Isolation and Characterization of a Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom Chaetoceros socialis

Diatoms are very significant primary producers in the world''s oceans. Various environmental factors affect the depletion of diatom populations. The importance of viruses as a potential mortality source has recently been recognized. We isolated and characterized a new diatom virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) causing the lysis of the bloom-forming species Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko. The virus infectious to C. socialis f. radians was isolated from water samples collected in Hiroshima Bay. Here we show the physiology, morphology, and genome characteristics of the virus clone. Virions were 22 nm in diameter and accumulated in the cytoplasm of the host cells. The latent period and the burst size were estimated to be <48 h and 66 infectious units per host cell, respectively. CsfrRNAV harbors a single-stranded RNA (ssRNA) genome and encodes at least three polypeptides of 32.0, 28.5, and 25.0 kDa. Sequencing analysis shows the length of the genome is 9,467 bases, excluding a poly(A) tail. The monophyly of CsfrRNAV and other diatom-infecting RNA viruses, Rhizosolenia setigera RNA virus and Chaetoceros tenuissimus RNA virus, was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA-dependent RNA polymerase domains. This suggested a new ssRNA virus family, Bacillariornaviridae. This discovery of CsfrRNAV may aid in further understanding the ecological dynamics of the C. socialis f. radians population in nature and the relationships between ssRNA diatom viruses and their hosts.Diatoms (Bacillariophyceae) account for a large part of the marine primary production, up to 35% in oligotrophic oceans and 75% in nutrient-rich systems (13). They play an important role in various marine systems as a food source for zooplankton and animal larvae. Moreover, diatoms are the primary oxygen producers for the atmosphere (25). Therefore, to understand diatom dynamics in nature is significant for biogeochemical science and fisheries studies. Phytoplankton population dynamics are the result of reproduction and losses. Losses include grazing, sinking, and natural mortality. Since the early 1990s, the importance of viruses infectious to microalgae is recognized as one of the principal causes of phytoplankton mortality. The direct evidence for the existence of diatom viruses was reported recently in 2004 (11). Since the discovery of the first diatom virus, the isolation and characterization of new viruses have been conducted. As a result, several new diatom viruses infecting ecologically important diatom members have been successfully isolated and reported.The first diatom virus, Rhizosolenia setigera RNA virus (RsRNAV), is a small icosahedral virus (32 nm) with a single-stranded RNA (ssRNA) genome at 8,877 nucleotides (nt), excluding a poly(A) tail (11, 15). Thereafter, two Chaetoceros-infecting single-stranded DNA (ssDNA) viruses were isolated and characterized: Chaetoceros salsugineum nuclear inclusion virus (CsNIV), a small (38-nm) virus harboring a covalently closed circular ssDNA (6,000 nt) and a segment of linear ssDNA (997 nt) (12) (H. Mizumoto, unpublished data), and Chaetoceros debilis DNA virus, whose partial genome sequence is highly similar to that of CsNIV (22). The genome analyses of the two ssDNA viruses showed that they are distinctive from previously reported viruses. The isolation of Chaetoceros nuclear inclusion virus (CspNIV) infectious to Chaetoceros cf. gracilis (a Chaetoceros sp. that looks like Chaetoceros gracilis) was also reported (1); however, its nucleic acid type is still unknown. A recent study reports the isolation of the second ssRNA diatom virus infectious to Chaetoceros tenuissimus (CtenRNAV). A phylogenetic analysis showed a putative RNA-dependent RNA polymerase (RdRp) domain from a genome sequence of CtenRNAV is highly similar to RsRNAV but less similar to other marine stramenopile organism viruses (16): Schizochytrium single-stranded RNA virus (SssRNAV) infecting a fungoid protist Aurantiochytrium sp. (formerly Schizochytrium sp.) (19) and Heterosigma akashiwo RNA virus (HaRNAV; Marnaviridae) infecting the bloom-forming raphidophyte H. akashiwo (7, 8). The ssRNA diatom viruses are unlike other known viruses at the family level. These reports suggest that the diatom viruses are an exclusively unique group distinct from previously described viruses where further study of diatom virus biology is significant to understand diatom ecology.Here we report the isolation and characterization of a new ssRNA virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) infecting Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko, one of the dominant phytoplankton species in the marine environments in especially productive areas during spring blooms; e.g., in the North Water polynya, the maximum concentration of C. socialis was as high as 3.0 × 10⁴ cells ml⁻¹ (2). Here, we also propose a new ssRNA virus family (Bacillariornaviridae), composed of three diatom-infecting ssRNA viruses based on phylogenetic analysis using the RdRp domain and other genomic characters.     

