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Host range encoded by the Agrobacterium tumefaciens tumor-inducing plasmid pTiAg63 can be expanded by modification of its T-DNA oncogene complement.

Agrobacterium tumefaciens harboring pTiA6 incite unorganized tumors on Nicotiana rustica, sunflowers, carrots, and tomatoes, whereas isogenic strains of agrobacteria harboring pTiAg63 form "rooty" tumors on N. rustica and are essentially avirulent on sunflowers, carrots, and tomatoes. In this report we show that the different host range characteristics of these two plasmids were due, in part, to differences in the T-DNA oncogene complements of the plasmids. Specifically, we constructed derivatives of pTiAg63 that contained pTiA6 oncogenes 4, 6a, and 6b inserted into the TB-DNA region and found that agrobacteria harboring these plasmids could incite unorganized tumors on N. rustica, tomatoes, carrots, and the inbred sunflower line HA202R. Undefined host factors, however, also appeared to be involved in determining A. tumefaciens host range since three inbred sunflower lines, HA303B, HA89B, and HA290B, were susceptible to tumor formation by agrobacteria harboring pTiA6 but not by strains harboring pTiAg63 or the modified pTiAg63 plasmids.

Base Sequence↗

Bovine parainfluenza virus type 3 (BPIV3) fusion and hemagglutinin-neuraminidase glycoproteins make an important contribution to the restricted replication of BPIV3 in primates.

This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F(H)HN(H)) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F(H)HN(H) replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F(H)HN(H) conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F(H)HN(H) as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.

Animals↗

Myxoma virus infection of primary human fibroblasts varies with cellular age and is regulated by host interferon responses.

Recent studies have indicated a critical role for interferon (IFN)-mediated antiviral responses in the host range of myxoma virus (MV), a pathogenic poxvirus of rabbits. To investigate the contribution of IFN to MV tropism in nonleporine cells, primary human dermal fibroblasts (HDFs) were tested for permissiveness to MV infection. Low-passage HDFs that underwent fewer than 25 population doublings (PD) were fully permissive for MV infection, supporting productive virus replication and cell-to-cell spread. In contrast, early and late viral gene expression was detectable in high-passage HDF (>75 PD), but MV failed to generate infectious progeny and could not form foci in these cells. Vesicular stomatitis virus (VSV) plaque reduction assays confirmed that constitutive IFN production progressively increased as HDFs were passaged, concurrent with an increase in the expression of transcripts for type I IFN and IFN-responsive genes involved in antiviral responses. These findings correlated with the enhanced sensitivity of higher-passage HDF to inducers of type I IFN responses, such as dsRNA. Furthermore, pretreatment of low-passage HDF with type I IFN abrogated MV spread and replication while treatment of mature HDF with neutralizing antibodies to IFN-beta, but not IFN-alpha, restored the capacity to form foci. These findings emphasize the importance of post-entry events in determining the permissiveness of human cells to MV infection and support a critical role for innate type I IFN responses as key determinants of poxvirus host range and species restriction.

Cell Line↗

Genetic analysis of host range mutant viruses suggests an uncoating defect in simian virus 40-resistant monkey cells.

Host range mutations that permit simian virus 40 (SV40) to grow with increased efficiency on SV40-resistant monkey cells have been positioned within the viral B/C gene by a mapping method that relies on the coupling of specific DNA fragments. Pairs of restriction endonucleases that each cleave SV40 DNA at only one site were used to generate pairs of specific DNA fragments. Corresponding pairs of fragments were purified from host range mutant and wild-type DNA and joined in known combinations to determine the location of the host range mutations. The map position of the host range mutations was confirmed by using the same technique to generate and couple genetically marked viral DNA fragments to produce the predicted double mutants. Three different double mutants were constructed that carry both host range and temperature-sensitive A mutations. The mutations in three independently isolated host range mutant viruses are located at very close, perhaps identical, sites, because no wild type viruses were produced from the cell-mediated repair of pairwise heteroduplexes between them. The location of these host range mutations suggests that their phenotype results from mutational alteration of the major capsid protein, the product of the B/C gene. In addition it was demonstrated that monkey cells can efficiently join appropriate pairs of restriction endonuclease fragments intracellularly to produce infectious genomes. That reaction has been partially characterized. The general utility of fragment coupling (in vitro and in vivo) and heteroduplex repair for constructing and analyzing multiple mutants of SV40 is discussed.

