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HIV-1 replication is tightly regulated in host cells, and various restriction factors have important roles in inhibiting viral replication. SAMHD1, a well-known restriction factor, suppresses HIV-1 replication by hydrolyzing intracellular dNTPs, thereby limiting the synthesis of viral cDNA in quiescent cells. In this study, we revealed an additional and distinct mechanism of SAMHD1 inhibition during the postviral cDNA synthesis stage. Using immunoprecipitation and mass spectrometry analysis, we demonstrated the interaction between SAMHD1 and MX2/MxB, an interferon-induced antiviral factor that inhibits HIV-1 cDNA nuclear import. The disruption of endogenous MX2 expression significantly weakened the ability of SAMHD1 to inhibit HIV-1. The crucial region within SAMHD1 that binds to MX2 has been identified. Notably, we found that SAMHD1 can act as a sensor that recognizes and binds to the incoming HIV-1 core, subsequently delivering it to the molecular trap formed by MX2, thereby blocking the nuclear entry of the HIV-1 core structure. SAMHD1 mutants unable to recognize the HIV-1 core showed a substantial decrease in antiviral activity. Certain mutations in HIV-1 capsids confer resistance to MX2 inhibition while maintaining susceptibility to suppression by the SAMHD1-MX2 axis. Overall, our study identifies an intriguing antiviral pattern wherein two distinct restriction factors, SAMHD1 and MX2, collaborate to establish an alternative mechanism deviating from their actions. These findings provide valuable insight into the complex immune defense networks against exogenous viral infections and have implications for the development of targeted anti-HIV therapeutics., Importance: In contrast to most restriction factors that directly bind to viral components to exert their antiviral effects, SAMHD1, the only known deoxynucleotide triphosphate (dNTP) hydrolase in eukaryotes, indirectly inhibits viral replication in quiescent cells by reducing the pool of dNTP substrates available for viral cDNA synthesis. Our study provides a novel perspective on the antiviral functions of SAMHD1. In addition to its role in dNTP hydrolysis, SAMHD1 cooperates with MX2 to inhibit HIV-1 nuclear import. In this process, SAMHD1 acts as a sensor for incoming HIV-1 cores, detecting and binding to them, before subsequently delivering the complex to the molecular trap formed by MX2, thereby immobilizing the virus. This study not only reveals a new antiviral pathway for SAMHD1 but also identifies a unique collaboration and interaction between two distinct restriction factors, establishing a novel line of defense against HIV-1 infection, which challenges the traditional view of restriction factors acting independently. Overall, our findings further indicate the intricate complexity of the host immune defense network and provide potential targets for promoting host antiviral immune defense., Competing Interests: The authors declare no conflict of interest.

Two different BC recombinant HIV-1 strains have arisen and begun to circulate among intravenous drug users in China. The recombinants are mostly subtype C with a few small subtype B segments. Additional full-genome sequences of the two recombinants, termed CRF07_BC and CRFO&_BC, are now available for analysis. Four CRF07_BC strains, including c54, 97CNU01, 98CN009, and a new strain CNGL-179, described here, and four CRF08_BC strains, including 97CNGX-6, 97CNGX-7, 97CNGX-9, and 98CN006, were compared for their recombination breakpoints by bootscanning and software for fine mapping of recombinants. The four CRF07_BC strains shared an identical recombination structure and the four CRF08_BC strains shared an identical, but different, recombination structure. The two CRFs share five precise subtype B/C boundaries, although although other segments differ between them, suggesting that they shared a common ancestor, itself a BC recombinant that separately "back-crossed" onto different subtype C strains. Both CRFs are broadly distributed from north to south in western China and have maintained low interpatient diversity.

Guangxi Province, China recently experienced an outbreak of HIV-1 infection among heroin users. We studied HIV-1 incidence rates and associated risk factors for HIV-1 infection among heroin users residing in Pingxiang City. A total of 318 heroin users were followed from February 1998 through January 1999 (median follow-up: 8.1 months). Of these, 130 were prospectively followed from January through September 1999 (median follow-up: 8.3 months). HIV-1 and hepatitis C virus (HCV) incidence rates for each period were calculated. A generalized estimating equation approach was implemented to identify independent risk factors associated with HIV-1 infection across both periods. Among 318 study participants, 97.2% were men. The median age was 22 years. Approximately 60% reported sharing needles. HIV-1 prevalence at baseline was 15.4%. During the first follow-up period, HIV-1 incidence was 2.38 per 100 person years (py), and HCV incidence was 26.8 per 100 py. During the second follow-up period, HIV-1 incidence was 6.86 per 100 py, and HCV incidence was 28.9 per 100 py. After controlling for age and other factors, HCV seropositivity, history of sexually transmitted diseases, and sharing needles were independently associated with HIV-1 infection. These data suggest that HIV-1 incidence was rising over time in Pingxiang City, Guangxi Province. The high incidence of HCV heightens the importance of enhanced prevention programs to reduce injection and needle sharing among heroin users.