Dinoflagellates,diatoms, and their viruses

Since the first discovery of the very high virus abundance in marine environments, a number of researchers were fascinated with the world of "marine viruses", which had previously been mostly overlooked in studies on marine ecosystems. In the present paper, the possible role of viruses infecting marine eukaryotic microalgae is enlightened, especially summarizing the most up-to-the-minute information of marine viruses infecting bloom-forming dinoflagellates and diatoms. To author's knowledge, approximately 40 viruses infecting marine eukaryotic algae have been isolated and characterized to different extents. Among them, a double-stranded DNA (dsDNA) virus "HcV" and a single-stranded RNA (ssRNA) virus "HcRNAV" are the only dinoflagellate-infecting (lytic) viruses that were made into culture; their hosts are a bivalve-killing dinoflagellate Heterocapsa circularisquama. In this article, ecological relationship between H. circularisquama and its viruses is focused. On the other hand, several diatom-infecting viruses were recently isolated and partially characterized; among them, one is infectious to a pen-shaped bloom-forming diatom species Rhizosolenia setigera; some viruses are infectious to genus Chaetoceros which is one of the most abundant and diverse diatom group. Although the ecological relationships between diatoms and their viruses have not been sufficiently elucidated, viral infection is considered to be one of the significant factors affecting dynamics of diatoms in nature. Besides, both the dinoflagellate-infecting viruses and diatom-infecting viruses are so unique from the viewpoint of virus taxonomy; they are remarkably different from any other viruses ever reported. Studies on these viruses lead to an idea that ocean may be a treasury of novel viruses equipped with fascinating functions and ecological roles.     

Isolation and characterization of a novel single-stranded RNA Virus infectious to a marine fungoid protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea)

Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decomposers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems. SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist Schizochytrium sp. (Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000, and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and it was also concentrated along the intracellular membrane structures. By means of one-step growth experiments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 x 10(3) to 6.4 x 10(4) infectious units per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was considered to have some similarities with invertebrate viruses belonging to the family Dicistroviridae based on its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these organisms.     

The use of a complementary DNA probe to detect accumulation of mengo RNA in infected cells pretreated with interferon.

Complementary DNA (cDNA) from Mengo virus RNA has been synthesized and used as a probe to measure the synthesis and accumulation of viral RNA in Mengo infected L cell cultures, treated or untreated with interferon. Under experimental conditions used (200 units interferon/ml and 50 virus plaque-forming units/cell) results show that there is some synthesis of Mengo virus RNA in cells treated with interferon. One hour after infection, treated cells contain three times less viral RNA than untreated cells; five hours after infection, this difference has increased to ten fold. As in the control, no fragmented Mengo virus RNA molecules were found in interferon treated cells. The smaller recovery of infectious particles from interferon treated cells as compared to RNA accumulation suggests that not only RNA accumulation is inhibited but also a step posterior in viral maturation.     

The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and Primer-Independent Polymerases

Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5’-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different substrates. The enzyme arranges as a stable dimer maintained by mutual interactions between the active site cleft of one molecule and the flexible N-terminal tail of the symmetrically related RdRP. The latter, partially mimicking the RNA template backbone, is involved in regulating the polymerization activity. As expected from previous sequence-based bioinformatics predictions, the overall architecture of the TaV enzyme shows important resemblances with birnavirus polymerases. In addition, structural comparisons and biochemical analyses reveal unexpected similarities between the TaV RdRP and those of Flaviviruses. In particular, a long loop protruding from the thumb domain towards the central enzyme cavity appears to act as a platform for de novo initiation of RNA replication. Our findings strongly suggest an unexpected evolutionary relationship between the RdRPs encoded by these distant ssRNA virus groups.     

The impact of local assembly rules on RNA packaging in a T = 1 satellite plant virus

The vast majority of viruses consist of a nucleic acid surrounded by a protective icosahedral protein shell called the capsid. During viral infection of a host cell, the timing and efficiency of the assembly process is important for ensuring the production of infectious new progeny virus particles. In the class of single-stranded RNA (ssRNA) viruses, the assembly of the capsid takes place in tandem with packaging of the ssRNA genome in a highly cooperative co-assembly process. In simple ssRNA viruses such as the bacteriophage MS2 and small RNA plant viruses such as STNV, this cooperative process results from multiple interactions between the protein shell and sites in the RNA genome which have been termed packaging signals. Using a stochastic assembly algorithm which includes cooperative interactions between the protein shell and packaging signals in the RNA genome, we demonstrate that highly efficient assembly of STNV capsids arises from a set of simple local rules. Altering the local assembly rules results in different nucleation scenarios with varying assembly efficiencies, which in some cases depend strongly on interactions with RNA packaging signals. Our results provide a potential simple explanation based on local assembly rules for the ability of some ssRNA viruses to spontaneously assemble around charged polymers and other non-viral RNAs in vitro.     

Isolation and characterization of a novel single-stranded RNA virus infecting the bloom-forming diatom Rhizosolenia setigera

A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15 degrees C under a 12-h-12-h light-dark cycle of ca. 110 micro mol of photons m(-2) s(-1) with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.     

