Nilambar Biswal

Temperature-sensitive mutants of herpes simplex virus type 1: Isolation, complementation and partial characterization

Abstract Twenty-two temperature-sensitive mutants of herpes simplex virus type 1 isolated following mutagenesis with 5-bromodeoxyuridine, nitrosoguanidine, and ultraviolet light were classified according to their ability to complement each other at 39°, the nonpermissive temperature. Fifteen nonoverlapping complementation groups have been identified. Partial biochemical characterization of mutants has demonstrated that members of 4 groups were unable to synthesize viral DNA at 39°. Several mutants were found to differ from the wild-type virus with regard to thermal stability at 39°, suggesting that they may possess altered structural proteins.

Physicochemical properties of the DNA of herpes viruses.

Abstract The biochemical and biophysical properties of the DNA of herpes simplex virus type 1 (herpes virus-1) and type 2 (herpes virus-2), infectious bovine rhinotracheitis and pseudorabies viruses were compared. Buoyant densities in CsCl were 1.725, 1.727, 1.730 and 1.731 g/cm 3 for the DNAs of herpes virus-1, -2, infectious bovine rhinotracheitis virus and pseudorabies virus, respectively. Base composition analysis was conducted by enzymatically hydrolyzing viral DNA and calculating the nucleotide composition from elution patterns obtained from anion-exchange column. From these patterns, the guanine plus cytosine content was calculated to be 69.9% for herpes virus-1 DNA, 72.0% for herpes virus-2 DNA, and 76.7% for pseudorabies virus DNA. When thermally denatured in 0.015 M NaCl-0.0015 M sodium citrate, the DNAs of herpes virus-1, -2, infectious bovine rhinotracheitis virus and pseudorabies virus yielded average T m values of 82.6°, 82.7°, 85.6° and 86.6°, respectively. The DNAs of herpes virus-1 and -2 had the same sedimentation coefficient (56 S) in neutral Tris-saline buffer, corresponding to a molecular weight of 88·10 6 . Pseudorabies virus DNA sedimented as two components with sedimentation coefficient values of 26 S and 31 S.

Defective Virions of Herpes Simplex Viruses

Serial undiluted passage of clonally purified herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in 4 different types of cells resulted in (i) partial loss (84–98%) of infectivity and (ii) the app

Studies on the relatedness of herpesviruses through DNA-DNA hybridization

The relatedness of the DNAs of three different herpesviruses, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and pseudorabies (Pr) virus was compared by DNA-DNA hybridization on nitrocellulose membrane filters. Reciprocal and competition hybridization experiments performed under saturation conditions demonstrated a maximum homology of 70% between the DNAs of HSV-1 and HSV-2. Under similar conditions, Pr virus DNA had 8–10% complementarity with either HSV-1 or HSV-2 DNA.

Cyclic Appearance of Defective Interfering Particles of Herpes Simplex Virus and the Concomitant Accumulation of Early Polypeptide VP175

Serial passage of undiluted herpes simplex virus types 1 and 2 resulted in cyclic production of infectious and defective virions. Defective virus production was characterized by the appearance of a new species of viral DNA with a higher bouyant density in CsCl than standard viral DNA. Measurement of the infectivity titer and DNA synthesis revealed that the defective particles interfered with the replication of standard virions and stimulated the overproduction of a large molecular weight (175,000 daltons) polypeptide.

Studies on the relatedness of herpes viruses through DNA · RNA hybridization

Abstract DNA · RNA hybridization was employed to investigate homology between three strains of herpes simplex virus type 1 (herpes virus-1; oral isolates), two strains of herpes simplex virus type 2 (herpes virus-2; genital isolates, infectious bovine rhinotracheitis virus and pseudorabies.virus. Saturation hybridization demonstrated essentially complete homology between the three strains of herpes virus-1, and between the two strains of herpes virus-2, and indicated a maximum of 40% homology between herpes virus-1 and -2. These procedures also indicated that pseudorabies virus and infectious bovine rhinotracheitis virus have no significant genetic relatedness to each other or to the two herpes simplex viruses. Competition hybridization demonstrated 8% homology between pseudorabies virus and herpes virus-1.

