Infection with a particularly neurovirulent strain of HIV has been hypothesized to explain why only a subset of patients develops HIV encephalitis. We studied the third hypervariable region (V3) of multiple clones from both brains and spleens of three patients who died with HIV encephalitis, to see if there was a molecular signature associated with neurological disease. Clones from the spleen and brain of individual patients showed significant nucleic acid homology and had envelope sequences characteristic of macrophage-tropic viruses. No brain-specific unique sequences were observed, suggesting that while CNS virus is macrophage tropic there is no evidence in the V3 envelope region studied to suggest a specific neurotropic variant.

The transcriptional induction of the a or immediate-early gene class of herpes simplex virus type 1 effected by the a trans-induction factor (aTIF, ICP25, VP16, Vmw65) requires an a-specific cis-acting site. Increased transcription does not result from the direct, independent binding of aTIF, but rather from an aTIFdependent formation of a protein-DNA complex containing, in addition to aTIF, at least one host cell factor. One of the host factors is a POU domain protein which recognizes an octamer element in the a-specific consensus. There is evidence that aTIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors. Previously, the gene products of UL46 and UL47 have been implicated in modulating the aTIF-dependent transcriptional induction of a genes. Our current studies have extended these analyses from a transient-expression system to a series of viral deletion mutants. In these studies we demonstrate that neither UL46nor UL47-encoded gene product, either separately or in combination, is required for viral growth in cell culture. The absence of UL47 reduces by up to 80% the ability of the virus to induce an a-regulated thymidine kinase reporter gene resident in 143TK-cells. Autoradiograms of [35S]methionine pulse-labeled infected cell proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, show that deleting UL46 and/or UL47 has no discernable effect on the synthesis of aTIF or aTIF-containing proteins. Subsequent Western immunoblot analysis, with rabbit anti-aTIF antibodies made to an otTIF-Staphylococcus aureus protein A fusion, demonstrated that the accumulation and steady-state levels of aTIF or aTIF-containing proteins was indistinguishable from that of the thymidine kinase-negative isogenic parental virus, RA305. * Corresponding author. able to complex with aTIF (3,12,21,23,24,32,37,44,49). Complex formation appears to be mediated through the POU domain present in these proteins, which recognizes an octamer element contained within the a element (gyATGN TAATgarattcyttgnggg) (3,12,18,21,24,36). A recent report suggests that aTIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors, one of which may be cell type specific (21).Previous work has identified a role for at least two additional viral factors, encoded by HSV-1 UL46 and UL47, in aTIF-dependent transcriptional induction (33,34). In transient-expression assays the gene product of UL46 was able to enhance aTIF-mediated induction of an aTK reporter construct 2to 3-fold, while the gene product of UL47 acted to reduce the levels of aTIF-mediated induction 2.5to 3-fold in the presence or absence of UL46. These results were the first report of additional viral factors involved in a-gene induction, and they predicted the presence of a regulatory gene cluster composed of UL46, UL47, and aTIF (UL48) (33,34).The goals of our current study were to investigate the roles of UL46 and UL47 in the context of the virus to determi...

Certain antibody neutralization escape mutants of HIV-1 map outside of the antibody recognition epitope, thereby suggesting the presence of nonlinear conformational domains. In an effort to begin to define the interacting regions of the HIV envelope proteins, a neutralization-sensitive clone of HIV-1, HXB2/BH10Sal-Bam, was passaged in the presence of the V3-specific monoclonal antibody 0.5beta. DNA sequence analysis of the V3 domain of the breakthrough viral populations revealed one population that retained the parental V3 genotype. Quantitative DNA sequence analysis of this breakthrough population revealed the presence of mutational "hotspots" in several envelope domains that are noncontiguous with V3. Mutations were seen throughout gp41 and the C1, V1/V2, C2, and C5 domains of gp120. In contrast, other regions of gp120, C3, V4, C4, and V5 remained totally unchanged. Within V1, three residues within a 14-amino acid stretch experienced five substitutions and in C5 three residues within a 7-amino acid stretch experienced four substitutions. This finding, that certain residues clustered within particular domains (V1/V2, C5, and gp41) experienced multiple substitutions under a defined environmental stressor, suggests that the degree of adaptive plasticity exhibited by the HIV envelope is limited. Based on this observation it may be possible, using a set of antibodies to various envelope epitopes, to discern a set of rules which explain the interactions of the various envelope domains with each other and with their environment. The insight gained into the physiologic constraints that the envelope proteins are subject to may be useful in developing therapeutic and vaccination strategies.

Recent studies using the polymerase chain reaction (PCR) have identified bacterial and viral genomic sequences in culture-negative pediatric middle ear effusions. To evaluate this technique in adults, 19 effusions were analyzed to compare bacterial and viral culture and PCR detection of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and adenovirus. Effusions from 4 subjects positive for human immunodeficiency virus (HIV) were analyzed by PCR for HIV virus. Three of 19 effusions were culture-positive for bacteria, and 0 of 19 for viruses. Fifteen of 19 effusions were PCR-positive for bacterial genomic sequences, and 0 of 19 for adenovirus. Thirteen of 15 PCR-positive specimens demonstrated S pneumoniae, 5 of 15 H influenzae, and 0 of 13 M catarrhalis. All 4 effusions from HIV-positive subjects were PCR-positive for HIV. No effusion was culture-positive and PCR-negative. These results confirm that culture-negative middle ear effusions contain genomic sequences from bacterial pathogens. Finding of HIV RNA and DNA in effusion from HIV-positives suggests replicating virus in this fluid.