Cell Transformation, Viral↗

Genomic polymorphism in the T-even bacteriophages.

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.

Amino Acid Sequence↗

Folding and function of repetitive structure in the homotrimeric phage P22 tailspike protein.

The Salmonella bacteriophage P22 recognizes its host cell receptor, lipopolysaccharide, by means of six tailspikes, thermostable homotrimers of 72-kDa polypeptides. Biophysical results on the binding reaction, together with high-resolution structural information from X-ray crystallography, have shed light on the interactions determining the viral host range. Folding and assembly of the tailspike protein in vitro have been analyzed in detail, and the data have been compared with observations on the in vivo assembly pathway. Repetitive structural elements in the tailspike protein, like a side-by-side trimer of parallel beta-helices, a parallel alpha-helical bundle, a triangular prism made up from antiparallel beta-sheets, and a short segment of a triple beta-helix can be considered building blocks for larger structural proteins, and thus, the results on P22 tailspike may have implications for fibrous protein structure and folding.

Bacteriophage P22↗

Comparative analysis of the pC194 group of rolling circle plasmids.

pC194-type plasmids have been isolated from widely divergent species of bacteria: Gram positive, Proteobacteria, Spirochaetes and Cyanobacteria. We have examined the three essential replication elements of these plasmids, i.e., the Rep protein, and the origins of double and single stranded synthesis. Comparative analysis of Rep protein sequences from these plasmids indicates that they are highly divergent. Those isolated from Gram positive species fall into five groups: a Bacillus group, a Lactobacillus group, a Streptococcus group and two Staphylococcus aureus groups. The two S. aureus clusters are quite separate, suggesting that there has been at least one plasmid transfer between divergent Gram positive species. The double stranded origin of replication and the active site of the Rep protein display similarities across species indicating that these motifs can function in very divergent hosts. In contrast the single stranded origin of replication is typical of the host from which the plasmid is isolated. This is exemplified by (i) pKYM where the single stranded origins are similar to the minus origins found on the single-stranded coliphages, and (ii) pTD1 (isolated from a Spirochaete), pNostoc, pMA1 and pRF1 (all isolated from Cyanobacteria) which have no sequence homology to the minus origins identified in Gram positive or Gram negative species. This points to the single stranded origin as a feature critical to the determination of the host range of the plasmid.

Amino Acid Sequence↗

In vitro differentiation of monocytoid THP-1 cells affects their permissiveness for HIV strains: a model system for studying the cellular basis of HIV differential tropism.

The prototypic macrophage-tropic HIV-1 isolate, HIV-1BaL, cannot replicate in the monocytoid cell line THP-1. After induction of differentiation by a phorbol diester, a fraction of THP-1 cells became permissive to HIV-1BaL. In contrast, this treatment decreased permissiveness for the lymphotropic isolate HIV-1LAI. Viral DNA was not synthesized in unstimulated THP-1 cells, as determined with PCR, suggesting that the block to HIV-1BaL replication in these cells occurred at an early step of the virus replicative cycle prior to or at the level of reverse transcription. Virus binding studies showed that differences in cell permissiveness for HIV-1BaL were not due to altered virus binding. Substantial amounts of HIV-1BaL bound to both undifferentiated and differentiated THP-1 cells, and this binding could not be prevented by blocking with the anti-CD4 antibody Leu3a, which did prevent the binding of HIV-1LAI to CEM T lymphoid cells. While Leu3a was very effective at preventing the infection by HIV-1LAI in CEM cells, it was less effective in preventing HIV-1BaL infection of differentiated THP-1 cells or primary macrophages. Although it is likely that molecules other than CD4 on monocytic cells can mediate binding of macrophage-tropic HIV, the binding of HIV-1BaL to THP-1 cells was not sufficient for infection, because binding was the same in nonpermissive undifferentiated cells as in permissive differentiated cells. Thus, the restriction of viral replication in this model cell system occurs at some step after virion binding. Comparison of differentiated THP-1 cells with their undifferentiated counterparts may provide an approach to defining cellular determinants of HIV host range other than CD4 expression and to characterizing the incompletely defined steps of viral entry.

