Transcription of the human immunodeficiency virus (HIV)-1 is controlled by the cooperation of virally encoded and host regulatory proteins. The Tat protein is essential for viral replication, however, expression of Tat after virus entry requires HIV-1 promoter activation. A sequence in the 5′ HIV-1 LTR, containing a binding site for transcription factors of the interferon regulatory factors (IRF) family has been suggested to be critical for HIV-1 transcription and replication. Here we show that IRF-1 activates HIV-1 LTR transcription in a dose-dependent fashion and in the absence of Tat. This has biological significance since IRF-1 is produced early upon virus entry, both in cell lines and in primary CD4+ T cells, and before expression of Tat. IRF-1 also cooperates with Tat in amplifying virus gene transcription and replication. This cooperation depends upon a physical interaction that is blocked by overexpression of IRF-8, the natural repressor of IRF-1, and, in turn is released by overexpression of IRF-1. These data suggest a key role of IRF-1 in the early phase of viral replication and/or during viral reactivation from latency, when viral transactivators are absent or present at very low levels, and suggest that the interplay between IRF-1 and IRF-8 may play a key role in virus latency.

The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.Components of the transcriptional apparatus that mediate the function of the transactivation domain (AD) of bovine papillomavirus type 1 (BPV1) E2 have not been characterized. The E2 AD encompasses its N-terminal 215 amino acids, and the C-terminal 125 amino acids bind the DNA sequence ACCGN 4 CGGT. Separating the AD and the DNA binding domain (DBD) is a region of approximately 100 amino acids termed the hinge. In addition to the full-length 410-amino-acid E2 protein (E2TA), the C-terminal 248 amino acids encoded by the E2 open reading frame (ORF) constitute a species (E2TR) lacking most of the AD, which retains DNA binding and represses transcription by E2TA.Within the E2 AD are two regions of net negative charge (14), a motif common in viral and cellular transcriptional activators. Specific amino acids in these regions are essential for transcriptional activation, although the distal portion of the E2 AD (amino acids 100 to 215) is also required for activity in yeast and mammalian cells (14,20,23,28). Whereas other ADs have been shown to interact with the basal factors TATA binding protein (TBP) and TFIIB, these associations have been reported not for the E2 AD but rather for its C-terminal DNA binding and dimerization domain (34, 39). These Cterminal interactions are dispensable for the function of E2, since it was observed that chimeric E2 proteins in which the E2 DBD was replaced with the GAL4 or LexA DBD activate transcription in vivo (7, 46). E2 cooperatively stimulates transcription with several cellular transcription factors, including Sp1 (27) and USF and CTF (8,19,42). Of these factors, only Sp1 has been reported to physically interact with E2 (27).E2TA also cooperates with the papillomavirus E1 protein to initiate papillomavirus DNA replication. E1 is a site-specific DNA binding protein that can unwind DNA and associate with DNA polymerase (5,36,41,48). The viral origin of replication contains binding sites for both E1 and E2 proteins (43). Although BPV1 ...

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

The N-terminal glycine of the VP4 capsid subunit of poliovirus is covalently modified with myristic acid (C14 saturated fatty acid). To investigate the function of VP4 myristoylation in poliovirus replication, amino acid substitutions were placed within the myristoylation consensus sequence at the alanine residue (4003A) adjacent to the N-terminal glycine by using site-directed mutagenesis methods. Mutants which replace the alanine residue with a small hydrophobic residue such as leucine, valine, or glycine displayed normal levels of myristoylation and normal growth kinetics. Replacement with the polar amino acid histidine (4003A.H) also resulted in a level of myristoylation comparable to that of the wild type. However, replacement of the alanine residue with aspartic acid (4003A.D) caused a dramatic reduction (about 40 to 60%) in myristoylation levels of the VP4 precursors (P1 and VPO). In contrast, no differences in modification levels were found in either VPO and VP4 proteins isolated from mature mutant virions, indicating that myristoylation is required for assembly of the infectious virion. The myristoylation levels of the VPO proteins found in capsid assembly intermediates indicate that there is a strong but not absolute preference for myristoyl-modified subunits during pentamer formation. Complete myristoylation was observed in mature virions but not in assembly intermediates, indicating that there is a selection for myristoyl-modified subunits during stable RNA encapsidation to form the mature virus particle. In addition, even though mutant infectious virions are fully modified, the severe reduction in specific infectivity of both 4003A.D and 4003A.H purified viruses indicates that the amino acid residue adjacent to the N-terminal glycine apparently has an additional role early during viral infection and that mutations at this position induce pleiotropic effects.