The infectivity of adenovirus genomes lacking DNA sequences from their left-hand termini.

Deletions extending various distances into the left-hand terminal DNA sequences of the adenovirus type 2 (Ad2) genome were generated in a plasmid containing a cloned fragment spanning from 0 to 4.9 map units. The altered Ad2 DNA sequences were introduced into viral genomes by ligating a plasmid-derived fragment, which included the sequences extending to 3.8 map units, to the 3.8-100 map unit fragment generated by XbaI cleavage of the DNA of the Ad5 variant, d1309 (N.Jones and T.Shenk, Cell 17 683-689, 1979). The infectivity of the ligation products was studied by transfection of line 293 cells. Genomes lacking 11, 40, or 51 nucleotides from their left-hand termini, or containing an additional 18dG residues linked to this position were infectious, and analysis of the progeny virus genomes demonstrated that the structure of these modified termini had been restored to normal. In contrast, genomes from which the first 160 base pairs (bp), including the entire 102 bp left hand inverted terminal repeat (ITR), had been removed were non-infectious. The results indicate that the ITRs present at the opposite ends of transfecting DNA molecules are able to interact in vivo, and enable the production of viable viruses containing corrected left-hand terminal sequences. Possible mechanisms for this interaction are discussed.     

Human cytomegalovirus DNA. I. Molecular weight and infectivity.

Human cytomegalovirus DNA (strain AD 169) was isolated from purified virions and further purified by sucrose density gradient centrifugation. The viral DNA molecules were studied by electron microscopy and found to be linear and to have a length of 76.22 +/- 5.22 micron, corresponding to a molecular weight of 147 +/- 6.2 x 10(6). The DNA was infectious when tested in human embryonic lung cells.     

Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex.

The infectivity of adenovirus type 2 DNA and a DNA-protein complex was studied in 293 cells, a human embryonic kidney cell line transformed by sheared adenovirus type 5 DNA, and in human KB cells. Adenovirus type 2 DNA was more infectious (up to about 40-fold) in 293 cells than in KB cells, whereas a DNA-protein complex (prepared by a rapid procedure) had about the same infectivity in both cell lines. These data may mean that a factor present in 293 cells (perhaps a viral-coded protein) enhances the infectivity of free viral DNA. The infectivity of DNA and the DNA-protein complex was increased up to fivefold by brief treatment of cell monolayers with 25% dimethyl sulfoxide after transfection. Under these conditions, (i) the infectivity of native adenovirus type 2 DNA ranged from 400 to 1,300 PFU/microgram of DNA in 293 cells and from about 9 to 14 PFU/microgram of DNA in KB cells, and (ii) the infectivity of the DNA-protein complex was 6 X 10(3)to 2 X 10(4) PFU/microgram in 293 cells and 1.4 X 10(4) to 1.6 X 10(4) PFU/microgram in KB cells.     

DNA infectivity and the induction of host DNA synthesis with temperature-sensitive mutants of simian virus 40.

Host DNA synthesis is induced when CV-1 (monkey kidney) cell cultures are infected at 40 C with wild-type virions or with temperature-sensitive Simian virus 40 mutants of the "early" complementation group A. Host DNA synthesis is not induced when cultures are infected with mutants of the late complementation group D. The simplest explanation for these observations, that induction depends not upon the expression of some early gene function but rather on the presence of an active D protein in the infecting virion, has been examined. Indirect experiments suggest that this explanation is not correct. Moreover, the induction of host DNA synthesis is impaired when cultures are infected with mutants of the A group at 42.5 C rather than 40 C, suggesting that the A function may be responsible for host induction. The inability of D virions to induce host DNA synthesis may reflect their inability to "uncoat" at 40C.     

The specificity of Helicoverpa armigera stunt virus infectivity.