Heroin users from Guangxi province, a southern province of China that borders Vietnam in the south and Yunnan province in China in the west, were studied for prevalence and risk factors for HIV-1 infection. Viral env sequences from HIV-1-positive individuals were also determined for subtypes of HIV-1. The overall HIV prevalence among 227 heroin users was 40%. Most had used drugs for < or = 3 years. Sharing of injection equipment and unprotected sex were significantly associated with HIV-1 infection. Subtypes C and E HIV-1 were detected in infected heroin users and were sharply segregated in two geographic locations: only subtype C was found in a border city with Yunnan province, whereas only subtype E was found in a city bordering northern Vietnam. HIV-1 strains within each subtype were remarkably homogenous, with a mean intersubject DNA distance of 2.32% for subtype E and 1.13% for subtype C, respectively. Phylogenetic analysis of C2-V5 region of Guangxi subtype E env sequences revealed significant clustering with subtype E sequences from southern Vietnam and Cambodia. These results suggest that HIV-1 infection among heroin users in Guangxi represents two emerging epidemics initiated from distinct sources: one from Vietnam and another from Yunnan province. Factors associated with HIV-1 infection were not restricted to injection practices. Unprotected sexual behaviors are likely to increase the probability of HIV transmission beyond this high-risk population. Designing and implementing effective intervention strategies targeted toward both injection drug use and high risk sexual behavior are urgently needed to further reduce HIV-1 spread in China.

Evolution of HIV-1 env sequences was studied in 15 seroconverting injection drug users selected for differences in the extent of CD4 T cell decline. The rates of increase of either sequence diversity at a given visit or divergence from the first seropositive visit were both higher in progressors than in nonprogressors. Viral evolution in individuals with rapid or moderate disease progression showed selection favoring nonsynonymous mutations, while nonprogressors with low viral loads selected against the nonsynonymous mutations that might have resulted in viruses with higher levels of replication. For 10 of the 15 subjects no single variant predominated over time. Evolution away from a dominant variant was followed frequently at a later time point by return to dominance of strains closely related to that variant. The observed evolutionary pattern is consistent with either selection against only the predominant virus or independent evolution occurring in different environments within the host. Differences in the level to which CD4 T cells fall in a given time period reflect not only quantitative differences in accumulation of mutations, but differences in the types of mutations that provide the best adaptation to the host environment.

For type-C and lentiviruses, including human immunodeficiency virus type 1 (HIV-1), the pathway of virus assembly remains poorly defined, and the assembly and budding of capsids are believed to occur simultaneously at the plasma membrane of the infected cell. We have now identified two putative HIV-1 assembly intermediate complexes in infected CD4+ T cells. The first of these intermediates, a detergent-resistant complex (DRC), was identified as a large oligomer that had a density of 1.10-1.13 g/ml and was primarily composed of Pr55Gag and Pr160Gag-Pol precursors. The other putative intermediate was a detergent-sensitive complex (DSC) with a density of 1.15-1.17 g/ml, which apparently represented the products of extensive proteolytic processing of both the Pr55Gag and Pr160Gag-Pol precursors. Both complexes could be distinguished from released mature virions as well as immature viral particles. Surprisingly, the formation of DRC was not dependent upon the myristylation at the N-terminus of the Gag proteins, a signal required for plasma membrane targeting and virus production. However, the myristic acid modification was essential for the formation of DSC. These data suggest that interactions between individual Gag molecules and between Gag and Gag-Pol precursors may occur before their targeting to the plasma membrane during HIV-1 assembly. However, formation of the late virus assembly complex and productive processing of Pr55Gag and Pr160Gag-Pol precursors apparently do not occur until these precursors are targeted to the plasma membrane.

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.

The relative infectiousness of laboratory and primary human immunodeficiency virus type 1 (HIV-1) variants was evaluated in in vitro cell cultures of peripheral blood mononuclear cells or MT-2 cells and in Hu-PBL-SCID mice. HIV(MN) and syncytium-inducing primary isolates were preferentially transmitted to cells in tissue culture. HIV(Ba-L) and non-syncytium-inducing (NSI) primary isolates were more infectious in Hu-PBL-SCID mice. Phylogenetic analysis of env sequences derived from the primary isolates, from the cell cultures, and from five Hu-PBL-SCID mice was performed by using methods designed for resolving differences among closely related sequence pairs. This analysis demonstrated preferential transmission of an evolutionarily related subset of NSI variants to Hu-PBL-SCID mice. The pattern of selective transmission of a restricted range of NSI variants that is observed in the clinical setting is maintained in Hu-PBL-SCID mice and not in tissue culture systems. The Hu-PBL-SCID mouse model system, when used with appropriate phylogenetic analysis methodologies, will be useful for identifying and characterizing the more infectious HIV-1 variants that should be targeted for vaccine development.