Cucumber mosaic virus mutants with altered physical properties and defective in aphid vector transmission

Two mutant strains of cucumber mosaic virus (CMV) were investigated with respect to virion stability and molecular determinants of aphid vector transmission. The mutant 2A1-MT-60x, derived from the mechanically passaged wild type 2A1-AT, is poorly transmissible by the aphid Aphis gossypii and not transmissible by the aphid Myzus persicae, whereas the wild type virus is transmissible by both aphid species. The mutant phenotype was shown to be conferred by a single encoded amino acid change of alanine to threonine at position 162 of the coat protein (CP). Modifying the mutant CP gene to encode the wild type sequence (alanine) at position 162 restored aphid transmission. To test for a correspondence between changes in the physical stability of virions and defects in aphid transmission, a urea disruption assay was developed. Virions of aphid-transmissible strains 2A1-AT and CMV-Fny were stable with treatments of up to between 3 and 4 M urea. In this assay mutant viruses 2A1-MT-60x and CMV-M were less stable, as they were completely disrupted at urea concentrations of 2 and 1 M urea, respectively. The mutant 2A1-MT-60x also accumulated at a reduced level in infected squash relative to the wild type virus. These studies suggest that a primary factor in the loss of aphid transmissibility of some strains of CMV is a reduction in virion stability.     

One more probable structural transition in potato virus X virions and a revised model of the virus coat protein structure

We found that a 2-h incubation of potato virus X (PVX) virions in 10 mM Tris-HCl buffer pH 7.5 at -20 degrees C results in a strong but reversible drop in virion stability. Under these conditions, the PVX virions are completely disrupted by low (starting from 50 mM) concentrations of LiCl and CaCl(2) but not of NaCl. Incubation of PVX samples with 0.05-2 M LiCl at +4 degrees C did not result in virion disassembly and the virions were not disrupted upon incubation at -20 degrees C in 10 mM Tris-HCl buffer pH 7.5 without LiCl. We suggest that a 2-h incubation of the PVX virions at -20 degrees C in 10 mM Tris-HCl pH 7.5 results in a structural transition in the virus particles. A revised model of the three-dimensional organization of coat protein subunits in the PVX virions is proposed. This two-domain model explains better the high plasticity of the PVX CP structure.     

The readthrough domain of pea enation mosaic virus coat protein is not essential for virus stability in the hemolymph of the pea aphid

A fraction of the coat protein (CP) subunits in virions of members of the family Luteoviridae contain a C-terminal extension called the readthrough domain (RTD). The RTD is necessary for persistent aphid transmission, but its role is unknown. It has been reported to be required for virion stability in the hemolymph. Here, we tested whether this was the case for pea enation mosaic virus (PEMV) virions in the pea aphid (Acyrthosiphon pisum) using RNA1Δ, a natural deletion mutant lacking the middle portion of the RTD ORF, and CPΔRTD, in which the entire RTD ORF was deleted. In infected plants, RNA1Δ virions were as abundant and stable as wild-type (WT) virions, while CPΔRTD virions were unstable. No RTD of any size was translated from artificial subgenomic mRNA of CPΔRTD or RNA1Δ in vitro. Thus, only the major CP was present in the mutant virions. Using real-time RT-PCR to detect virion RNA, no significant differences in the concentration or stability of WT and RNA1Δ virions were detected in the aphid hemolymph at much longer times than are necessary for virus transmission. Thus, the RTD is not necessary for stability of PEMV RNA in the aphid hemolymph, and it must play another role in aphid transmission.     

Human parainfluenza virus 3: Purification and characterization of subviral components,viral proteins and viral RNA

A simple method was established that allowed large quantities of human parainfluenza 3 (PF3) virions to be isolated from tissue culture cells. The purity of the virus was sufficient for biochemical analysis of virion proteins. The density of PF3 virions was 1.18–1.20. Purified virions contained seven viral proteins with estimated molecular weights of: L, 180000; P, 83000; HN, 69000; NP, 66000; f₀, 60000; F₁, 51000; and M, 38000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. There were three phosphoproteins, P, NP and M, and two glycoproteins, HN and F (includes f₀ and f₁). F_1,2, the activated, cleaved, fusion glycoprotein (60000 Da), consisting of two disulfide-linked subunits, f₁ and F₂, was seen only under nonreducing conditions. Because of its small size (approximately 9000 Da) F₂ could be seen only on gels with high acrylamide concentrations. As in other enveloped viruses, cellular actin (43 000 Da) was present in purified virions. Several minor bands migrating between NP and M represented breakdown products of NP.Solubilization of the virion membrane in low salt buffer with non-ionic detergent resulted in the loss of HN and F. In high salt buffer, the M protein was also removed. Nucleocapsids isolated by CsCl centrifugation contained L, P, NP and small amounts of M. Nucleocapsids isolated in the presence of the ionic detergent, sarcosyl, contained only the NP protein. The density of nucleocapsids was 1.29–1.30. Genomic 50S RNA isolated from nucleocapsids had an estimated molecular weight of 5 × 10⁶.     

