion was composed of the same type of subunits, which were arranged in threads twisted into a figure 8 configuration. The distribution of basic proteins was also investigated with the acrolein silver-methenamine technique. In the viral particles only nucleoids were stained; a uniformly distributed positive reaction was observed in the cytoplasmic factories.     

Isolation and complete nucleotide sequence of the measles virus IMB-1 strain in China

The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%–5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.