Helicoverpa armigera stunt virus (HaSV) is a member of the Tetraviridae family of RNA viruses whose replication and expression strategies are not well understood due to the absence of an in vitro cell culture system. We set out to find such a system for HaSV by screening an array of 13 insect and 1 mammalian cell culture lines with both virus particle infection and genomic RNA transfection. No cell line was found to be permissive for replication, although entry of genomic RNA was verified. The apparent specificity of this virus for its in vivo midgut target site was strongly corroborated by studies involving Northern blots of RNA extracted from infected insects. Only larval midgut RNA showed the presence of virus after hosts were infected per os or by injection which exposed other host cell types to the virus. The absence of replication in cell culture was due to a lack, or presence, of host factors important to replicase activity and also the likely absence of virus particle binding and entry. We thus provide both in vitro- and in vivo-based evidence demonstrating that this virus is extremely specific in the type of cells in which it will initiate an infection.     

Animal infectivity of Encephalitozoon cuniculi.

Rabbits, mice, rats and Rhesus monkeys were infected experimentally with a rabbit isolate of the mammalian microsporidan Encephalitozoon cuniculi. The lesions produced were typical of those occurring in spontaneous encephalitozoonosis in rabbits, mice, and rats, respectively. Viable E. cuniculi were recovered from tissues of injected animals with and without lesions. Titration of rabbit, mouse, and hamster isolates of E. cuniculi in mice and in rabbit choroid plexus cell cultures showed that the rabbit isolate was equally infectious for mice and cell cultures. Mouse and hamster isolates were less infectious for cell cultures than for mice. The results provide further evidence that the mouse, hamster, and rabbit isolates of E. cuniculi are identical.     

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.     

Lack of infectivity of the endogenous avian leukosis virus-related genes in the DNA of uninfected chicken cells.

The infectivity of the avian leukosis virus-related genes in the DNA of four genetically distinct types of chicken cells was determined. Infectious DNA of Rous-associated virus-O(RAV-O) was obtained from V- chicken cells which were experimentally infected with RAV-O and from V+tvbs chicken cells, which spontaneously produced RAV-O and were sensitive to exogenous RAV-O infection. However, infectious DNA of RAV-O was not obtained from uninfected V- chicken cells or from V+tvbr chicken cells, which spontaneously produced a low titer of RAV-O but were resistant to exogenous RAV-O infection. No detectable amplification of the RAV-O related DNA sequences in the V+tvbs cells was found by hybridization of RAV-O 125I-labeled RNA to the DNAs of V+tvbs and uninfected V- cells. These results indicate that the endogenous avian leukosis virus-related genes in uninfected V- and V+tvbr cells differ from the RAV-O proviruses in RAV-O-infected V- and V+tvbs cells. The lack of infectivity of the DNA of V+tvbr cells is consistent with the hypothesis that the endogenous RAV-O genome in V+tvbr cells is linked to a cis-acting control element, which results in its inefficient expression.     

The cytoplasmic domain of simian immunodeficiency virus transmembrane protein modulates infectivity.

A striking characteristic of the simian immunodeficiency virus (SIV) and of the human immunodeficiency virus type 2 (HIV-2) is the presence of a nonsense mutation in the env gene resulting in the synthesis of a truncated transmembrane protein lacking the cytoplasmic domain. By mutagenesis of an infectious molecular clone of SIVmac142, we investigated the function of the cytoplasmic domain and the significance of the env nonsense mutation. When the nonsense codon (TAG) was replaced by a glutamine codon (CAG), the virus infected HUT78 cells with markedly delayed kinetics. This negative effect was counterselected in vitro as reversion of the slow phenotype frequently occurred. The sequencing of one revertant revealed the presence of a new stop codon three nucleotides 5' to the original mutation. Deletions or an additional nonsense mutation introduced 3' to the original stop codon did not modify SIV infectivity. In contrast, the same deletions or nonsense mutation introduced in the clone in which the stop codon was replaced by CAG abolished infectivity. These results indicated that the envelope domain located 3' to the stop codon is not necessary for in vitro replication. However, the presence of this domain in SIV transmembrane protein leads to a reduced infectivity. This negative effect might correspond to a function controlling the rate of spread of the virus during in vivo infection.     

