Characterization of endonuclease activities in Moloney murine leukemia virus and its replication-defective mutants

To study Moloney murine leukemia virus (M-MulV) proteins associated with the integration of proviral DNA into the host chromosome, we isolated endonuclease activities from purified virion preparations of the wild type and two of its replication mutants. A major endonuclease activity was identified in virions of M-MuLV; the enzyme catalyzed nicks in double-stranded DNA in the presence of either Mn2+ or Mg2+ and was stimulated by ATP. The endonuclease nicked DNA adjacent to all four nucleotides with some preference for G and C. The same enzyme, and in comparable amounts, was isolated from two virus replication mutants: dl2905, deficient in the processing of Pr65gag and Pr200gag-pol, and dl50401, deficient for the virus integration function. In the process of these experiments, the residual reverse transcriptase in mutant dl2905 was shown to be the mature size, implying that the uncleaved precursor lacks enzymatic activity. It appears that the major endonuclease activity found in virions of M-MuLV is not encoded by either the gag or pol genes

Time Frames for Neutralization during the Human Immunodeficiency Virus Type 1 Entry Phase, as Monitored in Synchronously Infected Cell Cultures

Characterization of the neutralizing interaction between antibody and virus is hindered by the nonsynchronized progression of infection in cell cultures. Discrete steps of the viral entry sequence cannot be discerned, and thus, the mode of antibody-mediated interference with virus infectivity remains undefined. Here, we magnetically synchronize the motion and cell attachment of human immunodeficiency virus type 1 (HIV-1) to monitor the progression of neutralization, both in solution and following virus attachment to the cell. By simultaneous transfer of all viral particles from reaction solution with antibody to the cell-bound state, the precise rate of neutralization of cell-free virus could be determined for each antibody. HIV-1 neutralization by both monoclonal and polyclonal antibody preparations followed distinct pseudo-first-order kinetics. For all antibodies, cell types, and HIV-1 strains examined, postattachment interference served a major role in the neutralizing effect. To monitor the progression of postattachment interference, we synchronized the entry process at initiation and measured the escape of cell-bound virus from antibody. We found that different antibodies neutralized the virus over different time frames during the entry phase. Virus was observed to progress through a sequence of shifting sensitivities to different antibodies during entry, suggested here to correlate with the exposure time of the target epitope on receptor-activated viral envelope proteins. Thus, by monitoring the progression of HIV-1 entry under synchronized conditions, we identify a new and significant determinant of antibody neutralization capacity, namely, the time frames for neutralization during the course of the viral entry phase

Transgenic Mouse with the Herpes Simplex Virus Type 1 Latency-Associated Gene: Expression and Function of the Transgene

During herpes simplex virus type 1 (HSV-1) latent infection in human peripheral sensory ganglia, the major viral gene transcribed is the latency-associated transcript (LAT) gene. In order to facilitate the study of this gene, we generated a transgenic mouse that contains the DNA fragment that transcribes the LAT RNAs (2.0 kb and its 1.5-kb spliced transcript) under control of the cytomegalovirus promoter. The tissue distribution of these transcripts and their effects upon HSV-1 replication, latency, and reactivation in the transgenic-mouse model were examined. Different steady-state amounts of both transcripts were found in various tissues. While the highest levels of the 2.0-kb RNA were detected in heart and skeletal muscle, the 1.5-kb transcript was found at elevated levels in the brain and at much higher levels in the trigeminal ganglia (TG). Replication of both the wild-type and a LAT-negative mutant virus was suppressed in primary embryonic fibroblasts obtained from LAT-expressing transgenic mice compared to that in cells obtained from normal mice. HSV-1 DNA amounts in latently infected TG of transgenic mice were similar to those in normal mice. Reactivation of latent HSV-1 LAT-negative mutants by explant cocultivation of TG from transgenic mice was more efficient than reactivation from normal-mouse TG. Considering our present and previous results, we propose that the significantly higher steady-state level of the 1.5-kb RNA in the TG may link this transcript to latency functions and that by inhibition of virus replication, the LAT gene may protect ganglion cells and thereby increase the probability of reactivation

Ordered transcription of RNA tumor virus genomes

Recent improvements in the reaction conditions have allowed a detailed characterization of the DNA products synthesized in vitro by the reverse transcriptase of RNA tumor viruses from the 70 S genome. We have investigated the size distribution of DNA products synthesized in vitro under different reaction conditions using both Rous sarcoma virus (RSV) and Moloney murine leukemia virus (M-MuLV). Analysis of the reaction products on polyacrylamide gels shows that the majority of DNA synthesized, especially at high concentrations of dNTPs, comprised a set of DNA chains of discrete length the longest of which cover a major fraction of the genome. The effect of varying the time of incubation was to increase the fraction of the total DNA in longer chains. However, small DNA chains less than 200 nucleotides long were still abundant after 500 minutes. By varying which of the four dNTPs was at low concentration we revealed two classes of DNA fragments of discrete length, those present regardless of which dNTP was at low concentration (structural stops) and those which were made only when a specific dNTP was at a low concentration (sequence stops). A strong structural stop which gave a fragment 135 nucleotides long (M-MuLV) or 100 nucleotides long (RSV) comprised more than half of the population of DNA molecules even at the highest dNTP concentrations. The DNA products of the endogenous and exogenous (reconstructed) reactions were identical. The DNA chains of discrete length were initiated with a tRNA (tRNA^(Trp) RSV, tRNA^(Pro) M-MuLV). The DNA fragments of both RSV and M-MuLV have a common initiation sequence. We have sequenced the first 18 nucleotides of M-MuLV. The initial sequences of RSV (AATGAAGC) and M-MuLV (AATGAAAGA) are remarkably similar suggesting a common evolutionary ancestry. We analyzed the pyrimidine tracts of DNA chains of increasing length by electrophoresis followed by homochromatography. The pyrimidine tracts of the shorter pieces form a subset of the pyrimidine tracts of the longer pieces. These data demonstrate that the in vitro products of discrete length were initiated with a tRNA primer at a unique site along the 35 S genome and grew by linear extension