A highly sensitive single-round infection assay using a bacterial chloramphenicol acetyltransferase was developed to analyze an early stage of human immunodeficiency virus type 1 replication. By a combination of transfection and single-round infection assay, a virus with a vifmutation, depending on host cells from which the virus was derived, was demonstrated to be defective at the early phase of infection cycle. Analysis of viral proteins synthesized in cells indicated that incorporation of the Env surface protein into virions of the vif mutant, again in a cell-dependent way, was greatly restricted. Taken together, it is concluded that the Vif protein acts through modulation of the Env protein in the virions, directly or indirectly, to enhance viral infectivity in a certain cell type. Of accessory genes (4) of human immunodeficiency virus type 1 (HIV-1), the vifgene is essential for virus growth in a

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.

The dimer initiation site/dimer linkage sequence (DIS/DLS) region in the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play important roles in various steps of the virus life cycle. However, due to the presence of a putative DIS/DLS region located within the encapsidation signal region (E/psi), it is difficult to perform a mutational analysis of DIS/DLS without affecting the packaging of RNA into virions. Recently, we demonstrated that duplication of the DIS/DLS region in viral RNA caused the production of partially monomeric RNAs in virions, indicating that the region indeed mediated RNA-RNA interaction. We utilized this system to assess the precise location of DIS/DLS in the 5 region of the HIV-1 genome with minimum effect on RNA packaging. We found that the entire lower stem of the U5/L stem-loop was required for packaging, whereas the region important for dimer formation was only 10 bases long within the lower stem of the U5/L stem-loop. The R/U5 stem-loop was required for RNA packaging but was completely dispensable for dimer formation. The SL1 lower stem was important for both dimerization and packaging, but surprisingly, deletion of the palindromic sequence at the top of the loop only partially affected dimerization. These results clearly indicated that the E/psi of HIV-1 is much larger than the DIS/DLS and that the primary DIS/DLS is completely included in the E/psi. Therefore, it is suggested that RNA dimerization is a part of RNA packaging, which requires multiple steps.

The dimerization initiation site (DIS) and the dimer linkage sequences (DLS) of human immunodeficiency virus type 1 have been shown to mediate in vitro dimerization of genomic RNA. However, the precise role of the DIS-DLS region in virion assembly and RNA dimerization in virus particles has not been fully elucidated, since deletion or mutation of the DIS-DLS region also abolishes the packaging ability of genomic RNA. To characterize the DIS-DLS region without altering packaging ability, we generated mutant constructs carrying a duplication of approximately 1,000 bases including the encapsidation signal and DIS-DLS (E/DLS) region. We found that duplication of the E/DLS region resulted in the appearance of monomeric RNA in virus particles. No monomers were observed in virions of mutants carrying the E/DLS region only at ectopic positions. Monomers were not observed when pol or env regions were duplicated, indicating an absolute need for two intact E/DLS regions on the same RNA for generating particles with monomeric RNA. These monomeric RNAs were most likely generated by intramolecular interaction between two E/DLS regions on one genome. Moreover, incomplete genome dimerization did not affect RNA packaging and virion formation. Examination of intramolecular interaction between E/DLS regions could be a convenient tool for characterizing the E/DLS region in virion assembly and RNA dimerization within virus particles.Retrovirus RNAs packaged into virions are dimeric. The association between the two RNA molecules is noncovalent because the dimeric RNA dissociable into monomers under mild denaturing conditions, such as incubation at high temperature (ϳ70°C) or treatment with denaturing reagents (for a review, see references 14 and 24). Electron microscopic analysis of genomic RNA dimers obtained from several species of retroviruses reveals a symmetrical form with a contact point between the RNA situated in a region near the 5Ј end (4,5,19,27,31,32,37,48,57). It is likely that the presence of two genomes in single virus particles is advantageous for virus survival, facilitating recovery from physical damage to the RNA or providing genetic variety to the virus progeny (15, 28).Synthetic RNA fragments derived from the 5Ј region of retrovirus RNA can spontaneously dimerize in vitro upon incubation in appropriate buffer without protein factors (3, 6, 9, 11, 13, 16-18, 25, 26, 29, 33, 39, 51, 53, 56, 58). The contact point between the two monomers that constitute in vitro-synthesized dimers is referred to as the dimer linkage sequences (DLS). In human immunodeficiency virus type 1 (HIV-1), the 5Ј untranslated region just downstream of the splicing donor (s.d.) was first reported to be a DLS, because the RNA fragments harboring deletions in this region have formed dimers in vitro at significantly reduced efficiency (3,39,58). Recently, several groups reported that another site within the 5Ј untranslated region is also important for RNA dimerization in vitro. This site is located upstream of the 5Ј s.d. and designated the dim...