We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl 1 ]RANTES(2-68), known as AOP-RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure-activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES {N-nonanoyl, des-Ser 1 [L-thioproline 2 , L-cyclohexylglycine 3 ]-RANTES(2-68)}, which is 50 times more potent than AOP-RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.

Primary effusions presenting as the sole lymphoma localization are also known as body-cavity-based-lymphoma (BCBL), and have been shown to carry Kaposi's sarcoma herpesvirus (KSHV) DNA sequences. The aim of this study was a comparative analysis of the clinical, pathologic and molecular features of BCBL and lymphomatous effusions secondary to tissue-based lymphomas occurring both in the general population and in HIV-1-infected individuals. All the lymphomatous effusion samples (seven AIDS-related and nine AIDS-unrelated) were subjected to an identical multiparameter investigation, including collection of clinical data, analysis of morphology and immunophenotype, as well as the study of viral sequences and genetic lesions. In six cases defined as BCBL (four AIDS-related and two AIDS-unrelated), the patients exhibited exclusive or predominant involvement of the body cavities. BCBL tended to display indeterminate phenotypes (4/6), whereas all AIDS-related and AIDS-unrelated lymphomatous effusions secondary to tissue-based lymphomas consistently expressed B-cell phenotype. Detection of KSHV DNA sequences was restricted to cases of BCBL (3/4 AIDS-related and 1/2 AIDS-unrelated), whereas EBV association (3/4) and expression of EBV-encoded antigens (LMP-1, 2/3; EBNA-2, 1/3) were confined to the AIDS-related BCBL. Overall, our results confirm that both AIDS-related and AIDS-unrelated BCBL preferentially associate with peculiar clinical, immunophenotypic and molecular features among lymphomatous effusions and therefore should be singled out as a specific clinico-pathologic entity.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5-and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by sequential interaction of the envelope glycoprotein with CD4 and one of two chemokine receptors, CCR5 or CXCR4 (1, 5, 10, 11). Most primary infections involve transmission of viruses using CCR5 as the preferred coreceptor (8,25). Evolution of coreceptor use by HIV-1 from CCR5 to CXCR4 is known to be associated with poorer clinical prognosis (3,8) and can be assumed to be one pathway leading to resistance to CCR5 inhibitors currently in clinical trials (28,30,34). Moreover, treatment with CCR5 inhibitors may select for minor populations of viruses with the ability to utilize CXCR4. Understanding the evolution of coreceptor switching in terms of the fitness costs to the virus is thus important. Although the sequence of the V3 variable loop of HIV-1 gp120 envelope is known to contribute to coreceptor use (6,14,18,42,51), sequence variation in or near the V1/V2 loop is also an important influence on coreceptor choice (13,22,23,31,39,47,48,52,53). We have previously characterized coreceptor switch mutants selected by rapid substitution of U87-CD4-CXCR4 cells for U87-CD4-CCR5 cells in vitro (32). Mutations confined to the V3 region were sufficient to alter coreceptor use for some virus envelopes, but other viruses required additional mutations in or adjacent to the V1/V2 region for successful coreceptor switching. These prior studies allowed analysis of the starting virus, an occasional intermediate, and the final succ...

Fitness epistasis, the interaction among alleles at different loci in their effects on fitness, has potentially important consequences for adaptive evolution. We investigated fitness epistasis among amino acids of a functionally important region of the human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein (gp120). Seven mutations putatively involved in the adaptation of the second conserved to third variable protein region (C2-V3) to the use of an alternative host-cell chemokine coreceptor (CXCR4) for cell entry were engineered singly and in combinations on the wild-type genetic background and their effects on viral infectivity were measured. Epistasis was found to be common and complex, involving not only pairwise interactions, but also higher-order interactions. Interactions could also be surprisingly strong, changing fitness by more than 9 orders of magnitude, which is explained by some single mutations being practically lethal. A consequence of the observed epistasis is that many of the minimum-length mutational trajectories between the wild type and the mutant with highest fitness on cells expressing the alternative coreceptor are selectively inaccessible. These results may help explain the difficulty of evolving viruses that use the alternative coreceptor in culture and the delayed evolution of this phenotype in natural infection. Knowledge of common, complex, and strong fitness interactions among amino acids is necessary for a full understanding of protein evolution.

The natural evolution of human immunodeficiency virus type 1 infection often includes a switch in coreceptor preference late in infection from CCR5 to CXCR4, a change associated with expanded target cell range and worsened clinical prognosis. Why coreceptor switching takes so long is puzzling, since it requires as few as one to two mutations. Here we report three obstacles that impede the CCR5-to-CXCR4 switch. Coreceptor switch variants were selected by target cell replacement in vitro. Most switch variants showed diminished replication compared to their parental R5 isolate. Transitional intermediates were more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 virus or the final R5X4 (or rare X4) variant. The small number of mutations in viruses selected for CXCR4 use were distinctly nonrandom, with a dominance of charged amino acid substitutions encoded by G-to-A transitions, changes in N-linked glycosylation sites, and isolate-specific mutation patterns. Diminished replication fitness, less-efficient coreceptor use, and unique mutational pathways may explain the long delay from primary infection until the emergence of CXCR4-using viruses.The entry of human immunodeficiency virus type 1 (HIV-1) into target cells requires binding of the viral envelope glycoprotein gp120 to CD4 and one of two chemokine receptors, CCR5 or CXCR4 (1,9,15,17,19). Although other chemokine receptors have been identified as potential entry factors, only CCR5 and CXCR4 appear to be important for infection by clinical isolates of HIV-1 (53, 54). This observation is reflected in the current nomenclature for HIV-1 coreceptor use (E. A. Berger, R. W. Doms, E. M. Fenyo, B. T. Korber, D. R. Littman, J. P. Moore, Q. J. Sattentau, H. Schuitemaker, J. Sodroski, and R. A. Weiss, Letter, Nature 391:240, 1998): R5 for viruses that use only CCR5, X4 for viruses that use only CXCR4, and R5X4 for viruses that can use both receptors. R5 virus isolates are equivalent to macrophage-tropic, non-syncytium-inducing (NSI) viruses, and R5X4 or X4 isolates are T-cell-tropic, syncytium-inducing viruses (43,44,47), with only a few interesting exceptions (51, 52). R5 HIV-1 accounts for the vast majority of primary infections regardless of the route of transmission (12,43). During the evolution of virus populations (quasispecies) within an infected individual, coreceptor switching from R5 to X4 is common in clade B HIV-1 infection (12, 43) and less common in clade C infection (29,40). The R5-to-X4 coreceptor switch is a harbinger of accelerated clinical disease progression and typically occurs after 8 to 10 years of infection (6, 11). The issue of coreceptor switching has become relevant to drug resistance as coreceptor inhibitors enter clinical trials (18,37,48,49). Coreceptor switching is one route to resistance to these compounds (37, 49).Coreceptor use maps to the variable V3 and V2 loops of gp120, and the R5-to-X4 switch is often accompanied by an increase in charged residues in the V3 loop (7,8,20,25,27,42,46). As few as one or two amino...