Human immunodeficiency virus (HIV) infection of the central nervous system (CNS)is a significant cause of morbidity. The requirements for HIV adaptation to the CNS for neuropathogenesis and the value of CSF virus as a surrogate for virus activity in brain parenchyma are not well established. We studied 18 HIV-infected subjects, most with advanced immunodeficiency and some neurocognitive impairment but none with evidence of opportunistic infection or malignancy of the CNS. Clonal sequences of C2-V3 env and population sequences of pol from HIV RNA in cerebrospinal fluid (CSF) and plasma were correlated with clinical and virologic variables. Most (14 of 18) subjects had partitioning of C2-V3 sequences according to compartment, and 9 of 13 subjects with drug resistance exhibited discordant resistance patterns between the two compartments. Regression analyses identified three to seven positions in C2-V3 that discriminated CSF from plasma HIV. The presence of compartmental differences at one or more of the identified positions in C2-V3 was highly associated with the presence of discordant resistance (P ‫؍‬ 0.007), reflecting the autonomous replication of HIV and the independent evolution of drug resistance in the CNS. Discordance of resistance was associated with severity of neurocognitive deficits (P ‫؍‬ 0.07), while low nadir CD4 counts were linked both to the severity of neurocognitive deficits and to discordant resistance patterns (P ‫؍‬ 0.05 and 0.09, respectively). These observations support the study of CSF HIV as an accessible surrogate for HIV virions in the brain, confirm the high frequency of discordant resistance in subjects with advanced disease in the absence of opportunistic infection or malignancy of the CNS, and begin to identify genetic patterns in HIV env associated with adaptation to the CNS.

Human immunodeficiency virus type 1 (HIV-1) sequences were generated from blood and from brain tissue obtained by stereotactic biopsy from six patients undergoing a diagnostic neurosurgical procedure. Proviral DNA was directly amplified by nested PCR, and 8 to 36 clones from each sample were sequenced. Phylogenetic analysis of intrapatient envelope V3-V5 region HIV-1 DNA sequence sets revealed that brain viral sequences were clustered relative to the blood viral sequences, suggestive of tissue-specific compartmentalization of the virus in four of the six cases. In the other two cases, the blood and brain virus sequences were intermingled in the phylogenetic analyses, suggesting trafficking of virus between the two tissues. Slide-based PCR-driven in situ hybridization of two of the patients' brain biopsy samples confirmed our interpretation of the intrapatient phylogenetic analyses. Interpatient V3 region brain-derived sequence distances were significantly less than blood-derived sequence distances. Relative to the tip of the loop, the set of brain-derived viral sequences had a tendency towards negative or neutral charge compared with the set of blood-derived viral sequences. Entropy calculations were used as a measure of the variability at each position in alignments of blood and brain viral sequences. A relatively conserved set of positions were found, with a significantly lower entropy in the brain

During the course of adenovirus infection, the VAI RNA protects the translation apparatus of host cells by preventing the activation of host double-stranded RNA-activated protein kinase, which phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2. In the absence of VAI RNA, protein synthesis is drastically inhibited at late times in infected cells. The experimentally derived secondary structure of VAI RNA consists of two extended base-paired regions, stems I and III, which are joined by a short base-paired region, stem II, at the center. Stems I and II are joined by a small loop, A, and stem III contains a hairpin loop, B. At the center of the molecule and at the 3' side, stems II and III are connected by a short stem-loop (stem IV and hairpin loop C). A fourth, minor loop, D, exists between stems II and IV. To determine sequences and domains critical for function within this VAI RNA structure, we have constructed adenovirus mutants with linker-scan substitution mutations in defined regions of the molecule. Cells infected with these mutants were analyzed for polypeptide synthesis, virus yield, and eIF-2 alpha kinase activity. Our results showed that disruption of base-paired regions in the distal parts of the longest stems, I and III, did not affect function, whereas mutations causing structural perturbations in the central part of the molecule containing stem II, the proximal part of stem III, and the central short stem-loop led to loss of function. Surprisingly, one substitution mutant, sub742, although dramatically perturbing the integrity of the structure of this central portion, showed a wild-type phenotype, suggesting that an RNA with an alternate secondary structure is functional. On the basis of sensitivity to single-strand-specific RNases, we can derive a novel secondary structure for the mutant RNA in which a portion of the sequences may fold to form a structure that resembles the central part of the wild-type molecule, which suggests that only the short stem-loop located in the center of the molecule and the adjoining base-paired regions may define the functional domain. These results also imply that only a portion of the VAI RNA structure may be recognized by the host factor(s).

Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV‐1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor‐α stimulated and unstimulated ACH‐2 cells demonstrated the ability to detect <5% HIV RNA positive cells with a sensitivity of <30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV‐seropositive patients on no, single, combination, or triple drug therapy and 8 HIV‐seronegative patients were examined. The majority of HIV‐positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV‐infected cell populations in the peripheral blood of HIV‐positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease. Cytometry 31:265–274, 1998. © 1998 Wiley‐Liss, Inc.

The rate of disease progression varies considerably among human immunodeficiency virus type 1 (HIV-1)infected individuals. Several cross-sectional studies have shown an association between the stage of HIV-1 disease and the viral burden or the relative levels of viral gene expression. To study the extent of HIV-1 transcription and replication and its correlations with disease progression, we quantified serial, longitudinal samples of blood cells from 10 HIV-1-infected individuals with markedly different rates of CD4؉ T-cell number decline following seroconversion. After normalization for the input nucleic acid content, multiply spliced viral mRNA and unspliced viral RNA were quantified by competitive reverse transcription-PCR using oligonucleotide primers which flank the major tat/rev/nef splice junction and span an internal region of the gag open reading frame, respectively. Coamplification of internal cRNA template controls was used to normalize for variation in the efficiency of reverse transcription and in vitro enzymatic amplification. Similarly, proviral DNA was also quantified by competitive PCR performed within the linear range of amplification. Viral RNA was detected in the blood cells of each individual from all time points regardless of the rate of CD4 ؉ T-cell decline. Unspliced genomic viral RNA rapidly increased in the blood cells from HIV-1-infected individuals who had a precipitously declining CD4 ؉ T-cell number. In contrast, both unspliced and multiply spliced viral mRNAs remained relatively stable in the blood cells from HIV-1-infected individuals who have had a relatively benign clinical course. These data demonstrate that the extent of viral transcription and replication correlates with the rate of CD4 ؉ T-cell number decline and that quantifying intracellular viral RNA is of potential prognostic value.