Temperature-induced structural stabilization of the encapsidated RNA of southern bean mosaic virus and its biologic significance

Virions of southern bean mosaic virus (SBMV) show two distinct sensitivity patterns upon heating. Infectivity loss and capsid denaturation occur concurrently if virions are exposed at 50-55 degrees in 0.1 M sodium phosphate buffer, pH 7.5, or 0.1 M glycine-phosphate buffer, pH 9.0. Contrastingly, virions are inactivated without any detectable capsid damage at 65-70 degrees in 0.1 M sodium phosphate buffer, pH 6.0, 0.1 M Tris-HCI buffer, pH 7.5, or 0.1 M glycine-NaOH buffer, pH 9.0. Heat treatment causes no genomic degradation in these two situations; the divergent sensitivity of virions is due, apparently, to a differential thermal tolerance of the capsid protein to the buffer components and/or pH. SBMV-RNA isolated from virions inactivated at 65 degrees in 0.1 M Tris-HCl buffer, pH 7.5, possesses low infectivity (1-2%). Observations based upon sucrose gradient sedimentation, temperature: absorbance relationship, and sensitivity to ribonuclease T(1) suggest that such RNA is structurally more compact and stable relative to that of the RNA from the nonheated virions. Neither the capsid protein nor the genome protein plays a direct role in the temperature-induced structural stabilization of SBMV-RNA in situ. If treated with 8 M urea, 50% formamide or exposed at 55 degrees , the infectivity of RNA from the heat-inactivated SBMV is restored and is comparable to that of the RNA isolated from the nonheated virions. The observed mode of SBMV thermal inactivation, i.e., stabilization of RNA structure in situ, is unique among the viruses. Furthermore, these results suggest that the ability to initiate infection depends upon the secondary structure of the SBMV genome.     

Generation and mucosal transmissibility of emtricitabine- and tenofovir-resistant SHIV162P3 mutants in macaques

Transmission of drug-resistant HIV has been widely documented. We generated tenofovir (TFV)- and emtricitabine (FTC)-resistant SHIV162P3 mutants that can be used to investigate the transmission efficiency of drug-resistant viruses and their impact on the efficacy of pre-exposure prophylaxis. Both SHIV162p3_M184V and SHIV162p3_K65R replicated in vitro at high titers. Drug resistance profiles were similar to those seen in HIV. Virus infectivity to virion particle ratios were 4- and 10-fold lower in SHIV162p3_M184V and SHIV162p3_K65R, compared to a concurrently generated WT SHIV162p3, respectively. Mucosal transmissibility studies using a repeat low-dose macaque model of rectal and vaginal transmission showed that both mutants were able to efficiently infect macaques only after the dose was increased to adjust for fitness reductions due to K65R and M184V. Our results in limited number of macaques suggest that the reduction in fitness due to M184V and K65R decreases virus transmissibility, and identify in vitro infectivity parameters that associate with mucosal transmissibility.     

Rack-1, GAPDH3, and actin: proteins of Myzus persicae potentially involved in the transcytosis of beet western yellows virus particles in the aphid

Beet western yellows virus (BWYV) is a Polerovirus that relies on the aphid Myzus persicae for its transmission, in a persistent-circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary glands (ASG) epithelial barriers in a transcytosis mechanism where vector receptors interact with virions. In this paper, we report in vitro interaction experiments between BWYV and aphid components. Using the M. persicae clone from Colmar, we showed that a set of aphid polypeptides, separated by SDS-PAGE or 2D electrophoresis (2DE), can bind in vitro to purified wild type or mutant particles. Using subcellular fractionation, we showed that the 65-kDa polypeptide identified as symbionin is a soluble protein whereas the other polypeptides seem to be associated more or less strongly to the membrane. We hypothesize that three polypeptides, identified by mass spectrometry as Rack-1, GAPDH3, and actin, may be involved in the epithelial transcytosis of virus particles in the aphid vector.     