Isolation and Characterization of a Novel Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom Rhizosolenia setigera

A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15°C under a 12-h-12-h light-dark cycle of ca. 110 μmol of photons m⁻² s⁻¹ with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.     

The introduction of poliovirus RNA into cells via lipid vesicles (liposomes).

Large unilamellar vesicles (LUV) composed of phosphatidylserine are capable of encapsulating poliovirus ribonucleic acid (RNA) and delivering it efficiently to cells in an infectious form. The biological activity of vesicle-entrapped poliovirus RNA was 1-2 x 10(4) plaque forming units/nanogram (pfu/ng) and appeared to be enhanced by ribonuclease treatment of the vesicle preparations (infectivity = 1-2 x 10(5) pfu/ng). Vesicle-mediated RNA infection produced equivalent titers in primate and nonprimate cells. Moreover, the data strongly suggest that the ratio of molecules per infectious unit is close to one when the RNA is properly delivered to the cell. A comparative study of LUV and multilamellar vesicles (MLV) indicates that LUV deliver their contents to the cell cytoplasm much more efficiently than MLV. LUV-entrapped poliovirus RNA produced infectious titer 10-100 fold higher than comparable RNA preparations delivered to cells by other techniques.     

Deletion and mutational analyses of bluetongue virus NS2 protein indicate that the amino but not the carboxy terminus of the protein is critical for RNA-protein interactions.

Genome segment 8 (S8) of bluetongue virus serotype 10 (BTV-10) encodes the nonstructural protein NS2. This protein, which has single-stranded RNA (ssRNA) binding capacity, is found in BTV-infected cells in the form of virus inclusion bodies (VIBs). To identify the domain(s) important for RNA binding and oligomerization of the protein, a number of deletions were made in regions of the gene that code for either the amino or carboxy terminus of the protein. The modified genes were cloned into and expressed from baculovirus vectors based on Autographa californica nuclear polyhedrosis virus. Truncated NS2 proteins were individually analyzed for the ability to bind ssRNA and to form VIBs. The results indicated that the carboxy terminus of the protein is involved neither in RNA binding nor in the formation of VIBs. The amino terminus of NS2 was shown to be essential for ssRNA binding but not for NS2 protein oligomerization. Point mutations that involved the substitution of various charged residues at the amino terminus of NS2 were generated and tested for the ability to bind ssRNA. The results showed that the arginines at amino acid residues 6 and 7 and the lysine at residue 4, but not the glutamic acid at residue 2, are involved in ssRNA binding.     

Sequence specificity in the interaction of Bluetongue virus non-structural protein 2 (NS2) with viral RNA

The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.     

Virion nucleic acid of Ebola virus.

The virion nucleic acid of Ebola virus consists of a single-stranded RNA with a molecular weight of approximately 4.0 x 10(6). The virion RNA did not bind to oligodeoxythymidylic acid-cellulose under conditions known to bind RNAs rich in polyadenylic acid and was not infectious under conditions which yielded infectious RNA from Sindbis virus, suggesting that Ebola virus virion nucleic acid is a negative-stranded RNA.     

Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

Binding of APOBEC3G to the nucleocapsid (NC) domain of the human immunodeficiency virus (HIV) Gag polyprotein may represent a critical early step in the selective packaging of this antiretroviral factor into HIV virions. Previously, we and others have reported that this interaction is mediated by RNA. Here, we demonstrate that RNA binding by APOBEC3G is key for initiation of APOBEC3G:NC complex formation in vitro. By adding back nucleic acids to purified, RNase-treated APOBEC3G and NC protein preparations in vitro, we demonstrate that complex formation is rescued by short (> or =10 nucleotides) single-stranded RNAs (ssRNAs) containing G residues. In contrast, complex formation is not induced by add-back of short ssRNAs lacking G, by dsRNAs, by ssDNAs, by dsDNAs or by DNA:RNA hybrid molecules. While some highly structured RNA molecules, i.e., tRNAs and rRNAs, failed to rescue APOBEC3G:NC complex formation, other structured RNAs, i.e., human Y RNAs and 7SL RNA, did promote NC binding by APOBEC3G. Together, these results indicate that ternary complex formation requires ssRNA, but suggest this can be presented in the context of an otherwise highly structured RNA molecule. Given previous data arguing that APOBEC3G binds, and edits, ssDNA effectively in vitro, these data may also suggest that APOBEC3G can exist in two different conformational states, with different activities, depending on whether it is bound to ssRNA or ssDNA.     

