West Nile virus envelope protein glycosylation is required for efficient viral transmission by Culex vectors

Many, but not all, strains of West Nile virus (WNV) contain a single N-linked glycosylation site on their envelope (E) proteins. Previous studies have shown that E-glycosylated strains are more neuroinvasive in mice than non-glycosylated strains. E protein glycosylation also appears to play a role in attachment and entry of WNV into host cells in vitro; however, studies examining how E protein glycosylation affects the interactions of WNV with its mosquito vectors in vivo have not yet been performed. We mutated the E protein glycosylation site from NYS to IYS in a previously described full-length clone of the NY99 genotype of WNV (WT), resulting in a virus that lacked the glycan at aa154. WNV-N154I replicated less efficiently than WNV-WT in Culex mosquito tissues, although the extent of the decrease was greater in Cx. pipiens than in Cx. tarsalis. Following peroral infection, mosquitoes infected with WNV-N154I were less likely to transmit virus than those infected with WNV-WT. Interestingly, all but one of the mosquitoes infected with WNV-N154I transmitted a revertant virus, suggesting that there is strong selective pressure toward E protein glycosylation. Together these data suggest that loss of the glycan at aa154 on the WNV E protein can severely restrict viral spread in the mosquito vector.     

The Red clover necrotic mosaic virus origin of assembly is delimited to the RNA-2 trans-activator

The bipartite RNA genome of Red clover necrotic mosaic virus (RCNMV) is encapsidated into icosahedral virions that exist as two populations: i) virions that co-package both genomic RNAs and ii) virions packaging multiple copies of RNA-2. To elucidate the packaging mechanism, we sought to identify the RCNMV origin of assembly sequence (OAS). RCNMV RNA-1 cannot package in the absence of RNA-2 suggesting that it does not contain an independent packaging signal. A 209 nt RNA-2 element expressed from the Tomato bushy stunt virus CP subgenomic promoter is co-assembled with genomic RNA-1 into virions. Deletion mutagenesis delimited the previously characterized 34 nt trans-activator (TA) as the minimal RCNMV OAS. From this study we hypothesize that RNA-1 must be base-paired with RNA-2 at the TA to initiate co-packaging. The addition of viral assembly illustrates the critical importance of the multifunctional TA element as a key regulatory switch in the RCNMV life cycle.     

Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.     

Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2

The sequence of Lettuce chlorosis virus (LCV) (genus Crinivirus) was determined and found to contain unique open reading frames (ORFs) and ORFs similar to those of other criniviruses, as well as 3′ non-coding regions that shared a high degree of identity. Northern blot analysis of RNA extracted from LCV-infected plants identified subgenomic RNAs corresponding to six prominent internal ORFs and detected several novel LCV-single stranded RNA species. Virus replication in tobacco protoplasts was investigated and results indicated that LCV replication proceeded with novel crinivirus RNA accumulation kinetics, wherein viral genomic RNAs exhibited a temporally similar expression pattern early in the infection. This was noticeably distinct from the asynchronous RNA accumulation pattern previously observed for Lettuce infectious yellows virus (LIYV), the type member of the genus, suggesting that replication of the two viruses likely operate via dissimilar mechanisms.     

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.     

Lack of evidence for an interaction between Buchnera GroEL and Banana bunchy top virus (Nanoviridae)

Circulative plant viruses such as luteovirids and geminiviruses have been shown to bind to GroEL proteins produced by endosymbiotic bacteria harboured within hemipteran vectors. These interactions seem to prevent the degradation of the viral particles in the aphid's haemocoel. Similarly to luteovirids and geminiviruses, Banana bunchy top virus (BBTV), a member of the Nanoviridae family, is transmitted in a persistent, circulative manner and can be detected in the haemolymph of the aphid vector, Pentalonia nigronervosa. To date, it is not known if BBTV can interact with GroEL. In this study, we localised and inferred the phylogeny of a Buchnera aphidicola endosymbiont inhabiting P. nigronervosa. Furthermore, we predicted the 3D structure of Buchnera GroEL and detected the protein in the haemolymph of P. nigronervosa. Interactions were tested using 3 different assays: immunocapture PCR, dot blot, and far-western blot assays; however, none of them showed evidence of a BBTV–GroEL interaction. We concluded that it was unlikely that BBTV interacted with Buchnera GroEL either in vitro or in vivo and we discuss possible alternatives by which BBTV viral particles are able to avoid the process of degradation in the aphid haemocoel.     

Evaluation of the minimal replication time of Cauliflower mosaic virus in different hosts

Though the duration of a single round of replication is an important biological parameter, it has been determined for only few viruses. Here, this parameter was determined for Cauliflower mosaic virus (CaMV) in transfected protoplasts from different hosts: the highly susceptible Arabidopsis and turnip, and Nicotiana benthamiana, where CaMV accumulates only slowly. Four methods of differing sensitivity were employed: labelling of (1) progeny DNA and (2) capsid protein, (3) immunocapture PCR,, and (4) progeny-specific PCR. The first progeny virus was detected about 21 h after transfection. This value was confirmed by all methods, indicating that our estimate was not biased by the sensitivity of the detection method, and approximated the actual time required for one round of CaMV replication. Unexpectedly, the replication kinetics were similar in the three hosts; suggesting that slow accumulation of CaMV in Nicotiana plants is determined by non-optimal interactions in other steps of the infection cycle.     