In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses

Summary.  The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding. Received July 30, 1998 Accepted November 23, 1998     

Equine herpesvirus 1 (EHV-1) glycoprotein M: effect of deletions of transmembrane domains

Equine herpesvirus 1 (EHV-1) recombinants that carry either a deletion of glycoprotein M (gM) or express mutant forms of gM were constructed. The recombinants were derived from strain Kentucky A (KyA), which also lacks genes encoding gE and gI. Plaques on RK13 cells induced by the gM-negative KyA were reduced in size by 80%, but plaque sizes were restored to wild-type levels on gM-expressing cells. Electron microscopic studies revealed a massive defect in virus release after the deletion of gM in the gE- and gI-negative KyA, which was caused by a block in secondary envelopment of virions at Golgi vesicles. Recombinant KyA expressing mutant gM with deletions of predicted transmembrane domains was generated and characterized. It was shown that mutant gM was expressed and formed dimeric and oligomeric structures. However, subcellular localization of mutant gM proteins differed from that of wild-type gM. Mutant glycoproteins were not transported to the Golgi network and consequently were not incorporated into the envelope of extracellular virions. Also, a small plaque phenotype of mutant viruses that was indistinguishable from that of the gM-negative KyA was observed. Plaque sizes of mutant viruses were restored to wild-type levels by plating onto RK13 cells constitutively expressing full-length EHV-1 gM, indicating that mutant proteins did not exert a transdominant negative effect on wild-type gM.     

A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus

A nonaphid transmissible (NAT) variant of tobacco vein mottling virus (TVMV) was used to test the hypothesis that the viral coat protein (CP) plays a role in determining aphid transmissibility. Comparison of the nucleotide sequences in the coat protein cistron of an aphid transmissible isolate (TVMV-AT) with that of TVMV-NAT revealed a single nucleotide difference (G----A) at position 8445; this alters a single amino acid residue (G----E) at position 2747. A cDNA fragment representing the CP region of TVMV-NAT was substituted into the CP region of a full-length cDNA clone of TVMV-AT, and transcribed RNA was inoculated to tobacco plants. Aphids were unable to transmit the resultant hybrid virus which had the TVMV-NAT coat protein, although the concentration and infectivity of the hybrid virus in the source plants were similar to those of TVMV-AT. This is the first direct demonstration that a CP mutation affects aphid transmissibility of a potyvirus.     

Amino acid substitutions within the Cys-rich domain of the tobacco etch potyvirus HC-Pro result in loss of transmissibility by aphids

Summary.  We examined the role of several amino acid residues located at the N-terminus of the tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro) in virus transmissibility by aphids. Site-directed mutagenesis resulted in changes affecting amino acids that appear highly conserved among a number of potyviruses. The TEV HC-Pro amino acid residues Gly343, Val345, Ala346, Ile348, Pro355, Lys358, and Ile359 were arranged within a Cys-rich domain in a region dispensable for TEV infectivity. Two HC-Pro mutants (TEV-P355R and -K358N) exhibited a drastically reduced rate of aphid transmission whereas other mutants (TEV-G343D, -V345E, -A346H, -I348D, and -P355L) were completely unable to be aphid transmitted. In contrast, the I359M mutation had no effect on aphid transmissibility of TEV. This lack of transmissibility did not appear to be due to large differences in the amounts of both coat protein (CP) and HC-Pro in infected tobacco plants. Our results indicated that these amino acid residues likely play a highly conserved role in aphid transmission among potyviruses. Present address: Center for Gene Research and Biotechnology, and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, U.S.A. Received May 5, 2001; accepted June 26, 2002     

Potyvirus aphid transmission requires helper component and homologous coat protein for maximal efficiency

