clinicaltrials.gov Identifier: NCT00197587.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gpl20 and gp4l, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.

South Africa is one of the countries most severely affected by HIV/AIDS. At the peak of the epidemic, the government, going against consensus scientific opinion, argued that HIV was not the cause of AIDS and that antiretroviral (ARV) drugs were not useful for patients and declined to accept freely donated nevirapine and grants from the Global Fund. Using modeling, we compared the number of persons who received ARVs for treatment and prevention of mother-to-child HIV transmission between 2000 and 2005 with an alternative of what was reasonably feasible in the country during that period. More than 330,000 lives or approximately 2.2 million person-years were lost because a feasible and timely ARV treatment program was not implemented in South Africa. Thirty-five thousand babies were born with HIV resulting in 1.6 million person-years lost by not implementing a mother-to-child transmission prophylaxis program using nevirapine. The total lost benefits of ARVs are at least 3.8 million person-years for the period 2000-2005.

The matrix domain of human immunodeficiency virus type 1 Gag polyprotein was studied for its role in virus assembly. Deletion and substitution mutations caused a dramatic reduction in virus production. Mutant Gag polyproteins were myristoylated and had a high affinity for membrane association. Immunofluorescence staining revealed a large accumulation of mutant Gag precursors in the cytoplasm, while wild-type Gag proteins were primarily associated with the cell surface membrane. These results suggest a defect in intracellular transport of the mutant Gag precursors. Thus, in addition to myristoylation, the N-terminal region of the matrix domain is involved in determining Gag protein transport to the plasma membrane. Wild-type Gag polyproteins interacted with and efficiently packaged mutant Gag into virions. This finding is consistent with the hypothesis that intermolecular interaction of Gag polyproteins might occur in the cytoplasm prior to being transported to the assembly site on the plasma membrane. The Gag protein of human immunodeficiency virus type 1 (HIV-1), like those of other retroviruses, is initially synthesized as a polyprotein precursor. Gag precursors assemble at the cytoplasmic face of the plasma membrane (8). No other virion components are required for HIV-1 capsid formation (10, 19, 22, 40, 41). Concomitant with or soon after HIV-1 virions bud from infected cells, the virions undergo morphological maturation. The Gag precursor is then cleaved into smaller polypeptides by a virally encoded protease (5, 23). Mature Gag products include the matrix (MA) protein (p17), capsid (CA) protein (p24), nucleocapsid (NC) protein (P9), and a prolinerich protein (p6) (17, 18, 24, 44). Capsid proteins form the conical core structure characteristic of HIV (9). Domains within the capsid protein are critical for Gag polyprotein interaction and retrovirus particle formation (15, 39, 48). Moreover, HIV-1 p24 can self-assemble in vitro (4). p24 is phosphorylated and constitutes a major antigenic determinant (43). HIV-1 NC protein contains two Cys-His boxes which are conserved among all retroviruses. Mutations which disrupt the Cys-His motif appeared to impair packaging of the viral RNA genome into mature virions (1). Recent evidence suggests that Cys-His boxes of murine leukemic virus NC proteins are required for NC protein binding to RNA, an interaction important for complete virion maturation, protection of the encapsidated RNA from degradation, and subsequent infectivity (2). Mutations in the p6 domain do not affect virus assembly, but they were reported to block the release of assembled particles from the cell surface (12). Matrix proteins form a shell underneath the envelope in the mature virion. The HIV-1 MA protein is important for incorporation of viral envelope protein (50). It is widely believed that the MA protein provides a targeting signal for Gag

ADARs (adenosine deaminases that act on double-stranded RNA) are RNA editing enzymes that catalyze a change from adenosine to inosine, which is then recognized as guanosine by translational machinery. We demonstrate here that overexpression of ADARs but not of an ADAR mutant lacking editing activity could upregulate human immunodeficiency virus type 1 (HIV-1) structural protein expression and viral production. Knockdown of ADAR1 by RNA silencing inhibited HIV-1 production. Viral RNA harvested from transfected ADAR1-knocked-down cells showed a decrease in the level of unspliced RNA transcripts. Overexpression of ADAR1 induced editing at a specific site in the env gene, and a mutant with the edited sequence was expressed more efficiently than the wild-type viral genome. These data suggested the role of ADAR in modulation of HIV-1 replication. Our data demonstrate a novel mechanism in which HIV-1 employs host RNA modification machinery for posttranscriptional regulation of viral protein expression.Human immunodeficiency virus (HIV) uses many host cellular machineries for its replication. Posttranscriptional modifications of the HIV type 1 (HIV-1) RNA, i.e., alternative splicing, capping, and poly(A) synthesis of the viral pre-mRNA, have been well described (15). However, the process of RNA editing, another subtle type of pre-mRNA modification, is still not well defined, and its role in HIV-1 replication is in doubt.RNA editing was found to be an important process in regulating gene expression in many eukaryotes and viruses. In hepatitis D virus, the process of RNA editing has been well studied (reviewed in reference 3). A-to-I editing converts a stop codon into a Trp codon, providing envelope protein for virion assembly (4, 25). In the hepatitis C replicon, ADAR1 was shown to abolish replication through editing (33). Hypermutation by RNA editing was found in measles virus mRNA. It is speculated that this process leads to persistence of the virus (5). The editing has also been found in other viruses, such as parainfluenza virus 3 (9), mumps virus (22), and Ebola virus (28).Evidence of RNA editing in HIV-1 has also been demonstrated. Edited adenosine in the (transactivating response region) (TAR) when the virus is injected into Xenopus oocytes has been reported (31), though the functional consequences of this conversion are not known. It is hypothesized that the phenomenon caused a change in the secondary structure of TAR, which may affect transcription and translation of the viral RNA. Base modification of A to G and C to U has also been shown in the HIV-1 transcript (1). The modification was proposed to play a role in modulation of viral gene expression.There are many data suggesting that RNA editing might be involved in HIV-1 gene expression. ADARs are the RNA editing enzymes of interested that may be involved in modulation of HIV-1 expression. It was demonstrated that mouse ADAR1 but not ADAR2 is upregulated in lymphocytes in response to inflammation (34). In this study, we demonstrated that ADARs could enhance H...