The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

The present study was undertaken to examine structural features of L-chicoric acid (3) which are important for potency against purified HIV-1 integrase and for reported cytoprotective effects in cell-based systems. Through a progressive series of analogues, it was shown that enantiomeric D-chicoric acid (4) retains inhibitory potency against purified integrase equal to its L-counterpart and further that removal of either one or both carboxylic functionalities results in essentially no loss of inhibitory potency. Additionally, while two caffeoyl moieties are required, attachment of caffeoyl groups to the central linking structure can be achieved via amide or mixed amide/ester linkages. More remarkable is the finding that blockage of the catechol functionality through conversion to tetraacetate esters results in almost no loss of potency, contingent on the presence of at least one carboxyl group on the central linker. Taken as a whole, the work has resulted in the identification of new integrase inhibitors which may be regarded as bis-caffeoyl derivatives of glycidic acid and amino acids such as serine and beta-aminoalanine. The present study also examined the reported ability of chicoric acid to exert cytoprotective effects in HIV-infected cells. It was demonstrated in target and cell-based assays that the chicoric acids do not significantly inhibit other targets associated with HIV-1 replication, including reverse transcription, protease function, NCp7 zinc finger function, or replication of virus from latently infected cells. In CEM cells, for both the parent chicoric acid and selected analogues, antiviral activity was observable under specific assay conditions and with high dependence on the multiplicity of viral infection. However, against HIV-1- and HIV-2-infected MT-4 cells, the chicoric acids and their tetraacetylated esters exhibited antiviral activity (50% effective concentration (EC50) ranging from 1.7 to 20 microM and 50% inhibitory concentration (IC50) ranging from 40 to 60 microM).

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The human parvovirus B19 is now divided into three genotypes: type 1 (prototype), type 2 (A6-and LaLi-like), and type 3 (V9-like). In overall DNA sequence, the three virus types differ by ϳ10%. The most striking DNA dissimilarity, of >20%, is observed within the p6 promoter region. Because of the scarcity of data on the biological activities and pathogenetic potentials of virus types 2 and 3, we examined the functional characteristics of these virus types. We found the activities of the three p6 promoters to be of equal strength and to be most active in B19-permissive cells. Virus type 2 capsid protein VP2, alone or together with VP1, was expressed with the baculovirus system and was shown to assemble into icosahedral parvovirus-like particles, which were reactive in the hemagglutination assay. Furthermore, sera containing DNA of any of the three B19 types were shown to hemagglutinate. The infectivities of these sera were examined in two B19-permissive cell lines. Reverse transcription-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS and VP proteins in the infected cells. All three genotypes showed similar functional characteristics in all experiments performed, showing that the three virus types indeed belong to the same species, i.e., human parvovirus B19. Additionally, the antibody activity in sera from B19 type 1-or type 2-infected subjects (long-term immunity) was examined with homo-and heterologous virus-like particles. Cross-reactivity of 100% was observed, indicating that the two B19 genotypes comprise a single serotype.Human parvovirus B19, a member of the genus Erythrovirus within the subfamily Parvovirinae, has long been considered the only human pathogen of its family, in which the adeno-associated viruses of the genus Dependovirus conceivably are apathogenic. However, new parvoviruses distinct from the genus Erythrovirus were recently detected in plasma (PARV4 and PARV5) (20, 28) and in nasopharyngeal aspirates (human bocavirus) (1), the last of which is supposedly associated with severe respiratory illness in small children.Although infection with parvovirus B19 typically results in erythema infectiosum or fifth disease (4), more severe or even lethal manifestations can occur among predisposed individuals. The virus replicates in erythroid progenitor cells of bone marrow (49, 64), causing aplastic crisis in patients with hemolytic anemia of various etiologies (2, 53, 56). During pregnancy, B19 can be transmitted from the infected mother to the fetus and cause fetal hydrops and death (9). In the immunocompromised, B19 infection may remain persistently productive, leading to chronic anemia (31).The B19 virus is small and nonenveloped and encapsidates a linear single-stranded DNA genome of ϳ5.6 kb. The two genomic ends contain identical inverted terminal repeats of ϳ380 nucleotides that are imperfect palindromes and form hairpin loops (13). The genome contains only one functional promoter, p6, located in the 3Ј palindrome (15). The p6 promoter regulates...

The cellular peptidyl-prolyl isomerase cyclophilin A is incorporated into human immunodeficiency virus type 1 virions via contacts with the proline-rich domain of the Gag polyprotein. Cyclosporine A and nonimmunosuppressive analogs bind with high affinity to cyclophilin A, compete with Gag for binding to cyclophilin A, and prevent incorporation of cyclophilin A into virions; in parallel with the disruption of cyclophilin A incorporation into virions, there is a linear reduction in the initiation of reverse transcription after infection of a T cell. Passage of human immunodeficiency virus type 1 in the presence of the drug selects one of two mutations, either of which alters the proline-rich domain of Gag and is sufficient to confer drug resistance on the cloned wild-type provirus. Neither mutation alters Gag's cyclophilin A-binding properties in vitro, and cyclophilin A incorporation into drug-resistant virions is effectively disrupted by cyclosporine A, indicating that the drugresistant mutants do not require virion-associated cyclophilin A to initiate infection. That Gag's functional dependence on cyclophilin A can be differentiated genetically from its ability to bind cyclophilin A is further demonstrated by the rescue of a mutation precluding cyclophilin A packaging by a mutation conferring cyclosporine A resistance.These experiments demonstrate that, in addition to its ability to package cyclophilin A into virions, gag encodes the functional target of cyclophilin A.