Ribonucleotides in newly synthesized DNA of herpes simplex virus

Abstract Newly synthesized DNA of herpes simplex virus type 1 (HSV-1), obtained from primary rabbit kidney cells pulse-labeled with [ 3 H]thymidine or [ 3 H]uridine at 6 hr postinfection, was purified by two cycles of centrifugation in CsCl density gradients. These intracellular viral DNA preparations hybridized specifically with homologous HSV-1 DNA but not with host cell DNA or E. coli DNA. Upon denaturation by alkali, the [ 3 H]thymidine-labeled HSV-1 DNA cleaved to smaller pieces. The alkali-labile material in the viral DNA was identified as ribonucleotides on the basis of the following observations: (1) When labeled with [ 3 H]uridine for short periods, the labeled viral “DNA” was susceptible to RNase and NaOH, and all the radioactivity was confined to the nucleoside [ 3 H]uridine; however, upon longer labeling periods (up to 20 hr), the [ 3 H]uridine-labeled viral “DNA” became more susceptible to DNase, as most but not all of the [ 3 H]uridine was converted to deoxyribonucleosides. (2) Denaturation of [ 3 H]uridine-labeled double-stranded HSV-1 “DNA” ( ϱ Cs 2 SO 4 = 1.45 g/cm 3 ) by heat shifted the buoyant density to single-stranded DNA region ( ϱ Cs 2 SO 4 = 1.48−1.50 g/cm 3 ) but not to single-stranded RNA region; however, treatment with hot NaOH considerably reduced the radioactivity of this “DNA.” Treatment with DNase, but not with pronase, shifted the buoyant density to the heavier RNA region of the gradient. Heat-denatured DNA but not the native DNA was susceptible to single-strand specific nuclease S1.

Studies on the intracellular replicating DNA of herpes simplex virus type 1

Abstract Intracellular replicating molecules of herpes simplex virus type 1 (HSV-1) DNA were analyzed by use of restriction endonucleases and electron microscopy. HSV-1-infected rabbit kidney cells were either pulse-labeled with [ 3 H]thymidine ([ 3 H]dT) or density-labeled with 5-bromodeoxyuridine (BUdR). Replicating [ 3 H]dT-labeled HSV-1 DNA (ϱ CsCl = 1.725 g/cm 3 ) or BUdR-labeled HSV-1 DNA (ϱ CsCl = 1.750 g/cm 3 ) were isolated by CsCl density gradient centrifugation. Viral molecules with sedimentation coefficients of 56 to 84 S were isolated, concentrated, mixed with 32 P-labeled marker 56 S DNA from purified virions, and cleaved with Endo R· Eco R1 or Hind III. The highest 3 H to 32 P ratio for replicating HSV-1 DNA was found in fragments constituting the joint region of HSV-1 DNA. Electron microscopic observations of replicating molecules revealed the presence of both internal replication loops and lariat-type molecules. Though the origin and function of the lariat molecules is not known, the results presented suggest that the origin of HSV-1 DNA synthesis is located in the joint region of the S arm of the molecule.

The DNA of murine sarcoma-leukemia virus.

Abstract A DNA wih a sedimentation coefficient of 7 S (ρc s2 so 4 : = 1.423) was isolated from DNase- and RNase-treated purified virions of each of three different oncogenic RNA viruses: murine sarcoma virus, Rauscher murine leukemia virus, and avian myeloblastosis virus. The naturally occurring DNA of murine sarcoma virus constituted about 2.5% of the total viral nucleic acid and resolved into two components (ρ = 1.698 and ρ = 1.678) when centrifuged to equilibrium in a CsCl density gradient. The viral DNA was complementary to the 18 S viral RNA subunit, but not to the 37 S or 4 S subunits.

Studies on the in vivo methylation of replicating herpes simplex virus type 1 DNA

Abstract Intracellular replicating DNA molecules ( g 9 CsC1 = 1.725 g/cm 3 ) of herpes simplex virus type 1 (HSV-1) were found to be transiently methylated. Primary rabbit kidney cells infected with HSV-1 (KOS strain) were labeled with l -[ methyl - 3 H]methionine at different times during the virus growth cycle. Viral DNA was deproteinized, separated from cellular DNA by centrifugation in CsCl density gradients, and treated with NaOH to avoid any RNA contamination. Analysis of this intracellular viral DNA indicated that it was maximally methylated during 4–9 hr postinfection and the methylation of viral DNA started to decrease during later stages of infection. DNA from mature virions was not found to be methylated. The methylated base was identified as 5-methylcytosine. Nicotinamide, a potent acceptor of methyl groups, effectively inhibited the production of infectious virus particles at a concentration of 50 m M. These results suggest that methylation of HSV-1 DNA during active viral DNA synthesis is a prerequisite for infectious virus production.