Antibodies, Monoclonal↗

Infectivity analysis of two variable DNA B components of Mungbean yellow mosaic virus-Vigna in Vigna mungo and Vigna radiata.

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), a Begomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85.6%) for KA27 DNA B. The Rep-binding domain had three complete 11-nt (5'-TGTATCGGTGT-3') iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5'-ATCGGTGT-3') had a 3-nt deletion. KA27 DNA B, which exhibited 93.9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94.1%) and BC1 (97.6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMVVig is an important determinant of host-range between V. mungo and V. radiata.

Amino Acid Motifs↗

Infection of immunosuppressed mice with the abomasal nematode parasite of ruminants, Haemonchus contortus.

Infective larvae of Haemonchus contortus established in mice either immunosuppressed with the corticosteroid, dexamethasone, or the cytotoxic drug, cyclophosphamide, or treated with the histamine H2 inhibitor, cimetidine. Infections persisted for as long as the immunosuppressive treatment (7 days) and growth of larvae was similar to that seen in sheep. Virtually no larvae survived in untreated mice. Accordingly, it would appear that adaptive immunity is an important barrier against primary infection by H. contortus in mice and is a determinant of host-range for this parasite. Antibody raised in either sheep or mice against soluble extracts of adult H. contortus precipitated with different but overlapping sets of worm antigens. This suggests that the unique antigens recognized by the mouse compared with the sheep are crucial for the rapid protective responses which prevent primary infection.

Abomasum↗

A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2.

Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SVap15/21) was isolated which in chick cells produced small plaques and was temperature sensitive (ts). At 34.5 degrees this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SVap15/21 was RNA+ at both 34.5 and 40 degrees and on the basis of complementation tests carried out at 40 degrees, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SVap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SVstd). Analysis of revertants of SVap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.

Aedes↗

Antigenic and molecular characterization of subtype H13 hemagglutinin of influenza virus.

Influenza A viruses with subtype H13 hemagglutinin display an unusual host range. Although common in shorebirds, they are very rare or absent in wild ducks; additionally, H13 viruses have been isolated from a whale. To study the molecular basis for this host range, we have determined the complete nucleotide sequences of the hemagglutinin genes of three H13 influenza viruses from different species or geographical areas: A/gull/Maryland/77, A/gull/Astrachan (USSR)/84, and A/pilot whale/Maine/84. Based on the deduced amino acid sequences, H13 hemagglutinin shares the basic structure of other type A hemagglutinin subtypes such as H3, but has clearly diverged from other completely sequenced subtypes. Unique features of H13 hemagglutinin include the occurrence, near the receptor binding pocket, of residues Arg/Lys-227 and Trp-229 (H3 numbering); the significance of these are unknown. The sequence of the HA1-HA2 cleavage site resembles those of avirulent avian influenza viruses. The whale H13 hemagglutinin is similar to those from gulls, supporting the hypothesis that influenza viruses from avian sources can enter marine mammal populations but are probably not permanently maintained there. Antigenic analysis using a panel of monoclonal antibodies suggests that, like other subtypes, H13 viruses are heterogeneous, with different antigenic variants predominating in the eastern versus the western hemispheres.

Amino Acid Sequence↗

Analysis of retroviral packaging lines for generation of replication-competent virus.