Summary.  Aphid transmission of potyviruses depends on the presence of specific sequence domains in two virus encoded proteins, the coat protein (CP) and helper component-proteinase (HC-Pro). Aphid transmissable peanut stripe virus (PStV), like most potyviruses, has an Asp-Ala-Gly (DAG) motif in the amino-terminal part of the CP. Peanut Mottle Virus (PeMoV) was determined to be highly aphid transmissible but has a unique Asp-Ala-Ala-Ala (DAAA) motif. To determine if the DAAA motif could functionally replace the DAG motif in PStV, mutations were made in a full-length cDNA clone of PStV. All of the mutations in the CP DAG motif abolished aphid transmissibility of PStV but did not affect virus infectivity. The aphid transmissibility of the PStV-DAAA mutant was partially restored by feeding aphids an artificial diet containing puri- fied virus and PeMoV HC-Pro. The PStV-DAAA virus was poorly transmitted by aphids in vitro with HC-Pro purified from PStV or tobacco vein mottling virus (TVMV) infected plants. These experiments support the theory that specific HC-Pro/CP interactions are required for efficient aphid transmission. Based upon the sequence comparisons of 16 potyviral HC-Pro proteins several conserved motifs and striking differences have been identified. PeMoV was determined to have an Ala-Ser-Cys (ASC) HC-Pro motif instead of a highly conserved Cys-Cys-Cys (CCC) motif. We have predicted that this CCC motif could play an important role in the specific interaction between the HC-Pro and the CP DAG motif. Received January 6, 1997 Accepted June 19, 1998     

Substitutions of conserved amino acids in the receptor-binding domain of the spike glycoprotein affect utilization of murine CEACAM1a by the murine coronavirus MHV-A59

The host range of the murine coronavirus (MHV) is limited to susceptible mice and murine cell lines by interactions of the spike glycoprotein (S) with its receptor, mCEACAM1a. We identified five residues in S (S33, L79, T82, Y162 and K183) that are conserved in the receptor-binding domain of MHV strains, but not in related coronaviruses. We used targeted RNA recombination to generate isogenic viruses that differ from MHV-A59 by amino acid substitutions in S. Viruses with S33R and K183R substitutions had wild type growth, while L79A/T82A viruses formed small plaques. Viruses with S33G, L79M/T82M or K183G substitutions could only be recovered from cells that over-expressed a mutant mCEACAM1a. Viruses with Y162H or Y162Q substitutions were never recovered, while Y162A viruses formed minute plaques. However, viruses with Y162F substitutions had wild type growth, suggesting that Y162 may comprise part of a hydrophobic domain that contacts the MHV-binding site of mCEACAM1a.     

Complete nucleotide sequence and genome organization of a Chinese isolate of <Emphasis Type="Italic">Tobacco vein distorting virus</Emphasis>

Tobacco bushy top disease is caused by tobacco bushy top virus (TBTV, a member of the genus Umbravirus) which is dependent on tobacco vein-distorting virus (TVDV) to act as a helper virus encapsidating TBTV and enabling its transmission by aphids. Isometric virions from diseased tobacco plants were purified and disease symptoms were reproduced after experimental aphid transmission. The complete genome of TVDV was determined from cloned RT-PCR products derived from viral RNA. It was 5,920 nucleotides (nts) long and had the six major open reading frames (ORFs) typical of a member of the genus Polerovirus. Sequence comparisons showed that it differed significantly from any of the other species in the genus and this was confirmed by phylogenetic analyses of the RdRp and coat protein. SDS-PAGE analysis of purified virions gave two protein bands of about 26 and 59 kDa both of which reacted strongly in Western blots with antiserum produced to prokaryotically expressed TVDV CP showing that the two forms of the TVDV CP were the only protein components of the capsid.     

Potentiation of the poliocidal effectiveness of free chlorine by a buffer

Poliovirus 1 was inactivated by free chlorine at pH 9.0 three times more rapidly in boric acid (0.05 M)-NaOH buffer than in purified (carbon-filtered, deionized) water. Thus, at a given concentration of free chlorine, it took three times longer to inactivate the same fraction of the poliovirus in purified water than in the boric acid-NaOH buffer. Conversely, in a given period of time, three times more chlorine was required to inactivate a given percentage of the virus in purified water than in the boric acid-NaOH buffer. Buffers are almost always used to control pH in disinfection studies with free chlorine and with other chlorine compounds also. The use of buffers for pH control in such disinfection studies may distort the resulting data and, at least for waters that contain little salt, may cause serious overestimation of the rates at which viruses are inactivated.     