Benzo[a]pyrene-7,8-dihydrodiol: selective binding to single stranded DNA and inactivation of 0X174 DNA infectivity.

When single-stranded ØX174 DNA is exposed to certain dihydrodiol derivatives of benzo[a]pyrene and benz[a]anthracene, inhibition of viral DNA infectivity is observed. Binding studies with labeled $\text{trans}$-7,8-dihydrodiol of benzo[a]pyrene and $\text{anti}$-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide indicate that the diol preferentially reacts with single-stranded DNA, whereas the diolepoxide reacts equally well with both single- and double-stranded DNA, as well as with RNA. Also, the diol and diolepoxide derivatives show a marked difference in their capacity to complex with specific deoxyhomopolymers, i.e., Poly dI. These observations suggest that the diol and diolepoxide derivatives recognize different binding sites in nucleic acids, and that the diol derivative may play an important role in mutagenesis and carcinogenesis induced by polycyclic aromatic hydrocarbons.     

The dimorphism and infectivity of Histoplasma capsulatum

Summary By examining mycelial cultures at regular intervals during incubation at 37° C in cysteine-enriched media, it was possible to detect differences in the conversion properties of six isolates ofHistoplasma capsulatum. The organisms varied according to conversion ability, rate and degree of transformation following yeast initiation, and cysteine sensitivity. These findings were unrelated to infectivity of the mycelial phase for mice as determined by percentage recovery of isolates from the cultured organs of inoculated animals.     

In vitro synthesis of a 9 kbp terminally redundant DNA carrying the infectivity of Moloney murine leukemia virus.

Detergent-disrupted virions of Moloney murine leukemia virus synthesize a 9 kbp double-stranded infectious DNA. It contains mainly full-length, single-stranded DNA, and its infectivity and size are insensitive to digestion by the single-strand-specific S1 nuclease. Analysis of fragmentation of the DNA using restriction endonucleases has shown that it is indistinguishable from the linear double-stranded DNA synthesized in infected cells. On the basis of the positions of the cleavage sites for a number of enzymes, the 9 kbp DNA has a 575 base direct terminal repetition. It is longer than the viral RNA at both ends, evidently due to repetitive copying of segments of the RNA. Virions also synthesize an 8.4 kbp double-stranded circular DNA that lacks one copy of the terminal repetition, as well as viral DNA longer than 9 kbp. The enzymatic machinery in the virions of retroviruses therefore appears to be responsible for all the steps involved in making fully double-stranded linear and one form of circular DNA.     

The effect of chloroquine treatment on the infectivity of Plasmodium chabaudi gametocytes.

The antimalarial drug chloroquine has been reported to increase the infectivity of the forms of blood-stage malaria parasites (gametocytes) that are capable of infecting mosquito vectors. This effect has been demonstrated convincingly in the short term (12 h post treatment), although several authors have suggested infectivity enhancement a week or more after treatment. We carried out experiments to investigate the effects of chloroquine on the longer-term infectivity of gametocytes of the rodent malaria parasite, Plasmodium chabaudi, to Anopheles stephensi mosquitoes. Gametocytes of chloroquine-treated infections were significantly more infectious than untreated infections 6 and 7 days post-treatment, although not on days 8 and 9. However, this effect was most likely the result of a reduction in infectivity in untreated infections, caused by immune activity which was not so pronounced in chloroquine-treated infections. Gametocytaemia (gametocytes per r.b.c.) showed a strong positive and linear relationship with infectivity. Infectivity was not influenced by either asexual parasitaemia, asexual density or anaemia. Parsimonious interpretations of the effect of chloroquine on gametocyte infectivity are discussed.