Amplification of retroviral vector sequences occurs in cocultures of ecotropic and amphotropic packaging cell lines. Mixed packaging line cocultures were used to determine both the host range and time of appearance of replication-competent virus after introduction of a retroviral vector. Replication-competent virus was generated at a characteristic time for a given ecotropic and amphotropic packaging line combination. The time required to generate replication-competent virus in a packaging line varied with the number of recombination events necessary to generate replication-competent virus from the retroviral sequences present in the line. The psi 2 packaging line generated replication-competent virus within 10 days after transfection of the N2 vector into a mixture of psi 2 (ecotropic) and PA317 (amphotropic) packaging cells. Under the same conditions, it took only 3 days to develop replication-competent virus in psi 2/PA12 cocultures. The host range of replication-competent virus was used to identify the packaging line that initially generates virus. Each packaging line combination generated replication-competent virus at a characteristic time and this time period can be used as a measure of the "safety" of the packaging line and vector combination.

Animals↗

Identification of domains within gene VI of cauliflower mosaic virus that influence systemic infection of Nicotiana bigelovii in a light-dependent manner.

Gene VI of cauliflower mosaic virus strains D4 and W260 is an important determinant of systemic infection in solanaceous species (Qiu and Schoelz, 1992). To investigate whether D4 and W260 share any sequences within gene VI that determine their solanaceous host range, we characterized more completely the regions of gene VI involved in systemic infection of Nicotiana bigelovii. We found that two domains within gene VI, which corresponded approximately to the 5' third and middle third of gene VI, influenced systemic infection of N. bigelovii. Exchange of these domains between D4, W260, and CaMV strain CM1841, a strain which is unable to systemically infect any solanaceous plant, revealed different virus combinations that could specify systemic infection of N. bigelovii when plants were grown under two different lighting conditions. Systemic infection of N. bigelovii by D4/CM1841 chimeric viruses required only the 5' third (domain 1) of gene VI of D4. In contrast, systemic infection of N. bigelovii by W260/CM1841 chimeric viruses required both the domain 1 and middle third of gene VI (domain 2) of W260, as well as two other regions primarily containing W260 genes I and II, and gene IV. The genetic requirements for systemic infection by chimeric viruses were not as stringent when plants were grown under low light conditions. Specifically, domain 2 of gene VI of D4 contained sequences sufficient for D4/CM1841 chimeric viruses to systemically infect N. bigelovii at low light intensity. We sequenced gene VI of strain W260 and compared differences in the deduced amino acid sequences between W260 and the previously published sequences of D4 and CM1841. There was only one amino acid position in domain 1 of gene VI, and no sites in domain 2, in which W260 and D4 agreed with each other and differed from CM1841. Consequently, the host range studies and sequence information indicate that different sequences within gene VI of CaMV strains W260 and D4 are responsible for systemic infection of N. bigelovii.

Amino Acid Sequence↗

The persistent variant of influenza C virus carries one characteristic point mutation in RNA segment 1.

Influenza C/ Ann Arbor/1/50-pi(C/AA-pi) virus causes persistent infections in MDCK and Wi38 cells, but sets limited, wild-type like infections in other cells. Concluding that persistence itself it dependent on the host environment, we determined the nucleotide sequence of the C/AA-pi analogous gene to the basic polymerase 2 (PB2) of influenza A virus, which is known to be a determinant for the host range. C/AA-pi and the parental wild-type virus (C/AA-wt) have 16 nucleotides in common that are different to a previously published PB2 sequence (C/JJ/50). These variations, which are probably due to divergent passages histories, are scattered along the sequence and are partially found in another published isolate (C/Berlin/1/85). One single mutation, however, is unique to the persistent variant. Nucleotide 28 mutated from T to C which leads to a change of amino acid 3 from Leu to Phe. This substitution is stably associated with the persistent phenotype throughout multiple passages in different cells lines and eggs and cannot be found in any other influenza C viruses.

Base Sequence↗

Size relationships between the parasitic copepod, Lernanthropus cynoscicola , and its fish host, Cynoscion guatucupa.