Cymbidium mosaic potexvirus isolate-dependent host movement systems reveal two movement control determinants and the coat protein is the dominant

Little is known about how plant viruses of a single species exhibit different movement behavior in different host species. Two Cymbidium mosaic potexvirus (CymMV) isolates, M1 and M2, were studied. Both can infect Phalaenopsis orchids, but only M1 can systemically infect Nicotiana benthamiana plants. Protoplast inoculation and whole-mount in situ hybridization revealed that both isolates can replicate in N. benthamiana; however, M2 was restricted to the initially infected cells. Genome shuffling between M1 and M2 revealed that two control modes are involved in CymMV host dependent movement. The M1 coat protein (CP) plays a dominant role in controlling CymMV movement between cells, because all chimeric CymMV viruses containing the M1 CP systemically infected N. benthamiana plants. Without the M1 CP, one chimeric virus containing the combination of the M1 triple gene block proteins (TGBps), the M2 5′ RNA (1-4333), and the M2 CP effectively moved in N. benthamiana plants. Further complementation analysis revealed that M1 TGBp1 and TGBp3 are co-required to complement the movement of the chimeric viruses in N. benthamiana. The amino acids within the CP, TGBp1 and TGBp3 which are required or important for CymMV M2 movement in N. benthamiana plants were mapped. The required amino acids within the CP map to the predicted RNA binding domain. RNA-protein binding assays revealed that M1 CP has higher RNA binding affinity than does M2 CP. Yeast two-hybrid assays to detect all possible interactions of M1 TGBps and CP, and only TGBp1 and CP self-interactions were observed.     

Infection of a restrictive cell line (XC cells) by intratypic recombinants of HSV-1: relationship between penetration of the virus and relative amounts of glycoprotein C

The ability to induce the synthesis of virus polypeptides in nonpermissive XC cells by several strains, variants, and intratypic recombinants of HSV-1 has been investigated. Our results show that (i) whereas HSV-1 strains mP and MP, and recombinants F(MP)A through D (Ruyechan et al., J. Virol. 29, 677-697, 1979) and HFEMtsB5/MP3 (Manservigi et al., Proc. Nat. Acad. Sci. USA 74, 3913-3917, 1977) induced in XC cells the synthesis of several virus polypeptides (the "positive" viruses), in the case of HSV-1 strain F, variants HFEMtsB5 and mPtsHA1 (both tested at 33 degrees), and recombinants F(MP)E and F(MP)F, no virus specific polypeptides could be detected in these cells (the "negative" viruses). (ii) Failure of the "negative" viruses to synthesize polypeptides in XC cells could be explained by failure of the virions to penetrate the cells since polyethylene glycol, a fusion promotor agent, enabled these viruses to overcome the blockage of their expression. (iii) The ability to modulate penetration into XC cells is genetically determined. A locus affecting this function maps between coordinates 0.70 to 0.82 of HSV-1 DNA and is closely linked to Cr, a locus controlling the synthesis or accumulation of virus glycoprotein C (gC) (Ruyechan et al., 1979). (iv) A decrease in the amount of gC, relative to gB, is associated with an enhanced ability to enter XC cells, suggesting that gC may control penetration into these restrictive cells by negatively modulating the gB promoted fusion between host cell and virion membranes.     

The nucleotide sequence and genome organization of Sclerophthora macrospora virus A

Sclerophthora macrospora virus A (SmV A) found in S. macrospora, the pathogenic fungus responsible for downy mildew of gramineous plants, is a small icosahedral virus containing three segments (RNAs 1, 2, and 3) of the positive-strand ssRNA genome. In the present study we report the complete nucleotide sequence of the SmV A genome. The viral genome RNA 1 consists of 2928 nucleotides (nt) and has two open reading frames (ORFs 1a and 1b). ORF 1a contains the motifs of RNA-directed RNA polymerase (RdRp). The function of ORF 1b is unknown. RNA 2 consists of 1981 nt and single ORF (ORF 2). ORF 2 encodes a capsid protein. RNA 3 consists of 977 nt but not any ORFs, suggesting it as a satellite RNA. The deduced amino acid sequence of ORF 1a shows some similarity to those of RdRp of certain positive-strand RNA viruses, especially to the members of the family Nodaviridae, and that of ORF 2 to CP of the members in the family Tombusviridae. The nucleotide sequence of RNA 3 shows a 40-nucleotide length of partial similarity to S. macrospora virus B (SmV B) RNA. The capsid of SmV A is composed of two capsid proteins, CP 1 (p43) and CP 2 (p39), both encoded in ORF 2. CP 2 is apparently derived from CP 1 via proteolytic cleavage at the N-terminus. The genome organization of SmV A is characteristic and distinct from those of other known fungal RNA viruses, including SmV B. These results suggest that SmV A should be classified into a new group of mycoviruses.