Infectious Rous Sarcoma Virus and Reticuloendotheliosis Virus DNAs

An efficient and quantitative assay for infectious Rous sarcoma virus and reticuloendotheliosis virus DNAs is described. The specific infectivities of viral DNA corresponded to one infectious unit per 10(5) to 10(6) viral DNA molecules. Infection with viral DNA followed one-hit kinetics. The minimal size of infectious Rous sarcoma virus DNA was approximately 6 million daltons, whereas the minimal size of infectious reticuloendotheliosis virus DNA was larger, 10 to 20 million daltons.     

The separation between the 5′-3′ ends in long RNA molecules is short and nearly constant

RNA molecules play different roles in coding, decoding and gene expression regulation. Such roles are often associated to the RNA secondary or tertiary structures. The folding dynamics lead to multiple secondary structures of long RNA molecules, since an RNA molecule might fold into multiple distinct native states. Despite an ensemble of different structures, it has been theoretically proposed that the separation between the 5′ and 3′ ends of long single-stranded RNA molecules (ssRNA) remains constant, independent of their base content and length. Here, we present the first experimental measurements of the end-to-end separation in long ssRNA molecules. To determine this separation, we use single molecule Fluorescence Resonance Energy Transfer of fluorescently end-labeled ssRNA molecules ranging from 500 to 5500 nucleotides in length, obtained from two viruses and a fungus. We found that the end-to-end separation is indeed short, within 5–9 nm. It is remarkable that the separation of the ends of all RNA molecules studied remains small and similar, despite the origin, length and differences in their secondary structure. This implies that the ssRNA molecules are ‘effectively circularized’ something that might be a general feature of RNAs, and could result in fine-tuning for translation and gene expression regulation.     

Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA

Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins.     

Fluorescent Cell Counting as an Assay Method for Respiratory Syncytial Virus

The fluorescent cell-counting technique was applied to the enumeration of cell-infecting units of respiratory syncytial (RS) virus in human fetal diploid (HFD) cover-slip cell cultures; it was a sensitive, precise, and rapid assay method. Approximately 2 hr was required for maximal adsorption of RS virus to HFD cell monolayers. However, about 15% of the infectious virus in the inoculum remained unadsorbed; this percentage was not significantly reduced even when the adsorption period was extended to 5 hr. A linear relationship between virus concentration and the number of fluorescent cells existed over a range of 1.2 log(10) units. Variation of the mean of replicate determinations in a single experiment was approximately 7.5%. The distribution of single infected HFD cells on cover-slip cell cultures corresponded with the calculated frequencies of the Poisson distribution. The Chi square test for the extent of fit was calculated for several experiments, and the value of P was never less than 0.5. The addition of immune serum after virus adsorption effectively inhibited the development of detectable levels of viral antigen in secondarily infected cells.     

The Cytoplasmic Tail Domain of Influenza B Virus Hemagglutinin Is Important for Its Incorporation into Virions but Is Not Essential for Virus Replication in Cell Culture in the Presence of Compensatory Mutations

Influenza B virus hemagglutinin (BHA) contains a predicted cytoplasmic tail of 10 amino acids that are highly conserved among influenza B viruses. To understand the role of this cytoplasmic tail in infectious virus production, we used reverse genetics to generate a recombinant influenza B virus lacking the BHA cytoplasmic tail domain. The resulting virus, designated BHATail⁻, had a titer approximately 5 log units lower than that of wild-type virus but grew normally when BHA was supplemented in trans by BHA-expressing cells. Although the levels of BHA cell surface expression were indistinguishable between truncated and wild-type BHA, the BHATail⁻ virus produced particles containing dramatically less BHA. Moreover, removal of the cytoplasmic tail abrogated the association of BHA with Triton X-100-insoluble lipid rafts. Interestingly, long-term culture of a virus lacking the BHA cytoplasmic tail in Madin-Darby canine kidney (MDCK) cells yielded a mutant with infectivities somewhat similar to that of wild-type virus. Sequencing revealed that the mutant virus retained the original cytoplasmic tail deletion but acquired additional mutations in its BHA, neuraminidase (NA), and M1 proteins. Viral growth kinetic analysis showed that replication of BHA cytoplasmic tailless viruses could be improved by compensatory mutations in the NA and M1 proteins. These findings indicate that the cytoplasmic tail domain of BHA is important for efficient incorporation of BHA into virions and tight lipid raft association. They also demonstrate that the domain is not absolutely required for virus viability in cell culture in the presence of compensatory mutations.     

Presence of free viral DNA in simian virus 40-transformed nonproducer cells.

Extracts from several simian virus 40 (SV40)-transformed nonproducer cells were prepared by the hot-phenol procedure normally used to extract cellular RNA. These extracts contained SV40 infectious units. Part of the infectious units were identified as SV40 form I DNA molecules. The results of reconstruction experiments suggest that SV40 form I DNA is extractable by the hot-phenol procedure because of its fast renaturation rate. The significance of the presence of free viral DNA in nonproducer transformed cells is discussed.