The effects of the size of Cynoscion guatucupa on the size and demographic parameters of their parasitic copepod Lernanthropus cynoscicola were evaluated. Prevalence of copepods increased with host size up to fish of intermediate length, then it decreased, probably because changes in size of gill filaments affect their attachment capability, enhancing the possibility of being detached by respiratory currents. Body length of copepods was significantly correlated with host length, indicating that only parasites of an 'adequate' size can be securely attached to a fish of a given size. The absence of relationship between the coefficient of variability in copepod length and both host length and number of conspecifics, together with the host-size dependence of both male and juvenile female sizes, prevent to interpret this relationship as a phenomenon of developmental plasticity. Therefore, the observed peak of prevalence could reflect the distribution of size frequencies in the population of copepods, with more individuals near the average length. Concluding, the 'optimum' host size for L. cynoscicola could merely be the adequate size for most individuals in the population, depending, therefore, on a populational attribute of parasites. However, its location along the host size range could be determined by a balance between fecundity and number of available hosts, which increases and decreases, respectively, with both host and parasite size.

Animals↗

Molecular anatomy of Tupaia (tree shrew) adenovirus genome; evolution of viral genes and viral phylogeny.

Adenoviruses are globally spread and infect species in all five taxons of vertebrates. Outstanding attention is focused on adenoviruses because of their transformation potential, their possible usability as vectors in gene therapy and their applicability in studies dealing with, e.g. cell cycle control, DNA replication, transcription, splicing, virus-host interactions, apoptosis, and viral evolution. The accumulation of genetic data provides the basis for the increase of our knowledge about adenoviruses. The Tupaia adenovirus (TAV) infects members of the genus Tupaiidae that are frequently used as laboratory animals in behavior research dealing with questions about biological and molecular processes of stress in mammals, in neurobiological and physiological studies, and as model organisms for human hepatitis B and C virus infections. In the present study the TAV genome underwent an extensive analysis including determination of codon usage, CG depletion, gene content, gene arrangement, potential splice sites, and phylogeny. The TAV genome has a length of 33,501 bp with a G+C content of 49.96%. The genome termini show a strong CG depletion that could be due to methylation of these genome regions during the viral replication cycle. The analysis of the coding capacity of the complete TAV genome resulted in the identification of 109 open reading frames (ORFs), of which 38 were predicted to be real viral genes. TAV was classified within the genus Mastadenovirus characterized by typical gene content, arrangement, and homology values of 29 conserved ORFs. Phylogenetic trees show that TAV is part of a separate evolutionary lineage and no mastadenovirus species can be considered as the most related. In contrast to other mastadenoviruses a direct ancestor of TAV captured a DUT gene from its mammalian host, presumably controlling local dUTP levels during replication and enhance viral replication in non-dividing host tissues. Furthermore, TAV possesses a second DNA-binding protein gene, that is likely to play a role in the determination of the host range. In view of these data it is conceivable that TAV underwent evolutionary adaptations to its biological environment resulting in the formation of special genomic components that provided TAV with the ability to expand its host range during viral evolution.

Adenoviridae Infections↗

The functions and inhibition of the membrane glycoproteins of paramyxoviruses and myxoviruses and the role of the measles virus M protein in subacute sclerosing panencephalitis.

The F glycoprotein of paramyxoviruses is responsible for cell fusion and hemolysis and for virus penetration via fusion of viral and cell membranes. These functions are activated by specific proteolytic cleavage of an inactive precursor (F0) into two disulfide-linked polypeptides (F1 and F2). The susceptibility of the F0 protein to cleavage by a host protease is a major determinant of virus host range and virulence. Synthetic oligopeptides that mimic the N-terminal region of the F1 polypeptide are specific inhibitors of paramyxoviruses, and oligopeptides that mimic the N-terminus of the HA2 polypeptide of influenza virus, also generated by cleavage, specifically inhibit that virus. Antibodies to F protein prevent the spread of paramyxovirus infection via membrane fusion, but antibodies to HN protein do not, although they neutralize released virus. These results and previous findings that formalin-treated virus does not induce antibodies to F protein provide an explanation for atypical measles. The HN protein has both receptor-binding and neuraminidase activities, and Cl- inhibition of neuraminidase may modulate these antagonistic activities. Studies in patients with subacute sclerosing panencephalitis (SSPE) suggest that there is a host restriction of synthesis of the M protein of measles virus in brain cells which is involved in the abortive, persistent infection that causes SSPE.

Antibodies, Viral↗