Ronald E. Unger

Simian immunodeficiency virus (SIVmac) exhibits complex splicing for tat, rev, and env mRNA

The simian immunodeficiency virus (SIV) is a T-lymphotropic lentivirus associated with a fatal AIDS-like disease in rhesus macaques. SIV has a complex genome encoding virion structural proteins, transactivators, and accessory genes. From lymphoid cells chronically infected with a biologically active molecular clone of SIV, SIVmac1A11, the polymerase chain reaction technique has been used to selectively amplify transcripts for viral transactivators and the envelope gene. Three species of mRNA encoding only rev, and three mRNA encoding both rev and tat were identified by nucleotide sequence analysis. They differed in the splice acceptor sites utilized upstream of the first coding exon, in the presence or the absence of noncoding exons between the major splice donor at the LTR and the splice acceptor at the first coding exons, and in the splicing pattern between the coding exons. Alternate splice acceptors were utilized between the coding exons of tat and rev, but the altered tat proteins did not differ in their ability to transactivate the SIV-LTR. The splicing for env mRNA is more complex than previously reported. Both singly and multiply spliced transcripts exist for env mRNA, and the same splice acceptor site is utilized by both rev and env mRNA.

The cloning of full-length genome segments 2, 5, 6, and 8 of bluetongue virus (BTV) serotype 17 and studies of their genetic relatedness to United States BTV serotypes.

The double-stranded (ds) RNA genome segments 2, 5, 6, and 8, which encode the outer capsid proteins P2 and P5, and the two nonstructural proteins, NS1 and NS2, respectively, of bluetongue virus (BTV) serotype 17 have been cloned into pBR322. The length of the cloned genes indicates that the entire respective dsRNA genome has been cloned in each case. The four cloned genes were used as 32P-labeled probes to hybridize to PAGE-separated United States BTV serotypes 2, 10, 11, 13, and 17. Of these, the segment 6 clone was identified as being highly conserved and hybridized to all serotypes. Segment 8 was less conserved and segment 5 even less. The segment 2 clone was serotype specific. None of the probes hybridized with dsRNA from epizootic hemorrhagic disease virus (EHDV).

Comparison of dot-blot and Northern blot hybridizations in the determination of genetic relatedness of United States bluetongue virus serotypes.

Dot-blot and Northern blot hybridization methods to determine the genetic relatedness of United States bluetongue virus serotypes 2, 10, 11, 13, and 17 were compared. Both plasmid and insert DNA probes from cloned BTV-17 dsRNA segments 2, 5, 6, and 8 were hybridized to dsRNA from the BTV serotypes and epizootic hemorrhagic disease virus (EHDV). Stringencies of hybridization were kept identical, and experiments differed only in the method in which dsRNA was applied to the membranes (dot-blot or Northern blot). The Northern blot hybridization method yielded more consistent results, and it was visually and unequivocally shown to which dsRNA segment a cDNA probe bound, since the dsRNA segments were separated by PAGE prior to blotting and hybridization. In contrast, the dot-blot hybridization method gave less consistent results. Identical results for plasmid and insert probes were obtained for Northern blot hybridizations but not for dot-blot hybridizations. At least two probes could be used simultaneously in Northern blot hybridizations.

Molecular interactions between human immunodeficiency virus type 1 and human cytomegalovirus.

In humans infected with human immunodeficiency virus type 1 (HIV-1) and in rhesus macaques infected with simian immunodeficiency virus (SIV), the cause of death appears not to be a direct result of infection by either lentivirus; instead, fatality is due to any of a variety of secondary infectious agents brought about by the ablation of the hosts immune system. Persistent infections by these heterologous viral, bacterial, or fungal pathogens are a characteristic of individuals suffering from acquired immune deficiency (AIDS).* One such viral pathogen, cytomegalovirus (CMV), is a member of the herpesvirus family of viruses and is a frequent pathogen in HIV-infected human^.^ A large percentage of dually infected individuals suffer life-threatening complications as a direct result of CMV pathogenesi~.~.~ A critical point that requires further investigation is whether infections by heterologous pathogens are merely an opportunistic response to deterioration of the hosts immune system, or whether these agents might be important cofactors in the onset of AIDS. The issue of cofactors is vitally important because therapeutic agents directed against these secondary agents will not only alleviate suffering of the individuals, but also might delay onset of frank AIDS. A potential role for heterologous cofactors is not unique to humans infected with HIV. Rhesus macaques experimentally infected with SIV suffer an AIDS-like disease,6 and rhesus CMV in terminally ill SIV-infected macaques appears to contribute to pathogenesis (A. Lackner 8c P. Vogel, unpublished results). We have been examining molecular interactions of CMV with HIV-1 and SIV using in vitro transient expression assays to

Colony hybridizations using nylon membranes and RNA probes. An improved method for screening bacterial clones containing bluetongue virus genome

Bluetongue virus (BTV) total genomic and isolated individual segment dsRNAs end-labeled with 32P were successfully used as probes in colony hybridization to detect clones of BTV genomic material. The RNA probes were highly specific for cloned BTV genomic material. DNA probes, however, gave false positive results. DNA from bacterial clones was fixed to nylon and nitrocellulose membranes. The hybridized nylon membranes could be stripped of probe and reprobed at least 6 times without loss of signal strength.