A highly basic KGKKGK sequence in the RNA-binding domain of the Cucumber necrosis virus coat protein is associated with encapsidation of full-length CNV RNA during infection

The Cucumber necrosis virus particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. The N-terminal 58 aa residue segment of the CP R domain is believed to bind viral RNA within virions and during assembly. We report results of in vivo experiments that examine the role of the R domain in assembly. Deletion analyses identified 3 conserved 5-10 aa regions as playing critical roles. A highly basic KGKKGK sequence was found to be both necessary and sufficient for encapsidation of the full-length genome and polymorphic virions were produced in mutants lacking the KGKKGK sequence. The amount of full-length RNA present in virions was substantially reduced in R domain mutants where 2 of the 4 lysine residues were substituted with alanine, whereas substitution of 4 lysines by arginine had only a modest effect. The potential role of the R domain in formation of a scaffold for particle assembly is discussed.     

Endoplasmic reticulum targeting of the Red clover necrotic mosaic virus movement protein is associated with the replication of viral RNA1 but not that of RNA2

Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome. The movement protein (MP) encoded by RNA2 is essential for viral movement. To obtain further insights into the viral movement mechanism, subcellular localizations of RCNMV MP fused with green fluorescent protein (MP:GFP) were examined in Nicotiana benthamiana epidermal cells and protoplasts. The MP:GFP expressed from the recombinant virus first appeared in the cell wall and subsequently was observed on the cortical endoplasmic reticulum (ER) as punctate spots. In contrast, the MP:GFP expressed transiently in the absence of other viral components was localized exclusively in the cell wall. Transient expression of the MP:GFP with a variety of RCNMV components revealed that the ER localization of the MP:GFP was associated with RNA1 replication, or its negative-strand RNA synthesis, but not those of RNA2 or replicase proteins per se. A model of RCNMV cell-to-cell movement is discussed.     

Lettuce infectious yellows virus (LIYV) RNA 1-encoded P34 is an RNA-binding protein and exhibits perinuclear localization

The Crinivirus, Lettuce infectious yellows virus (LIYV) has a bipartite, positive-sense ssRNA genome. LIYV RNA 1 encodes replication-associated proteins while RNA 2 encodes proteins needed for other aspects of the LIYV life cycle. LIYV RNA 1 ORF 2 encodes P34, a trans enhancer for RNA 2 accumulation. Here we show that P34 is a sequence non-specific ssRNA-binding protein in vitro. P34 binds ssRNA in a cooperative manner, and the C-terminal region contains the RNA-binding domain. Topology predictions suggest that P34 is a membrane-associated protein and the C-terminal region is exposed outside of the membrane. Furthermore, fusions of P34 to GFP localized to the perinuclear region of transfected protoplasts, and colocalized with an ER-specific dye. This localization was of interest since LIYV RNA 1 replication (with or without P34 protein) induced strong ER rearrangement to the perinuclear region. Together, these data provide insight into LIYV replication and possible functions of P34.     

A RGG motif protein is involved in Toxoplasma gondii stress-mediated response

The molecular mechanisms controlling gene expression are still poorly understood in apicomplexan parasites. Here, we report the characterization of a homolog of the single strand binding proteins (named TgSsossB) in Toxoplasma gondii. We previously showed that TgSsossB interacts with the TgAlba proteins that are involved in translation regulation. We examined the role of TgSsossB in stress-mediated response, and particularly the role of its arginine–glycine–glycine (RGG) repeats domain. TgSsossB recombinant protein is able to bind to single strand DNA and RNA in a sequence-independent manner, but not to double stranded DNA. We showed that the RGG motif is not involved in this ability to bind to nucleic acid. We produced a mutant tagged strain lacking the RGG motif of TgSsossB using the knock-in strategy. We observed that this strain exhibited a fitness defect compared with the parental parasites. Moreover, the mutant strain produced fewer plaques in stress conditions, a defect that is due to a slow growth phenotype when extracellular parasites are exposed to stress. At the molecular level, we showed that the TgSsossB protein lacking a RGG motif lost its ability to interact with TgAlba2 and an isoform of TgAlba1, indicating that the TgAlba complex is likely non-functional in those parasites. Thus, our findings define the RGG domain of TgSsossB as a protein–protein interaction platform and underline the role of the TgAlba–TgSsossB complex in stress-mediated response.     

Identification of a Hyphantria cunea nucleopolyhedrovirus (NPV) gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPV multiplication in a B. mori cell line

We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV, vHycuΔep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycuΔep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV.     

Beet soil-borne mosaic virus RNA-3 is replicated and encapsidated in the presence of BNYVV RNA-1 and -2 and allows long distance movement in Beta macrocarpa

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) belong to the Benyvirus genus. BSBMV has been reported only in the United States, while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae and possess similar host ranges, particle number and morphology. BNYVV and BSBMV are not serologically related but they have similar genomic organizations. Field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles in plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed that BSBMV and BNYVV are sufficiently different to be classified as two species. Complementary base changes found within the BSBMV RNA-3 5′ UTR made it resemble to BNYVV 5′ RNA-3 structure whereas the 3′ UTRs of both species were more conserved. cDNA clones were obtained, and allowed complete copies of BSBMV RNA-3 to be trans-replicated, trans-encapsidated by the BNYVV viral machinery. Long-distance movement was observed indicating that BSBMV RNA-3 could substitute BNYVV RNA-3 for systemic spread, even though the p29 encoded by BSBMV RNA-3 is much closer to the RNA-5-encoded p26 than to BNYVV RNA-3-encoded p25. Competition occurred when BSBMV RNA-3-derived replicons were used together with BNYVV-derived RNA-3 but not when the RNA-5-derived component was used. Exploitation of the similarities and divergences between BSBMV and BNYVV should lead to a better understanding of molecular interactions between Benyviruses and their hosts.